Document technical information

Format pdf
Size 1.6 MB
First found Jun 9, 2017

Document content analysis

not defined
no text concepts found


Henry Pownall
Henry Pownall

wikipedia, lookup




The Graduate School of Biomedical Sciences
Baylor College of Medicine
Thursday, October 23, 2014
The Graduate School of Biomedical Sciences
Baylor College of Medicine
Houston, Texas
October 23, 2014
Program ....................................................................................... iii
Awards Ceremony ...................................................................... iv
Welcome ........................................................................................ v
Graduate Student Council ......................................................... vi
Message from Dr. Klotman ....................................................... vii
Message from Dr. Johnson ....................................................... viii
The Joseph L. Melnick Lecturer ............................................... ix
Awards 2013-14 ............................................................................ x
Acknowledgements ................................................................... xiii
Cover Legend:
“Embryonic stem cell (ESC)-derived neurons on ESC-derived glial cells” Mouse embryonic
stem cells were genetically targetd to constitutively express a red fluorescent reporter, then
differentiated into neurons (green, stained with the neuronal marker beta III-tubulin) and glial
cells (red). Blue marks DAPI-positive nuclei.” Developmental Biology student Isabella
Garcia from Dr. Benjamin Arenkiel’s lab.
A publication of The Graduate School of Biomedical Sciences, Baylor College of Medicine, Houston, TX 77030
Oral Presentations – Cullen
Poster Presentations Rayzor Lounge
9:15 – 9:30
9:30 – 9:35
9:35 – 9:40
9:40 – 9:45
9:45 – 10:00
10:00 – 10:15
10:15 – 10:30
10:30 – 10:45
10:45 – 11:00
Breakfast (Coffee & Pastries provided)
Announce Poster Finalist
Sarah Hein
Deborah L. Johnson, Ph.D.
Gayle Slaughter, Ph.D.
Carolyn Adamski Biochemistry & Molecular Biology (pg 8)
Shrenik Mehta Pharmacology (pg 9)
Corey Hecksel Molecular Virology & Microbiology (pg 10)
Redwan Huq Molecular Physiology (pg11)
Break (Coffee & Snacks provided)
The Joseph L. Melnick Distinguished Guest Speaker
11:00 – 12:00
Mina Bissell, Ph.D.
“Why don't we get more cancer? The crucial role of Extracellular
Matrix and Microenvironment in metastasis and dormancy.”
12:00 – 1:15
1:15 – 1:30
1:30 – 1:45
1:45 – 2:00
2:00 – 2:15
2:15 – 2:30
2:30 – 2:45
2:45 – 3:00
3:00 – 3:15
3:15 – 3:30
3:30 – 3:45
3:45 – 4:00
4:00 – 5:00
Finalist Poster Session
Molecular & Human Genetics (pg 12)
Developmental Biology (pg 13)
Molecular & Cellular Biology (pg 14)
Clinical Scientist Traning Program (pg 15)
Structural & Comp. Biology (pg 16)
Jessica Sowa
Andrew Folick
Jonathan Shepherd
Maria Monica Gramatges
Corey Hryc
Break (Coffee & Snacks provided)
Integrative Molec & Biomed Sciences (pg 17)
Translational Biology & Mol. Med. (pg18)
Neuroscience (pg 19)
Immunology (pg 20)
Lauren Figard
Tabassum Majid
Daniel Zollinger
Xiaoyi Yuan
Break (Coffee & Snacks provided)
Awards Ceremony & Reception
Awards Ceremony & Reception
Thursday, October 23, 2014
4:00 – 5:00 PM
8-Stranded Beta-Barrel Jelly Roll Awards
Professor John J. Trentin Scholarship Awards
Deborah K. Martin Achievement Award in Biomedical Sciences
Marc Dresden Excellence in Graduate Education Award
Milton Gregory Poster Awards
Beckman Poster Awards
Mavis P. Kelsey Student Speaker Awards
Beckman Platform Award
Abstract Book can be located at:
Welcome back to the Annual GSBS Graduate Student Symposium! Thank you for
joining us to honor the graduate students performing exceptional research here at the Baylor
College of Medicine.
Every year, students from across the graduate school take a break from their busy
scientific schedules to showcase their work. These poster and speaker presentations promote
the free sharing of ideas and foster communication and collaboration between different lab
groups. Furthermore, each department has competitively selected an outstanding student for a
platform presentation. In addition to the exceptional student work, we are particularly excited
to have Dr. Mina Bissell, a distinguished scientist and senior advisor at the Lawrence Berkeley
National Laboratory, here to deliver the Joseph L. Melnick Distinguished Lecture.
We owe a special thanks to the students and faculty who have made today possible. The
members of the Graduate Student Council have spent many hours during the planning and
execution of the symposium and other student events throughout the year, and for their efforts
we are sincerely grateful. Special thanks are needed for Melissa Houghton and Dr. Gayle
Slaughter for their critical roles in planning and organizing the symposium today. It would not
be possible without their efforts every year. We also thank our poster and presentation judges
who volunteered their time today to make this a successful symposium.
In addition, we would like to extend our sincere gratitude to Dr. Deborah Johnson for
her mentorship of the GSC and the student body this year. We would like to thank the faculty
and staff members here at BCM who work tirelessly to ensure that our students receive the best
educational experience possible. We would especially like to recognize Dr. Gad Shaulsky, Dr.
Carolyn Smith, Dr. Rick Sifers, Emily Legler, Codysue Congdon and all of our faculty mentors
and program administrators for their continued dedication. And of course, we extend our
thanks to Dr. Mina Bissell for joining us here today to deliver the Distinguished Lecture.
It has been our privilege and pleasure to serve you over the last year and we hope that
you enjoy today’s Symposium.
Sarah Hein, GSC President
Timothy Dosey, GSC Vice President
Sarah Hein, President
Tim Dosey, Vice-President
Jessica Moore
Jessica Scott
Jordan Kho
Amulya Sreekumar
Ninad Oak
Jonathan Gallion
Jenny Sun
Biochemistry & Molecular Biology
Jessica Lunsford
Integrative Molec & Biomed Sciences
Shrenik Mehta
Developmental Biology
Cameron Landers
Molecular and Cellular Biology
Amanda Koire
Molecular and Human Genetics
Marissa Scavuzzo
Structural & Computational Biology
Chinh Nguyen
Molecular Virology & Microbiology
Translational Biol. & Molec.
1st year Representative
Each year the Graduate Student Symposium of the Graduate School of Biomedical
Sciences at Baylor College of Medicine takes us into a new era of research as the School’s
trainees present the results of their work in the laboratory. Their work demonstrates two of the
College’s important missions – research and education.
While each student presents the work he or she has done in the laboratory, that work
could not be accomplished without the input of their mentors, the scientists whose presence
makes Baylor College of Medicine one of the top research institutions in the United States and,
indeed, the world. The work of Baylor’s graduate students is an important part of the output of
each basic and translational laboratory in the College. Such work not only advances the mission
of Baylor, it also provides promising new information that can be translated into better diagnosis
and treatment for some of the most difficult diseases afflicting mankind around the globe.
In my tenure as President of Baylor College of Medicine, I have found that my
interactions with graduate students are among the most challenging and interesting. Our graduate
students hail from Texas and a variety of states and nations around the world, yet they work well
together in the College’s laboratories, setting up the possibility of collaborations for decades to
come. This is the kind of work and cooperation that results in answers to challenges in disease
and biology.
Fostering such work and training tomorrow’s scientists is the reason that the Baylor
Graduate School of Biomedical Sciences exists. Each year, the School’s leadership and I are
honored to take part in this important event that displays our students’ work.
On behalf of the Baylor College of Medicine faculty, I congratulate the students, their
faculty mentors and the leadership of the Graduate School for creating an outstanding Graduate
Student Symposium for 2014. Their continued success is important to the future of Baylor
College of Medicine, the Graduate School of Biomedical Sciences and our global community.
Paul Klotman, M.D.
President & CEO
Executive Dean
Baylor College of Medicine
The Graduate Student Research Symposium is a wonderful platform that highlights the
enormous breadth of research contributions made by our graduate students at Baylor College of
Medicine. It commemorates the hard work and diligence of our students that represent the next
generation of scientific leaders. The day fosters stimulating discussions that enhance our
students’ ability to articulate their work and presents opportunities for networking and new
I would like to thank the many individuals that made this Symposium possible. I would
like to specifically thank our judges for critiquing the platform presentations and posters,
Melissa Houghton for assembling the abstract book, and the Graduate Student Council who
planned the scientific program.
I am delighted to be part of this special event and hope you will take the time to
participate and celebrate the tremendous efforts and productivity of our students and their
Deborah Johnson, Ph.D.
Dean, The Graduate School of Biomedical Sciences
The William R. Brinkley BRASS Chair
Professor, Molecular & Cellular Biology
Baylor College of Medicine
Mina J. Bissell, Ph.D.
Faculty Member at University of California, Berkeley,
Distinguished Scientist and Senior Advisor at Lawrence
Berkeley National Laboratory
Dr. Bissell is a visionary and pioneer in the area of the role of extracellular matrix (ECM) and
microenvironment in regulation of tissue-specific gene expression with special emphasis in breast cancer,
where she has changed a number of established paradigms. She earned an A.B. with honors in chemistry
from Harvard College and a Ph.D. in bacterial genetics from Harvard University. She joined the Lawrence
Berkeley National Laboratory in 1972, became Director of Cell & Molecular Biology in 1988, and was
appointed Director of all of Life Sciences in 1992. Upon stepping down as the Life Sciences Division
Director, she was named Distinguished Scientist. She was also the OBER/DOE Distinguished Scientist
Fellow in Life Sciences.
Dr. Bissell has authored more than 380 publications, is a member of nine international scientific
boards, and is on the editorial board of a dozen scientific journals. She has given more than 120 ‘named
and distinguished’ lectures and has been both a Fogarty (1983) and Guggenheim (1992) Fellow. Her
awards include the E.O. Lawrence Award and Medal (1996), the Mellon Award from the University of
Pittsburgh (1998), the Clowes/Eli Lilly Award from AACR (1999), the first “Innovator Award” of the US
DOD for breast cancer research (2002), the Brinker Award from Komen Foundation (2003), the Discovery
Health Channel Medical Honor and Medal (2004), the H. Lee Moffitt Cancer Center Ted Couch
Lectureship and Award (2007), the Pezcoller Foundation–AACR International Award for Cancer Research
(2007), the 2007 Inserm/France Foreign Scientist of the Year Award , the 2007 FASEB Excellence in
Science Award, the 2008 American Cancer Society's Medal of Honor for Basic Research, a 2009
Rothschild-Mayent Fellowship award by Institut Curie, the 2010 Alexander Bodini Foundation Prize for
Scientific Excellence in Medicine from American-Italian Cancer Foundation's The. In 2011 Breast Cancer
Research Foundation's Jill Rose Award; the 2011 Susan Bulkeley Butler Leadership Excellence Award,
and the 2012 AACR Distinguished Lectureship in Breast Cancer Research as well as the Lifetime
Achievement Award, Lawrence Berkeley National Laboratory, and the California State Assembly STEM
Woman of the Year (2014) Award. In 2008, University of Porto, Portugal created the Mina J. Bissell Award
to be given every 2 years to a person who has changed a field.
Dr. Bissell has been elected a Fellow of the AAAS, the Institute of Medicine of the National
Academies, the American Academy of Arts and Sciences, the American Philosophical Society, The Royal
Society of Chemistry, the National Academy of Sciences, and in 2013 was inducted into the inaugural
class of Fellows in the AACR Academy. She has served as President of the American Society of Cell
Biology and the International Society of Differentiation. She has received honorary doctorates from Pierre
& Marie Curie University in Paris and the University of Copenhagen.
The Graduate Student Research Symposium’s Joseph L. Melnick Guest Lectureship is made possible
through a generous endowment from the late Joseph L. Melnick, Ph.D., the first Dean of the Graduate
School of Baylor College of Medicine. Dr. Melnick served in that capacity from 1968-1991.
Ryan Ash, Neuroscience
Abhisek Bhattacharya, Immunology (2nd place)
Viktor Feketa, Molecular Physiology (3rd place)
Fatih Semerci, Developmental Biology
1st Place
William Choi – Developmental Biology
Laura Heckman – Molecular & Human Genetics
Fatih Semerci– Developmental Biology
Liuliu Zheng– Biochemistry and Molecular Biology
2nd Place
Shawn Badal – Translational Biology & Molecular Medicine
Abhisek Bhattacharya – Immunology
Antentor Hinton– Integrative Molecular & Biomedical Sciences
Tongchao Li– Developmental Biology
3 Place
Alexander Herman – Developmental Biology
Teng-Wei Huang– Developmental Biology
Jennifer Johnson – Neuroscience
Stephanie Kyle – Molecular & Human Genetics
Honorable Mention
Stefanie Alexander – Integrative Molecular & Biomedical Sciences
Viktor Feketa – Molecular Physiology/Cardio. Sciences
Jeffrey Howard – Translational Biology & Molecular Medicine
Amy Ku– Translational Biology & Molecular Medicine
Aditya Kulkarni – Biochemistry and Molecular Biology
Antonina Kurtova – Integrative Molecular & Biomedical Sciences
awards are given by the first year graduate students at the end of their first year. The awards
are in recognition of outstanding educational achievement in the GSBS Service Curriculum.
John Trentin, former Professor and Head of the Division of Experimental Biology in the
Department of Surgery at Baylor from 1960-1992. These awards are given yearly to the
graduate students with the top GPA’s upon completion of their first year in graduate school.
SCIENCES—Established in 1996, this award is supported by the Deborah K. Martin fund in
memory of the late Debbie Martin, former administrator of the Cell and Molecular Biology
Program who died of breast cancer. This award is given to a graduate student in recognition of
his/her achievements in Graduate School.
memory of the late Marc Dresden, Ph.D. Dr. Dresden joined the Baylor faculty in 1968 and
was appointed Assistant Dean of The Graduate School in 1979. Dr. Dresden was Associate
Dean from 1986 until his death in 1990. This award is supported by the Marc Dresden
Excellence in Graduate Education Fund. The award recipient is selected each year by the
Graduate Student Council from nominations submitted by graduate students. The faculty
recipient is chosen for his/her outstanding contributions to graduate education as a
distinguished teacher and mentor.
John Wilson, Ph.D., Biochemistry & Molecular Biology
Kristin Karlin, Integrative Molecular & Biomedical Sciences
Ryan Gomoto -- Structural & Computational Biology & Molecular Medicine
Hsin-I Jen -- Developmental Biology
Kathleen Seger -- Integrative Molecular & Biomedical Sciences
Heidi Martini-Stoica -- Translational Biology & Molecular Medicine and
MD/PhD program
Nicholas Neill – Molecular and Human Genetics
Victor Onuchic -- Structural & Computational Biology & Molecular Medicine
Martin Powers – Developmental Biology
Jenny Sun – Neuroscience
Edgar Walker – Neuroscience
Xing Wei -- Integrative Molecular & Biomedical Sciences
Quan Zhou -- Structural & Computational Biology & Molecular Medicine
Dr. Andy Groves, Neuroscience
Dr. Joanna Jankowsky, Neuroscience
Dr. Steen Pedersen, Molecular Physiology
Abhisek Bhattacharya, Immunology
We are grateful to the following for their continued support of the Symposium:
JOSEPH L. MELNICK, Ph.D. whose endowment supports The Joseph L. Melnick
Distinguished Guest Lecturer and the Symposium Awards Reception. Dr. Melnick was the
first Dean of the Graduate School of Baylor College of Medicine and served in that capacity
from 1968 to 1991.
was the Michael E. DeBakey Professor of the Department of Pharmacology. Dr. Busch was
the Chairman of the Department of Pharmacology from July 1, 1960 to June 30, 1998.
MAVIS P. KELSEY AWARDS—The Mavis P. Kelsey fund supports outstanding research
by graduate students. This endowment will provide a monetary award for each graduate
student who is selected to speak for his or her program.
MILTON GREGORY AWARDS—The Milton Gregory endowment will support the
twelve poster awards at the annual symposium. The funds are in honor of outstanding research
by graduate students.
by Beckman Instruments, Inc. Beckman Instruments of Fullerton, California, operates on a
worldwide basis distributing products used in laboratories for biological analysis in all phases
of the battle against disease, from pioneering medical research through drug discovery to
clinical diagnostics.
established in 2013 by the BCM Alumni Affairs and will support the student speaker awards.
Table of Contents
Acevedo-Rodriguez, Alexandra ..................................................................................... 1
Department of Neuroscience
Advisor: Shailaja Mani, Ph.D. - Department of Molecular & Cellular Biology
Benjamin Arenkiel, Ph.D.-Department of Molecular & Human Genetics
Adamski, Carolyn Joy ..................................................................................................... 2
Department of Biochemistry & Molecular Biology
Advisor: Timothy Palzkill, Ph.D. - Department of Pharmacology
Ahmed, Saeed ................................................................................................................ 3
Clinical Scientist Training Program
Advisor: Thomas Giordano, M.D. - Department of Medicine
Elizabeth Chiao, M.D./M.P.H.-Department of Medicine
Akinfenwa, Patricia Y..................................................................................................... 4
Program in Translational Biology & Molecular Medicine
Advisor: Richard Hurwitz, M.D. - Department of Pediatrics
Donald Parsons, M.D./Ph.D.-Department of Pediatrics
Alexander, Stefanie ........................................................................................................ 5
Integrative Program in Molecular and Biomedical Sciences
Advisor: Brendan Lee, M.D./Ph.D. - Department of Molecular & Human Genetics
Allen, Hunter Marshall ................................................................................................... 6
Department of Neuroscience
Advisor: Hui-Chen Lu, Ph.D. - Department of Pediatrics
Amin, Samirkumar B ...................................................................................................... 7
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Lynda Chin, M.D. - Genomic Medicine
Amin, Viren R ................................................................................................................. 8
Department of Molecular & Human Genetics
Advisor: Aleksandar Milosavljevic, Ph.D. - Department of Molecular & Human Genetics
Araya, Mussie K ............................................................................................................. 9
Department of Molecular Physiology & Biophysics
Advisor: William Brownell, Ph.D. - Department of Otolaryngology-Head and Neck Surgery
Arnold, James Michael ................................................................................................. 10
Department of Biochemistry & Molecular Biology
Advisor: Arun Sreekumar, Ph.D. - Department of Molecular & Cellular Biology
Ash, Ryan Thomas ........................................................................................................ 11
Department of Neuroscience
Advisor: Stelios Smirnakis, M.D./Ph.D. - Department of Neurology
Atkins, Lisa Michelle .................................................................................................... 12
Department of Molecular Virology & Microbiology
Advisor: Joseph Petrosino, Ph.D. - Department of Molecular Virology & Microbiology
Atri, Benu ..................................................................................................................... 13
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Olivier Lichtarge, M.D./Ph.D. - Department of Molecular & Human Genetics
Bachman, Benjamin Judson .......................................................................................... 14
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Olivier Lichtarge, M.D./Ph.D. - Department of Molecular & Human Genetics
Badal, Shawn Samson ................................................................................................... 15
Program in Translational Biology & Molecular Medicine
Advisor: Farhad Danesh, M.D. - Department of Medicine
William Mitch, M.D.-Department of Medicine
Bader, David A ............................................................................................................. 16
Department of Molecular & Cellular Biology
Advisor: Sean Mcguire, M.D./Ph.D. - Department of Molecular & Cellular Biology
Baete, Dillon Patrick ..................................................................................................... 17
Department of Neuroscience
Advisor: Mauro Costa-Mattioli, Ph.D. - Department of Neuroscience
Bajaj, Lakshya ............................................................................................................. 18
Department of Molecular & Human Genetics
Advisor: Marco Sardiello, Ph.D. - Department of Molecular & Human Genetics
Balderas, Miriam ......................................................................................................... 19
Department of Molecular Virology & Microbiology
Advisor: Anthony Maresso, Ph.D. - Department of Molecular Virology & Microbiology
Barrasso, Anthony Patrick ............................................................................................ 20
Integrative Program in Molecular and Biomedical Sciences
Advisor: Loning Fu, Ph.D. - Department of Pediatrics
Barry, Meagan Amelia .................................................................................................. 21
Program in Translational Biology & Molecular Medicine
Advisor: Peter Hotez, M.D./Ph.D. - Department of Pediatrics
Laila Woc-Colburn, M.D.-Department of Medicine
Benton, Susan Michelle ................................................................................................ 22
Integrative Program in Molecular and Biomedical Sciences
Advisor: Jason Heaney, Ph.D. - Department of Molecular & Human Genetics
Bertolet, Grant Daniel ................................................................................................... 23
Department of Pathology & Immunology
Advisor: Dongfang Liu, M.D./Ph.D. - Department of Pediatrics
Birol, Onur ................................................................................................................... 24
Program in Developmental Biology
Advisor: Andrew Groves, Ph.D. - Department of Neuroscience
Bondar, Vitaliy V .......................................................................................................... 25
Department of Molecular & Human Genetics
Advisor: Huda Zoghbi, M.D. - Department of Pediatrics
Breaux, Meghan ........................................................................................................... 26
Integrative Program in Molecular and Biomedical Sciences
Advisor: Nikolaj Timchenko, Ph.D. - Department of Pathology & Immunology
Frederick Pereira, Ph.D.-Department of Molecular & Cellular Biology
Brewer III, Alex Jawann ............................................................................................... 27
Department of Pharmacology
Advisor: Richard De La Garza, Ph.D. - Department of Psychiatry & Behavioral Sciences
Brinegar, Amy Elizabeth .............................................................................................. 28
Department of Molecular & Cellular Biology
Advisor: Thomas Cooper, M.D. - Department of Pathology & Immunology
Brown, Rogers, II Milton .............................................................................................. 29
Program in Developmental Biology
Advisor: Andrew Groves, Ph.D. - Department of Neuroscience
Burton, Jason Christopher............................................................................................. 30
Integrative Program in Molecular and Biomedical Sciences
Advisor: Irina Larina, Ph.D. - Department of Molecular Physiology & Biophysics
Byrd, Tiara T................................................................................................................. 31
Program in Translational Biology & Molecular Medicine
Advisor: Nabil Ahmed, M.D. - Department of Pediatrics
Robert Grossman, M.D.-Department of Neurosurgery
Cadwell, Cathryn Rene ................................................................................................. 32
Department of Neuroscience
Advisor: Andreas Tolias, Ph.D. - Department of Neuroscience
Cai, Mingbo ................................................................................................................. 33
Department of Neuroscience
Advisor: David Eagleman, Ph.D. - Department of Neuroscience
Campbell, Ian Morgan .................................................................................................. 34
Department of Molecular & Human Genetics
Advisor: James Lupski, M.D./Ph.D. - Department of Molecular & Human Genetics
Pawel Stankiewicz, M.D./Ph.D.-Department of Molecular & Human Genetics
Campbell, James Christopher ....................................................................................... 35
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Choel Kim, Ph.D. - Department of Pharmacology
Cao, Zhijuan ................................................................................................................. 36
Program in Cardiovascular Sciences
Advisor: Sean Marrelli, Ph.D. - Department of Anesthesiology
Carter, Angela N ........................................................................................................... 37
Department of Neuroscience
Advisor: Anne Anderson, M.D. - Department of Pediatrics
Chaboub, Lesley S.M.................................................................................................... 38
Program in Developmental Biology
Advisor: Benjamin Deneen, Ph.D. - Department of Neuroscience
Chang, Chi-Hsuan ........................................................................................................ 39
Integrative Program in Molecular and Biomedical Sciences
Advisor: Jeffrey Rosen, Ph.D. - Department of Molecular & Cellular Biology
Chapple, Rich Harrison................................................................................................. 40
Department of Molecular & Human Genetics
Advisor: Daisuke Nakada, B.A.Sc. - Department of Molecular & Human Genetics
Chen, Bo ...................................................................................................................... 41
Department of Biochemistry & Molecular Biology
Advisor: Wah Chiu, Ph.D. - Department of Biochemistry & Molecular Biology
Chen, Chun-An ............................................................................................................ 42
Department of Molecular & Human Genetics
Advisor: Huda Zoghbi, M.D. - Department of Pediatrics
Chen, Elaine ................................................................................................................. 43
Department of Molecular & Cellular Biology
Advisor: Lawrence Chan, D.Sc. - Department of Medicine
Chen, Kuang-Yui Michael ............................................................................................ 44
Department of Biochemistry & Molecular Biology
Advisor: Patrick Barth, Ph.D. - Department of Pharmacology
Chen, Kuchuan ............................................................................................................. 45
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M. - Department of Molecular & Human Genetics
Cheng, Jinxuan ............................................................................................................. 46
Department of Biochemistry & Molecular Biology
Advisor: Kimberley Tolias, Ph.D. - Department of Neuroscience
Chiang, Angie Chi An .................................................................................................. 47
Department of Neuroscience
Advisor: Joanna Jankowsky, Ph.D. - Department of Neuroscience
Choi, Sungwoo ............................................................................................................. 48
Program in Developmental Biology
Advisor: David Moore, Ph.D. - Department of Molecular & Cellular Biology
Choi, William Tin-Shing .............................................................................................. 49
Program in Developmental Biology
Advisor: Mirjana Maletic-Savatic, M.D./Ph.D. - Department of Pediatrics
Chung, Hsiang-Ching .................................................................................................. 50
Integrative Program in Molecular and Biomedical Sciences
Advisor: Thomas Westbrook, Ph.D. - Department of Biochemistry & Molecular Biology
Ciupek, Andrew Mark Robinson .................................................................................. 51
Program in Translational Biology & Molecular Medicine
Advisor: Suzanne Fuqua, Ph.D. - Department of Medicine
Mothaffar Rimawi, M.D.-Department of Medicine
Ciupek, Sarah Margaret ................................................................................................ 52
Department of Neuroscience
Advisor: Daoyun Ji, Ph.D. - Department of Molecular & Cellular Biology
Collinson-Pautz, Matthew R ......................................................................................... 53
Program in Translational Biology & Molecular Medicine
Advisor: David Spencer, Ph.D. - Department of Pathology & Immunology
Kevin Slawin, M.D.-Department of Urology
Conley, Zachary Christopher ........................................................................................ 54
Department of Biochemistry & Molecular Biology
Advisor: E. Zechiedrich, Ph.D. - Department of Molecular Virology & Microbiology
Cullen, Sean Michael .................................................................................................... 55
Program in Developmental Biology
Advisor: Margaret Goodell, Ph.D. - Department of Pediatrics
Dai, Hang ..................................................................................................................... 56
Department of Molecular & Human Genetics
Advisor: Suzanne Leal, Ph.D. - Department of Molecular & Human Genetics
Darrow, Michele C. ...................................................................................................... 57
Department of Biochemistry & Molecular Biology
Advisor: Wah Chiu, Ph.D. - Department of Biochemistry & Molecular Biology
David, Gabriela Riva .................................................................................................... 58
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M. - Department of Molecular & Human Genetics
Davis, Shaun Michael ................................................................................................... 59
Department of Molecular & Human Genetics
Advisor: Herman Dierick, M.D. - Department of Molecular & Human Genetics
Dawson, Emily Packard ................................................................................................ 60
Integrative Program in Molecular and Biomedical Sciences
Advisor: Jason Heaney, Ph.D. - Department of Molecular & Human Genetics
De Maio, Antonia ........................................................................................................ 61
Program in Developmental Biology
Advisor: Huda Zoghbi, M.D. - Department of Pediatrics
Denfield, George Hilton ............................................................................................... 62
Department of Neuroscience
Advisor: Andreas Tolias, Ph.D. - Department of Neuroscience
Dharmadhikari, Avinash Vijay ..................................................................................... 63
Program in Translational Biology & Molecular Medicine
Advisor: Pawel Stankiewicz, M.D./Ph.D. - Department of Molecular & Human Genetics
Ignatia Van Den Veyver, M.D.-Department of Obstetrics & Gynecology
Dogruluk, Turgut ......................................................................................................... 64
Department of Molecular & Human Genetics
Advisor: Kenneth Scott, Ph.D. - Department of Molecular & Human Genetics
Dominguez-Vidana, Rocio .......................................................................................... 65
Integrative Program in Molecular and Biomedical Sciences
Advisor: Thomas Westbrook, Ph.D. - Department of Biochemistry & Molecular Biology
Dosey, Timothy Lloyd .................................................................................................. 66
Integrative Program in Molecular and Biomedical Sciences
Advisor: Theodore Wensel, Ph.D. - Department of Biochemistry & Molecular Biology
Egunsola, Adetutu Taiwo ............................................................................................ 67
Department of Molecular & Human Genetics
Advisor: Brendan Lee, M.D./Ph.D. - Department of Molecular & Human Genetics
Eisenhofer, Joel David .................................................................................................. 68
Department of Neuroscience
Advisor: Richard De La Garza, Ph.D. - Department of Psychiatry & Behavioral Sciences
Emerson, Charlene H .................................................................................................... 69
Department of Molecular & Human Genetics
Advisor: Alison Bertuch, M.D./Ph.D. - Department of Pediatrics
Fachini, Joseph Mario ................................................................................................... 70
Department of Molecular Physiology & Biophysics
Advisor: Joel Neilson, Ph.D. - Department of Molecular Physiology & Biophysics
Faille, River Uru ........................................................................................................... 71
Department of Pathology & Immunology
Advisor: Scott Wenderfer, M.D. - Department of Pediatrics
Farinholt, Timothy ....................................................................................................... 72
Department of Biochemistry & Molecular Biology
Advisor: Adam Kuspa, Ph.D. - Department of Biochemistry & Molecular Biology
Feketa, Viktor .............................................................................................................. 73
Program in Cardiovascular Sciences
Advisor: Sean Marrelli, Ph.D. - Department of Anesthesiology
Feng, Xiang .................................................................................................................. 74
Department of Pharmacology
Advisor: Patrick Barth, Ph.D. - Department of Pharmacology
Figard, Lauren Renee .................................................................................................... 75
Integrative Program in Molecular and Biomedical Sciences
Advisor: Anna Sokac, Ph.D. - Department of Biochemistry & Molecular Biology
Fitch, Olivia Michelle ................................................................................................... 76
Department of Neuroscience
Advisor: Michael Friedlander, Ph.D. - Department of Neuroscience
Mauro Costa-Mattioli, Ph.D.-Department of Neuroscience
Fleet, Tiffany C ............................................................................................................. 77
Program in Translational Biology & Molecular Medicine
Advisor: Bert O'Malley, M.D. - Department of Molecular & Cellular Biology
Clifford Dacso, M.D./M.P.H.-Department of Molecular & Cellular Biology
Foley, Christopher James .............................................................................................. 78
Department of Molecular & Cellular Biology
Advisor: Nicholas Mitsiades, M.D./Ph.D. - Department of Medicine
Folick, Andrew Kenji ................................................................................................... 79
Program in Developmental Biology
Advisor: Meng Wang, Ph.D. - Department of Molecular & Human Genetics
Fountain, Michael David .............................................................................................. 80
Program in Translational Biology & Molecular Medicine
Advisor: Christian Schaaf, M.D./Ph.D. - Department of Molecular & Human Genetics
Daryl Scott, M.D./Ph.D.-Department of Molecular & Human Genetics
Fousek, Kristen Nicole ................................................................................................. 81
Program in Translational Biology & Molecular Medicine
Advisor: Nabil Ahmed, M.D. - Department of Pediatrics
Helen Heslop, M.D.-Department of Pediatrics
Freire, Pablo Riera ........................................................................................................ 82
Department of Molecular & Cellular Biology
Advisor: Orla Conneely, Ph.D. - Department of Molecular & Cellular Biology
Froudarakis, Emmanouil .............................................................................................. 83
Department of Neuroscience
Advisor: Andreas Tolias, Ph.D. - Department of Neuroscience
Fuja, Daniel G ............................................................................................................... 84
Integrative Program in Molecular and Biomedical Sciences
Advisor: Jason Yustein, M.D./Ph.D. - Department of Pediatrics
Fujiwara, Kenichiro ..................................................................................................... 85
Integrative Program in Molecular and Biomedical Sciences
Advisor: Lawrence Donehower, Ph.D. - Department of Molecular Virology & Microbiology
Fullerton, Paul Thomas ................................................................................................. 86
Department of Molecular & Human Genetics
Advisor: Martin Matzuk, M.D./Ph.D. - Department of Pathology & Immunology
Fultz, Robert Steven ..................................................................................................... 87
Integrative Program in Molecular and Biomedical Sciences
Advisor: James Versalovic, M.D./Ph.D. - Department of Pathology & Immunology
Gala, Upasana .............................................................................................................. 88
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M. - Department of Molecular & Human Genetics
Galaz-Montoya, Monica Laura ..................................................................................... 89
Department of Biochemistry & Molecular Biology
Advisor: Theodore Wensel, Ph.D. - Department of Biochemistry & Molecular Biology
Gao, Chunxu ................................................................................................................ 90
Department of Molecular Virology & Microbiology
Advisor: James Versalovic, M.D./Ph.D. - Department of Pathology & Immunology
Garcia, Courtney Dawn ................................................................................................ 91
Department of Neuroscience
Advisor: Dora Angelaki, Ph.D. - Department of Neuroscience
Garcia, Melissa Nolan................................................................................................... 92
Clinical Scientist Training Program
Advisor: Kristy Murray, Ph.D. - Epidemiology
Garno, Sandy Lynn ....................................................................................................... 93
Integrative Program in Molecular and Biomedical Sciences
Advisor: Weiwei Dang, Ph.D. - Department of Molecular & Human Genetics
Gates, Leah Ashley ....................................................................................................... 94
Department of Molecular & Cellular Biology
Advisor: Bert O'Malley, M.D. - Department of Molecular & Cellular Biology
Gelowani, Violet .......................................................................................................... 95
Department of Molecular & Human Genetics
Advisor: Rui Chen, Ph.D. - Department of Molecular & Human Genetics
Ghosh-Choudhury, Triparna ........................................................................................ 96
Program in Translational Biology & Molecular Medicine
Advisor: Matthew Anderson, M.D./Ph.D. - Department of Obstetrics & Gynecology
Creighton Edwards, M.D.-Department of Obstetrics & Gynecology
Girard, Mary Elizabeth ................................................................................................. 97
Department of Molecular Virology & Microbiology
Advisor: Christophe Herman, Ph.D. - Department of Molecular & Human Genetics
Godoy, Guilherme ........................................................................................................ 98
Clinical Scientist Training Program
Advisor: Seth Lerner, M.D. - Department of Urology
Gokulakrishnan, Ganga ............................................................................................... 99
Clinical Scientist Training Program
Advisor: Steven Abrams, M.D. - Department of Pediatrics
Gramatges, Maria Monica .......................................................................................... 100
Clinical Scientist Training Program
Advisor: Alison Bertuch, M.D./Ph.D. - Department of Pediatrics
Graves, Joshua Daniel................................................................................................. 101
Integrative Program in Molecular and Biomedical Sciences
Advisor: Weei-Chin Lin, M.D./Ph.D. - Department of Medicine
Griffin, Deric Maurice ................................................................................................ 102
Program in Translational Biology & Molecular Medicine
Advisor: Robia Pautler, Ph.D. - Department of Molecular Physiology & Biophysics
Stephanie Abrams, M.D.-Department of Pediatrics
Grzeskowiak, Caitlin ................................................................................................. 103
Department of Molecular & Human Genetics
Advisor: Kenneth Scott, Ph.D. - Department of Molecular & Human Genetics
Haelterman, Nele ....................................................................................................... 104
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M. - Department of Molecular & Human Genetics
Haines, Katherine ....................................................................................................... 105
Department of Molecular & Human Genetics
Advisor: Sharon Plon, M.D./Ph.D. - Department of Pediatrics
Haller, Meade ............................................................................................................. 106
Department of Molecular & Cellular Biology
Advisor: Dolores Lamb, Ph.D. - Department of Urology
Hamilton, Mark Patrick .............................................................................................. 107
Department of Molecular & Cellular Biology
Advisor: Sean Mcguire, M.D./Ph.D. - Department of Molecular & Cellular Biology
Harrigal, Lindsay Jeanette .......................................................................................... 108
Integrative Program in Molecular and Biomedical Sciences
Advisor: Janet Butel, Ph.D. - Department of Molecular Virology & Microbiology
Hause, Anne M ........................................................................................................... 109
Program in Translational Biology & Molecular Medicine
Advisor: Pedro Piedra, M.D. - Department of Molecular Virology & Microbiology
Robert Atmar, M.D.-Department of Medicine
He, Zongxiao .............................................................................................................. 110
Department of Molecular & Human Genetics
Advisor: Suzanne Leal, Ph.D. - Department of Molecular & Human Genetics
Hecksel, Corey W ....................................................................................................... 111
Department of Molecular Virology & Microbiology
Advisor: Wah Chiu, Ph.D. - Department of Biochemistry & Molecular Biology
Hein, Sarah Marie ....................................................................................................... 112
Department of Molecular & Cellular Biology
Advisor: Yi Li, Ph.D. - Department of Molecular & Cellular Biology
Hendrix, Amanda Y. ................................................................................................... 113
Program in Translational Biology & Molecular Medicine
Advisor: Farrah Kheradmand, M.D. - Department of Medicine
Anita Sabichi, M.D.-Department of Medicine
Herman, Alexander Michael ....................................................................................... 114
Program in Developmental Biology
Advisor: Benjamin Arenkiel, Ph.D. - Department of Molecular & Human Genetics
Herrera, Jose A. .......................................................................................................... 115
Program in Translational Biology & Molecular Medicine
Advisor: Jeffrey Neul, M.D./Ph.D. - Department of Pediatrics
Xander Wehrens, M.D./Ph.D.-Department of Molecular Physiology & Biophysics
Hilton, Tyler John ....................................................................................................... 116
Integrative Program in Molecular and Biomedical Sciences
Advisor: Theodore Wensel, Ph.D. - Department of Biochemistry & Molecular Biology
John Wilson, Ph.D.-Department of Biochemistry & Molecular Biology
Hinton, Antentor Othrell ............................................................................................ 117
Integrative Program in Molecular and Biomedical Sciences
Advisor: Yong Xu, Ph.D. - Department of Pediatrics
Ho, Hsing-I ................................................................................................................ 118
Department of Molecular & Human Genetics
Advisor: Gad Shaulsky, Ph.D. - Department of Molecular & Human Genetics
Ho, Szu-Yu ................................................................................................................ 119
Program in Developmental Biology
Advisor: Matthew Rasband, Ph.D. - Department of Neuroscience
Holdman, Xue B ......................................................................................................... 120
Integrative Program in Molecular and Biomedical Sciences
Advisor: Jeffrey Rosen, Ph.D. - Department of Molecular & Cellular Biology
Holt, Matthew Valle ................................................................................................... 121
Department of Biochemistry & Molecular Biology
Advisor: Jun Qin, Ph.D. - Department of Biochemistry & Molecular Biology
Hong, Chuan .............................................................................................................. 122
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Wah Chiu, Ph.D. - Department of Biochemistry & Molecular Biology
Hooker, Stanley Eugene ............................................................................................. 123
Department of Molecular & Human Genetics
Advisor: Suzanne Leal, Ph.D. - Department of Molecular & Human Genetics
Hornstein, Benjamin Daniel ....................................................................................... 124
Department of Molecular Virology & Microbiology
Advisor: E. Zechiedrich, Ph.D. - Department of Molecular Virology & Microbiology
Hou, Jason K ............................................................................................................... 125
Clinical Scientist Training Program
Advisor: Hashem El-Serag, M.D./M.P.H. - Department of Medicine
Hryc, Corey F.............................................................................................................. 126
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Wah Chiu, Ph.D. - Department of Biochemistry & Molecular Biology
Hsu, Hsiang-Ting ....................................................................................................... 127
Department of Pathology & Immunology
Advisor: Jordan Orange, M.D. - Department of Pediatrics
Hsu, Teng-Kuei .......................................................................................................... 128
Department of Biochemistry & Molecular Biology
Advisor: Olivier Lichtarge, M.D./Ph.D. - Department of Molecular & Human Genetics
Hsu, Tiffany ............................................................................................................... 129
Integrative Program in Molecular and Biomedical Sciences
Advisor: Thomas Westbrook, Ph.D. - Department of Biochemistry & Molecular Biology
Huang, Gilbert ........................................................................................................... 130
Department of Biochemistry & Molecular Biology
Advisor: Choel Kim, Ph.D. - Department of Pharmacology
Huang, Longwen ........................................................................................................ 131
Department of Neuroscience
Advisor: Benjamin Arenkiel, Ph.D. - Department of Molecular & Human Genetics
Huang, Teng-Wei ....................................................................................................... 132
Program in Developmental Biology
Advisor: Jeffrey Neul, M.D./Ph.D. - Department of Pediatrics
Huang, Yu-Mei .......................................................................................................... 133
Department of Neuroscience
Advisor: Matthew Rasband, Ph.D. - Department of Neuroscience
Huq, Redwan .............................................................................................................. 134
Department of Molecular Physiology & Biophysics
Advisor: Christine Beeton, Ph.D. - Department of Molecular Physiology & Biophysics
Hurwitz, Amy Marie ................................................................................................... 135
Program in Translational Biology & Molecular Medicine
Advisor: Timothy Palzkill, Ph.D. - Department of Pharmacology
Robert Atmar, M.D.-Department of Medicine
Hutchinson, Diane Smith ............................................................................................ 136
Program in Translational Biology & Molecular Medicine
Advisor: Joseph Petrosino, Ph.D. - Department of Molecular Virology & Microbiology
Stephen Pflugfelder, M.D.-Department of Ophthalmology
James, Regis Aaron ..................................................................................................... 137
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Chad Shaw, Ph.D. - Department of Molecular & Human Genetics
Jarrett, Kelsey Elizabeth ............................................................................................. 138
Integrative Program in Molecular and Biomedical Sciences
Advisor: William Lagor, Ph.D. - Department of Molecular Physiology & Biophysics
Jen, Hsin-I .................................................................................................................. 139
Program in Developmental Biology
Advisor: Andrew Groves, Ph.D. - Department of Neuroscience
Jiang, Xiqian .............................................................................................................. 140
Department of Pharmacology
Advisor: Jin Wang, Ph.D. - Department of Pharmacology
Johnson, Jennifer Leigh .............................................................................................. 141
Department of Neuroscience
Advisor: Mauro Costa-Mattioli, Ph.D. - Department of Neuroscience
Johnston, Alyssa N ..................................................................................................... 142
Program in Translational Biology & Molecular Medicine
Advisor: Yi Li, Ph.D. - Department of Molecular & Cellular Biology
Kaelber, Jason T ......................................................................................................... 143
Department of Molecular Virology & Microbiology
Advisor: Wah Chiu, Ph.D. - Department of Biochemistry & Molecular Biology
Karandur, Deepti ........................................................................................................ 144
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: B. Pettitt, Ph.D. - Biochemistry
Jianpeng Ma, Ph.D.-Department of Biochemistry & Molecular Biology
Kaushik, Akash Kumar ............................................................................................... 145
Department of Biochemistry & Molecular Biology
Advisor: Arun Sreekumar, Ph.D. - Department of Molecular & Cellular Biology
Kee, Sara Elizabeth ..................................................................................................... 146
Department of Neuroscience
Advisor: Huda Zoghbi, M.D. - Department of Pediatrics
Kelly, Aaron Josef ...................................................................................................... 147
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Tsz-Kwong Man, Ph.D. - Department of Pediatrics
Kettner, Nicole M ....................................................................................................... 148
Department of Molecular & Cellular Biology
Advisor: Loning Fu, Ph.D. - Department of Pediatrics
Kho, Jordan ................................................................................................................ 149
Program in Developmental Biology
Advisor: Brendan Lee, M.D./Ph.D. - Department of Molecular & Human Genetics
Kim, Cynthia J ............................................................................................................ 150
Department of Molecular & Human Genetics
Advisor: Benjamin Arenkiel, Ph.D. - Department of Molecular & Human Genetics
Kim, Ik Sun ............................................................................................................... 151
Integrative Program in Molecular and Biomedical Sciences
Advisor: Xiang Zhang, Ph.D. - Department of Molecular & Cellular Biology
Kim, Maria Hyoun ...................................................................................................... 152
Clinical Scientist Training Program
Advisor: Elizabeth Chiao, M.D./M.P.H. - Department of Medicine
Knezevic, Jana ........................................................................................................... 153
Department of Molecular & Cellular Biology
Advisor: Jeffrey Rosen, Ph.D. - Department of Molecular & Cellular Biology
Kong, Kathleen .......................................................................................................... 154
Department of Molecular Virology & Microbiology
Advisor: Ronald Javier, Ph.D. - Department of Molecular Virology & Microbiology
Kruse, Robert Layne ................................................................................................... 155
Program in Translational Biology & Molecular Medicine
Advisor: Karl-Dimiter Bissig, M.D./Ph.D. - Department of Molecular & Cellular Biology
Fasiha Kanwal, M.D.-Department of Medicine
Ku, Amy Tsu ............................................................................................................... 156
Program in Translational Biology & Molecular Medicine
Advisor: Hoang Nguyen, Ph.D. - Department of Molecular & Cellular Biology
Abdul Diwan, Ph.D.-Department of Pathology & Immunology
Kurbanov, Suhrab ...................................................................................................... 157
Department of Pathology & Immunology
Advisor: Leonid Metelitsa, M.D./Ph.D. - Department of Pediatrics
Kurtova, Antonina V................................................................................................... 158
Program in Translational Biology & Molecular Medicine
Advisor: Keith Chan, Ph.D. - Department of Urology
Seth Lerner, M.D.-Department of Urology
Kyle, Stephanie Marie ................................................................................................ 159
Department of Molecular & Human Genetics
Advisor: Monica Justice, Ph.D. - Department of Molecular & Human Genetics
David Nelson, Ph.D.-Department of Molecular & Human Genetics
Laitman, Andrew Marc ............................................................................................... 160
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Mirjana Maletic-Savatic, M.D./Ph.D. - Department of Pediatrics
Lakshminarasimhan, Janakiraman Kausik ................................................................. 161
Department of Neuroscience
Advisor: Dora Angelaki, Ph.D. - Department of Neuroscience
Lam, Sharon ............................................................................................................... 162
Department of Pathology & Immunology
Advisor: Catherine Bollard, M.B.,B.Ch. - Department of Pediatrics
Cliona Rooney, Ph.D.-Department of Pediatrics
Laug, Dylan James ...................................................................................................... 163
Program in Developmental Biology
Advisor: Benjamin Deneen, Ph.D. - Department of Neuroscience
Leach, John Preston .................................................................................................... 164
Department of Molecular Physiology & Biophysics
Advisor: James Martin, M.D./Ph.D. - Department of Molecular Physiology & Biophysics
Lee, Yi-Chien ............................................................................................................. 165
Integrative Program in Molecular and Biomedical Sciences
Advisor: Brendan Lee, M.D./Ph.D. - Department of Molecular & Human Genetics
Lee, Yu-Ju .................................................................................................................. 166
Program in Translational Biology & Molecular Medicine
Advisor: Weei-Chin Lin, M.D./Ph.D. - Department of Medicine
Mothaffar Rimawi, M.D.-Department of Medicine
Lemon, Laramie Denise .............................................................................................. 167
Integrative Program in Molecular and Biomedical Sciences
Advisor: Alison Bertuch, M.D./Ph.D. - Department of Pediatrics
Lesteberg, Kelsey Elizabeth ....................................................................................... 168
Department of Pathology & Immunology
Advisor: George Makedonas, Ph.D. - Department of Pediatrics
Lewis, Kyle Joseph Edward........................................................................................ 169
Department of Molecular & Cellular Biology
Advisor: Nikolaj Timchenko, Ph.D. - Department of Pathology & Immunology
Richard Sifers, Ph.D.-Department of Pathology & Immunology
Lewis, Phoebe Elnora ................................................................................................. 170
Program in Translational Biology & Molecular Medicine
Advisor: Qizhi Yao, M.D./Ph.D. - Department of Surgery
Shital Patel, M.D.-Department of Medicine
Li, Cheng-Lin ............................................................................................................. 171
Department of Molecular & Human Genetics
Advisor: Gad Shaulsky, Ph.D. - Department of Molecular & Human Genetics
Li, Lele ....................................................................................................................... 172
Program in Cardiovascular Sciences
Advisor: James Martin, M.D./Ph.D. - Department of Molecular Physiology & Biophysics
Li, Tongchao .............................................................................................................. 173
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M. - Department of Molecular & Human Genetics
Andrew Groves, Ph.D.-Department of Neuroscience
Li, Zao ........................................................................................................................ 174
Department of Biochemistry & Molecular Biology
Advisor: Zheng Zhou, Ph.D. - Department of Biochemistry & Molecular Biology
Lian, Hong ................................................................................................................. 175
Department of Molecular & Human Genetics
Advisor: Hui Zheng, Ph.D. - Department of Molecular & Human Genetics
Liang, Dan .................................................................................................................. 176
Department of Pathology & Immunology
Advisor: William Decker, Ph.D. - Department of Pathology & Immunology
Lien, Steven Donald ................................................................................................... 177
Department of Neuroscience
Advisor: J. Dickman, Ph.D. - Department of Neuroscience
Lin, Angelique ........................................................................................................... 178
Integrative Program in Molecular and Biomedical Sciences
Advisor: Daisuke Nakada, B.A.Sc. - Department of Molecular & Human Genetics
Lin, Chih-Chun .......................................................................................................... 179
Department of Molecular & Human Genetics
Advisor: Meng Wang, Ph.D. - Department of Molecular & Human Genetics
Lingappan, Krithika ................................................................................................... 180
Clinical Scientist Training Program
Advisor: Bhagavatula Moorthy, Ph.D. - Department of Pediatrics
Litvinchuk, Alexandra ............................................................................................... 181
Integrative Program in Molecular and Biomedical Sciences
Advisor: Hui Zheng, Ph.D. - Department of Molecular & Human Genetics
Liu, Kuanqing ............................................................................................................ 182
Department of Molecular Virology & Microbiology
Advisor: Jue Wang, Ph.D. - Department of Molecular & Human Genetics
Anthony Maresso, Ph.D.-Department of Molecular Virology & Microbiology
Liu, Lucy .................................................................................................................... 183
Department of Neuroscience
Advisor: Hugo Bellen, Ph.D./D.V.M. - Department of Molecular & Human Genetics
Lo, Flora ..................................................................................................................... 184
Integrative Program in Molecular and Biomedical Sciences
Advisor: Xiang Zhang, Ph.D. - Department of Molecular & Cellular Biology
Lo, Yuan-Hung .......................................................................................................... 185
Integrative Program in Molecular and Biomedical Sciences
Advisor: Noah Shroyer, Ph.D. - Department of Pediatrics
Loehr, James Anthony ................................................................................................ 186
Department of Molecular Physiology & Biophysics
Advisor: George Rodney, Ph.D. - Department of Molecular Physiology & Biophysics
Lu, Hengyu ................................................................................................................ 187
Integrative Program in Molecular and Biomedical Sciences
Advisor: Kenneth Scott, Ph.D. - Department of Molecular & Human Genetics
Lu, Hsiang-Chih ......................................................................................................... 188
Program in Developmental Biology
Advisor: Huda Zoghbi, M.D. - Department of Pediatrics
Lu, Wen ...................................................................................................................... 189
Department of Pathology & Immunology
Advisor: David Corry, M.D. - Department of Medicine
Lu, Yang .................................................................................................................... 190
Integrative Program in Molecular and Biomedical Sciences
Advisor: Jeffrey Rosen, Ph.D. - Department of Molecular & Cellular Biology
Lumaban, Jeannette Galande ...................................................................................... 191
Department of Molecular & Human Genetics
Advisor: David Nelson, Ph.D. - Department of Molecular & Human Genetics
Lunsford, Jessica Leigh .............................................................................................. 192
Department of Pathology & Immunology
Advisor: Jonathan Levitt, Ph.D. - Department of Pathology & Immunology
Ma, Boxue .................................................................................................................. 193
Department of Biochemistry & Molecular Biology
Advisor: Wah Chiu, Ph.D. - Department of Biochemistry & Molecular Biology
Madan, Simran ........................................................................................................... 194
Program in Translational Biology & Molecular Medicine
Advisor: Brendan Lee, M.D./Ph.D. - Department of Molecular & Human Genetics
Brett Graham, M.D./Ph.D.-Department of Molecular & Human Genetics
Magnan, David Russell ............................................................................................... 195
Integrative Program in Molecular and Biomedical Sciences
Advisor: David Bates, Ph.D. - Department of Molecular & Human Genetics
Mahadevan, Sangeetha Kaveri ................................................................................... 196
Program in Translational Biology & Molecular Medicine
Advisor: Ignatia Van Den Veyver, M.D. - Department of Obstetrics & Gynecology
Carlos Bacino, M.D.-Department of Molecular & Human Genetics
Mahanic, Christina Susan ........................................................................................... 197
Integrative Program in Molecular and Biomedical Sciences
Advisor: Weei-Chin Lin, M.D./Ph.D. - Department of Medicine
Majid, Tabassum ........................................................................................................ 198
Program in Translational Biology & Molecular Medicine
Advisor: Robia Pautler, Ph.D. - Department of Molecular Physiology & Biophysics
Rachelle Doody, M.D./Ph.D.-Department of Neurology
Mak, Keng Hou .......................................................................................................... 199
Integrative Program in Molecular and Biomedical Sciences
Advisor: Jue Wang, Ph.D. - Department of Molecular & Human Genetics
Meng Wang, Ph.D.-Department of Molecular & Human Genetics
Manning, Kathleen Seger ........................................................................................... 200
Integrative Program in Molecular and Biomedical Sciences
Advisor: Thomas Cooper, M.D. - Department of Pathology & Immunology
Mao, Dongxue ............................................................................................................ 201
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M. - Department of Molecular & Human Genetics
Marin, Miguel ............................................................................................................ 202
Department of Neuroscience
Advisor: Matthew Rasband, Ph.D. - Department of Neuroscience
Martinez, Jarrod Don .................................................................................................. 203
Department of Molecular & Cellular Biology
Advisor: Jianming Xu, Ph.D. - Department of Molecular & Cellular Biology
Martini-Stoica, Heidi ................................................................................................. 204
Program in Translational Biology & Molecular Medicine
Advisor: Hui Zheng, Ph.D. - Department of Molecular & Human Genetics
Joshua Shulman, M.D./Ph.D.-Department of Neurology
Masand, Ruchi ........................................................................................................... 205
Department of Molecular & Human Genetics
Advisor: Brett Graham, M.D./Ph.D. - Department of Molecular & Human Genetics
Mata, Melinda ............................................................................................................ 206
Department of Pathology & Immunology
Advisor: Stephen Gottschalk, M.D. - Department of Pediatrics
Mayle, Allison ........................................................................................................... 207
Department of Molecular & Human Genetics
Advisor: Margaret Goodell, Ph.D. - Department of Pediatrics
Mayle, Ryan ............................................................................................................... 208
Department of Molecular & Human Genetics
Advisor: Grzegorz Ira, Ph.D. - Department of Molecular & Human Genetics
Maynard, Janielle P..................................................................................................... 209
Program in Translational Biology & Molecular Medicine
Advisor: Sundararajah Thevananther, Ph.D. - Department of Pediatrics
John Goss, M.D.-Department of Surgery
McCue, Tyler Jordan .................................................................................................. 210
Integrative Program in Molecular and Biomedical Sciences
Advisor: Joseph Petrosino, Ph.D. - Department of Molecular Virology & Microbiology
Mehta, Pooja Chetan ................................................................................................... 211
Department of Pathology & Immunology
Advisor: C Smith, M.D. - Department of Pediatrics
Mehta, Shrenik Chetan ............................................................................................... 212
Department of Pharmacology
Advisor: Timothy Palzkill, Ph.D. - Department of Pharmacology
Meng, Xiangling ........................................................................................................ 213
Department of Neuroscience
Advisor: Huda Zoghbi, M.D. - Department of Pediatrics
Minnick, Phillip Jay .................................................................................................... 214
Department of Biochemistry & Molecular Biology
Advisor: Susan Rosenberg, Ph.D. - Department of Molecular & Human Genetics
Minor, Marissa Mie Kehaulani ................................................................................... 215
Department of Molecular Virology & Microbiology
Advisor: Betty Slagle, Ph.D. - Department of Molecular Virology & Microbiology
Mitchell, Patrick Gerald .............................................................................................. 216
Integrative Program in Molecular and Biomedical Sciences
Advisor: Wah Chiu, Ph.D. - Department of Biochemistry & Molecular Biology
Mitra, Sayantan .......................................................................................................... 217
Department of Biochemistry & Molecular Biology
Advisor: B Prasad, Ph.D. - Department of Biochemistry & Molecular Biology
Monkkonen, Teresa ................................................................................................... 218
Department of Molecular & Cellular Biology
Advisor: Michael Lewis, Ph.D. - Department of Molecular & Cellular Biology
Monroe, Tanner Oliver ............................................................................................... 219
Department of Molecular Physiology & Biophysics
Advisor: James Martin, M.D./Ph.D. - Department of Molecular Physiology & Biophysics
George Rodney, Ph.D.-Department of Molecular Physiology & Biophysics
Moore, Jessica M ........................................................................................................ 220
Department of Biochemistry & Molecular Biology
Advisor: Susan Rosenberg, Ph.D. - Department of Molecular & Human Genetics
Philip Hastings, Ph.D.-Department of Molecular & Human Genetics
Morra, Christina Narie ................................................................................................ 221
Integrative Program in Molecular and Biomedical Sciences
Advisor: James Versalovic, M.D./Ph.D. - Department of Pathology & Immunology
Murray, Stephen Christopher ...................................................................................... 222
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Steven Ludtke, Ph.D. - Department of Biochemistry & Molecular Biology
Murry, Jaclyn Nicole .................................................................................................. 223
Department of Molecular & Human Genetics
Advisor: Ignatia Van Den Veyver, M.D. - Department of Obstetrics & Gynecology
Muscarella, Aaron M .................................................................................................. 224
Integrative Program in Molecular and Biomedical Sciences
Advisor: Xiang Zhang, Ph.D. - Department of Molecular & Cellular Biology
Myers, Christopher Gary ............................................................................................ 225
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: B. Pettitt, Ph.D. - Biochemistry
Nair, Amritha ............................................................................................................. 226
Department of Molecular & Human Genetics
Advisor: Thomas Westbrook, Ph.D. - Department of Biochemistry & Molecular Biology
Namwanje, Maria ....................................................................................................... 227
Department of Molecular & Human Genetics
Advisor: Chester Brown, M.D./Ph.D. - Department of Molecular & Human Genetics
Nash, Andrea Kathleen ............................................................................................... 228
Department of Molecular Virology & Microbiology
Advisor: Joseph Petrosino, Ph.D. - Department of Molecular Virology & Microbiology
Neill, Nicholas Jay ...................................................................................................... 229
Department of Molecular & Human Genetics
Advisor: Thomas Westbrook, Ph.D. - Department of Biochemistry & Molecular Biology
Nguyen, Chinh Thi Quynh .......................................................................................... 230
Department of Molecular Virology & Microbiology
Advisor: Anthony Maresso, Ph.D. - Department of Molecular Virology & Microbiology
Nguyen, Lena H .......................................................................................................... 231
Department of Neuroscience
Advisor: Anne Anderson, M.D. - Department of Pediatrics
Nikolai, Bryan C ......................................................................................................... 232
Department of Molecular & Cellular Biology
Advisor: Bert O'Malley, M.D. - Department of Molecular & Cellular Biology
Novikov, Ilya ............................................................................................................. 233
Department of Biochemistry & Molecular Biology
Advisor: Olivier Lichtarge, M.D./Ph.D. - Department of Molecular & Human Genetics
O’Neil, Derek Steven .................................................................................................. 234
Program in Translational Biology & Molecular Medicine
Advisor: Kjersti Aagaard, M.D./Ph.D. - Department of Obstetrics & Gynecology
William Gibbons, M.D.-Department of Obstetrics & Gynecology
Oak, Ninad Ramesh .................................................................................................... 235
Department of Molecular & Human Genetics
Advisor: Sharon Plon, M.D./Ph.D. - Department of Pediatrics
Oakes, Joshua Michael ............................................................................................... 236
Program in Cardiovascular Sciences
Advisor: Susan Hamilton, Ph.D. - Department of Molecular Physiology & Biophysics
Onuchic, Vitor ........................................................................................................... 237
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Aleksandar Milosavljevic, Ph.D. - Department of Molecular & Human Genetics
Oyola, Mario G ........................................................................................................... 238
Department of Neuroscience
Advisor: Shailaja Mani, Ph.D. - Department of Molecular & Cellular Biology
Mariella De Biasi, Ph.D.-Department of Neuroscience
Ozseker, Ayse-Sena ................................................................................................... 239
Program in Developmental Biology
Advisor: Meng Wang, Ph.D. - Department of Molecular & Human Genetics
Pankowicz, Francis .................................................................................................... 240
Department of Molecular & Cellular Biology
Advisor: Karl-Dimiter Bissig, M.D./Ph.D. - Department of Molecular & Cellular Biology
Park, Jiyoung ............................................................................................................. 241
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Stelios Smirnakis, M.D./Ph.D. - Department of Neurology
Patel, Maha Praful....................................................................................................... 242
Program in Translational Biology & Molecular Medicine
Advisor: Timothy Palzkill, Ph.D. - Department of Pharmacology
Robert Atmar, M.D.-Department of Medicine
Pejerrey, Sasha McKai ................................................................................................ 243
Department of Molecular & Cellular Biology
Advisor: Suzanne Fuqua, Ph.D. - Department of Medicine
Pew, Braden Kyle ....................................................................................................... 244
Department of Molecular & Cellular Biology
Advisor: Kjersti Aagaard, M.D./Ph.D. - Department of Obstetrics & Gynecology
Dennis Bier, M.D.-Department of Pediatrics
Piazza, Victor George ................................................................................................. 245
Program in Cardiovascular Sciences
Advisor: Mary Dickinson, Ph.D. - Department of Molecular Physiology & Biophysics
Pitcher, Meagan Rochelle ........................................................................................... 246
Program in Translational Biology & Molecular Medicine
Advisor: Jeffrey Neul, M.D./Ph.D. - Department of Pediatrics
Daniel Glaze, M.D.-Department of Pediatrics
Pohodich, Amy E ........................................................................................................ 247
Department of Neuroscience
Advisor: Huda Zoghbi, M.D. - Department of Pediatrics
Polleys, Erica Jean ...................................................................................................... 248
Integrative Program in Molecular and Biomedical Sciences
Advisor: Alison Bertuch, M.D./Ph.D. - Department of Pediatrics
Pradhan, Geetali ......................................................................................................... 249
Program in Translational Biology & Molecular Medicine
Advisor: Yuxiang Sun, M.D./Ph.D. - Department of Pediatrics
Susan Samson, M.D.-Department of Medicine
Premkumar, Muralidhar Hebbur ................................................................................. 250
Clinical Scientist Training Program
Advisor: Brendan Lee, M.D./Ph.D. - Department of Molecular & Human Genetics
Qin, Liying ................................................................................................................. 251
Department of Biochemistry & Molecular Biology
Advisor: Choel Kim, Ph.D. - Department of Pharmacology
Quick, Ann Pepper ...................................................................................................... 252
Program in Cardiovascular Sciences
Advisor: Xander Wehrens, M.D./Ph.D. - Department of Molecular Physiology & Biophysics
Quiros, Joel Patrick ..................................................................................................... 253
Integrative Program in Molecular and Biomedical Sciences
Advisor: Olivier Lichtarge, M.D./Ph.D. - Department of Molecular & Human Genetics
Raghavan, Adithya ..................................................................................................... 254
Department of Molecular & Human Genetics
Advisor: William Craigen, M.D./Ph.D. - Department of Molecular & Human Genetics
Rajasekharan, Vivek .................................................................................................. 255
Department of Molecular & Cellular Biology
Advisor: Frederick Pereira, Ph.D. - Department of Molecular & Cellular Biology
Ramachandran, Prasanna ........................................................................................... 256
Department of Molecular & Human Genetics
Advisor: Meng Wang, Ph.D. - Department of Molecular & Human Genetics
Regenbogen, Sam Julian ............................................................................................. 257
Department of Pharmacology
Advisor: Olivier Lichtarge, M.D./Ph.D. - Department of Molecular & Human Genetics
Renwick, Alexander ................................................................................................... 258
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Chad Shaw, Ph.D. - Department of Molecular & Human Genetics
Reyes, Natalie Michelle .............................................................................................. 259
Integrative Program in Molecular and Biomedical Sciences
Advisor: Suzanne Fuqua, Ph.D. - Department of Medicine
Rhee, Christopher J ..................................................................................................... 260
Clinical Scientist Training Program
Advisor: Kenneth Brady, M.D. - Department of Anesthesiology
Ridgeway, Alex David ................................................................................................ 261
Department of Molecular & Cellular Biology
Advisor: Dolores Lamb, Ph.D. - Department of Urology
Roberts, Justin Michael............................................................................................... 262
Department of Molecular & Cellular Biology
Advisor: Nancy Weigel, Ph.D. - Department of Molecular & Cellular Biology
Rodriguez, Amanda ................................................................................................... 263
Department of Molecular & Cellular Biology
Advisor: Stephanie Pangas, Ph.D. - Department of Pathology & Immunology
Rodriguez, Perla Janet ................................................................................................ 264
Integrative Program in Molecular and Biomedical Sciences
Advisor: Henry Pownall, Ph.D. - Department of Medicine
Roh, Soung Hun ......................................................................................................... 265
Department of Biochemistry & Molecular Biology
Advisor: Wah Chiu, Ph.D. - Department of Biochemistry & Molecular Biology
David Tweardy, M.D.-Department of Medicine
Roman-Sanchez, Ramon ............................................................................................ 266
Integrative Program in Molecular and Biomedical Sciences
Advisor: John Wilson, Ph.D. - Department of Biochemistry & Molecular Biology
Rosewell Shaw, Amanda Nicole................................................................................. 267
Department of Molecular & Human Genetics
Advisor: Philip Ng, Ph.D. - Department of Molecular & Human Genetics
Ross, Matthew Clayton ............................................................................................... 268
Department of Molecular Virology & Microbiology
Advisor: Joseph Petrosino, Ph.D. - Department of Molecular Virology & Microbiology
Rubin, Michelle Jenny ................................................................................................ 269
Integrative Program in Molecular and Biomedical Sciences
Advisor: Gad Shaulsky, Ph.D. - Department of Molecular & Human Genetics
Runquist, Ingrid Sophie .............................................................................................. 270
Department of Molecular & Cellular Biology
Advisor: Michael Lewis, Ph.D. - Department of Molecular & Cellular Biology
Rutledge, Eric Benjamin ............................................................................................. 271
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Zhandong Liu, Ph.D. - Department of Pediatrics
Sabharwal, Jasdeep Singh ........................................................................................... 272
Department of Neuroscience
Advisor: Samuel Wu, Ph.D. - Department of Ophthalmology
Safdar, Zeenat ............................................................................................................ 273
Clinical Scientist Training Program
Advisor: Mark Entman, M.D. - Department of Medicine
Saliba, Jason ............................................................................................................... 274
Department of Molecular & Human Genetics
Advisor: Sharon Plon, M.D./Ph.D. - Department of Pediatrics
Salvo, Jason Scott ....................................................................................................... 275
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Rui Chen, Ph.D. - Department of Molecular & Human Genetics
San Martin, Rebeca .................................................................................................... 276
Department of Molecular & Cellular Biology
Advisor: David Rowley, Ph.D. - Department of Molecular & Cellular Biology
Santhanam, Balaji ...................................................................................................... 277
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Gad Shaulsky, Ph.D. - Department of Molecular & Human Genetics
Satterfield, Laura Louise............................................................................................. 278
Integrative Program in Molecular and Biomedical Sciences
Advisor: Jason Yustein, M.D./Ph.D. - Department of Pediatrics
Scott, Jessica Diane..................................................................................................... 279
Integrative Program in Molecular and Biomedical Sciences
Advisor: David Moore, Ph.D. - Department of Molecular & Cellular Biology
Seilheimer, Robert Lionel ........................................................................................... 280
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Samuel Wu, Ph.D. - Department of Ophthalmology
Semerci, Fatih ............................................................................................................ 281
Program in Developmental Biology
Advisor: Mirjana Maletic-Savatic, M.D./Ph.D. - Department of Pediatrics
Senturk, Mumine ........................................................................................................ 282
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M. - Department of Molecular & Human Genetics
Seymour, Michelle L. ................................................................................................. 283
Department of Molecular & Cellular Biology
Advisor: Frederick Pereira, Ph.D. - Department of Molecular & Cellular Biology
Shafi, Ayesha ............................................................................................................. 284
Department of Molecular & Cellular Biology
Advisor: Nancy Weigel, Ph.D. - Department of Molecular & Cellular Biology
Shen, Shan .................................................................................................................. 285
Department of Neuroscience
Advisor: Andreas Tolias, Ph.D. - Department of Neuroscience
Shen, Ye ..................................................................................................................... 286
Integrative Program in Molecular and Biomedical Sciences
Advisor: H Lacorazza, Ph.D. - Department of Pathology & Immunology
Shepherd, Jonathan .................................................................................................... 287
Department of Molecular & Cellular Biology
Advisor: Powel Brown, M.D./Ph.D. - Department of Molecular & Cellular Biology
Shin, Hongsup ............................................................................................................ 288
Department of Neuroscience
Advisor: Whee Ma, Ph.D. - Department of Neuroscience
Michael Beauchamp, Ph.D.-Department of Neuroscience
Shivanna, Binoy ......................................................................................................... 289
Clinical Scientist Training Program
Advisor: Bhagavatula Moorthy, Ph.D. - Department of Pediatrics
Shum, Thomas ........................................................................................................... 290
Program in Translational Biology & Molecular Medicine
Advisor: Cliona Rooney, Ph.D. - Department of Pediatrics
Stephen Gottschalk, M.D.-Department of Pediatrics
Siehr, Meagan ............................................................................................................ 291
Department of Molecular & Human Genetics
Advisor: Jeffrey Noebels, M.D./Ph.D. - Department of Neurology
Simon, Lukas ............................................................................................................. 292
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Chad Shaw, Ph.D. - Department of Molecular & Human Genetics
Sinha, Papiya ............................................................................................................. 293
Department of Pathology & Immunology
Advisor: Jordan Orange, M.D. - Department of Pediatrics
Sivaramakrishnan, Priya ............................................................................................ 294
Department of Molecular & Human Genetics
Advisor: Christophe Herman, Ph.D. - Department of Molecular & Human Genetics
Soens, Zachry Tore ..................................................................................................... 295
Department of Molecular & Human Genetics
Advisor: Rui Chen, Ph.D. - Department of Molecular & Human Genetics
Sowa, Jessica Nichole ................................................................................................. 296
Department of Molecular & Human Genetics
Advisor: Meng Wang, Ph.D. - Department of Molecular & Human Genetics
Spike, Aaron Jonathon ................................................................................................ 297
Department of Molecular & Cellular Biology
Advisor: Jeffrey Rosen, Ph.D. - Department of Molecular & Cellular Biology
Sreekumar, Amulya ................................................................................................... 298
Department of Molecular & Cellular Biology
Advisor: Jeffrey Rosen, Ph.D. - Department of Molecular & Cellular Biology
Stay, Trace L ............................................................................................................... 299
Department of Neuroscience
Advisor: Dora Angelaki, Ph.D. - Department of Neuroscience
Stinnett, Gary R. ......................................................................................................... 300
Department of Molecular Physiology & Biophysics
Advisor: Robia Pautler, Ph.D. - Department of Molecular Physiology & Biophysics
Stoica, Loredana Georgiana ........................................................................................ 301
Department of Molecular & Cellular Biology
Advisor: Mauro Costa-Mattioli, Ph.D. - Department of Neuroscience
Stojanoski, Vlatko ...................................................................................................... 302
Department of Biochemistry & Molecular Biology
Advisor: Timothy Palzkill, Ph.D. - Department of Pharmacology
Stone, Adrianne Elayne .............................................................................................. 303
Program in Translational Biology & Molecular Medicine
Advisor: Brendan Lee, M.D./Ph.D. - Department of Molecular & Human Genetics
Carlos Bacino, M.D.-Department of Molecular & Human Genetics
Stuhlsatz, Danielle Dee ............................................................................................... 304
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: B. Pettitt, Ph.D. - Biochemistry
Su, Qingtai ................................................................................................................. 305
Integrative Program in Molecular and Biomedical Sciences
Advisor: Li Xin, Ph.D. - Department of Molecular & Cellular Biology
Sukumar, Ann Tabitha ............................................................................................... 306
Department of Molecular & Human Genetics
Advisor: Alison Bertuch, M.D./Ph.D. - Department of Pediatrics
Sukumaran, Sujita ...................................................................................................... 307
Program in Translational Biology & Molecular Medicine
Advisor: Ann Leen, Ph.D. - Department of Pediatrics
William Fisher, M.D.-Department of Surgery
Sun, Jenny J ................................................................................................................ 308
Department of Neuroscience
Advisor: Russell Ray, Ph.D. - Department of Neuroscience
Sun, Jiayi Monika ....................................................................................................... 309
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Ching Lau, M.D./Ph.D. - Department of Pediatrics
Sun, Yu ...................................................................................................................... 310
Integrative Program in Molecular and Biomedical Sciences
Advisor: Weiwei Dang, Ph.D. - Department of Molecular & Human Genetics
Sung, Yun-Min .......................................................................................................... 311
Department of Biochemistry & Molecular Biology
Advisor: Theodore Wensel, Ph.D. - Department of Biochemistry & Molecular Biology
Szwarc, Maria Magdalena .......................................................................................... 312
Department of Molecular & Cellular Biology
Advisor: Bert O'Malley, M.D. - Department of Molecular & Cellular Biology
Tackett, Bryan Christopher ......................................................................................... 313
Program in Translational Biology & Molecular Medicine
Advisor: Sundararajah Thevananther, Ph.D. - Department of Pediatrics
Saul Karpen, M.D./Ph.D.-Department of Pediatrics
Tajhya, Rajeev Babu ................................................................................................... 314
Department of Molecular Physiology & Biophysics
Advisor: Christine Beeton, Ph.D. - Department of Molecular Physiology & Biophysics
Tan, Kai Li ................................................................................................................. 315
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M. - Department of Molecular & Human Genetics
Tanner, Mark R. .......................................................................................................... 316
Program in Translational Biology & Molecular Medicine
Advisor: Christine Beeton, Ph.D. - Department of Molecular Physiology & Biophysics
David Corry, M.D.-Department of Medicine
Taylor, Aaron Michael ................................................................................................ 317
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Ching Lau, M.D./Ph.D. - Department of Pediatrics
Tepe, Burak ................................................................................................................ 318
Program in Developmental Biology
Advisor: Benjamin Arenkiel, Ph.D. - Department of Molecular & Human Genetics
Terrón-Díaz, María Elisa ............................................................................................ 319
Integrative Program in Molecular and Biomedical Sciences
Advisor: Olivier Lichtarge, M.D./Ph.D. - Department of Molecular & Human Genetics
Terwilliger, Austen Lee .............................................................................................. 320
Integrative Program in Molecular and Biomedical Sciences
Advisor: Anthony Maresso, Ph.D. - Department of Molecular Virology & Microbiology
Tian, Gengwen ........................................................................................................... 321
Department of Pathology & Immunology
Advisor: Leonid Metelitsa, M.D./Ph.D. - Department of Pediatrics
Tian, Lin ..................................................................................................................... 322
Department of Biochemistry & Molecular Biology
Advisor: Xiang Zhang, Ph.D. - Department of Molecular & Cellular Biology
Tran, Baouyen ............................................................................................................ 323
Department of Neuroscience
Advisor: Edward Cooper, M.D./Ph.D. - Department of Neurology
Tran, Linda Lien ......................................................................................................... 324
Integrative Program in Molecular and Biomedical Sciences
Advisor: David Rowley, Ph.D. - Department of Molecular & Cellular Biology
Tsai, Chang-Ru .......................................................................................................... 325
Program in Developmental Biology
Advisor: Michael Galko, Ph.D. - Biochemistry and Molecular Biology
Tsai, Wei-Chih ........................................................................................................... 326
Department of Molecular Virology & Microbiology
Advisor: Richard Lloyd, Ph.D. - Department of Molecular Virology & Microbiology
Tu, Yen-Kuei ............................................................................................................. 327
Integrative Program in Molecular and Biomedical Sciences
Advisor: Kimberley Tolias, Ph.D. - Department of Neuroscience
Tung, Hui-Ying .......................................................................................................... 328
Department of Pathology & Immunology
Advisor: David Corry, M.D. - Department of Medicine
Ugur, Berrak .............................................................................................................. 329
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M. - Department of Molecular & Human Genetics
Ung, Kevin ................................................................................................................. 330
Program in Developmental Biology
Advisor: Benjamin Arenkiel, Ph.D. - Department of Molecular & Human Genetics
Vasquez, Yasmin1 Margarita ..................................................................................... 331
Department of Molecular & Cellular Biology
Advisor: Francesco Demayo, Ph.D. - Department of Molecular & Cellular Biology
Villanueva, Hugo ....................................................................................................... 332
Department of Molecular & Cellular Biology
Advisor: Michael Lewis, Ph.D. - Department of Molecular & Cellular Biology
Vogt, Megan Brittany ................................................................................................. 333
Integrative Program in Molecular and Biomedical Sciences
Advisor: Rebecca Rico, M.P.H. - Department of Molecular Virology & Microbiology
Vue, Zer ..................................................................................................................... 334
Program in Developmental Biology
Advisor: Richard Behringer, - Molecular Genetics - M.D. Anderson
Walker, Edgar Yasuhiro.............................................................................................. 335
Department of Neuroscience
Advisor: Andreas Tolias, Ph.D. - Department of Neuroscience
Wang, Chih-Chuan .................................................................................................... 336
Integrative Program in Molecular and Biomedical Sciences
Advisor: Matthew Rasband, Ph.D. - Department of Neuroscience
Wang, Feng ................................................................................................................ 337
Department of Molecular & Human Genetics
Advisor: Rui Chen, Ph.D. - Department of Molecular & Human Genetics
Wang, Gao T. .............................................................................................................. 338
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Suzanne Leal, Ph.D. - Department of Molecular & Human Genetics
Wang, Li .................................................................................................................... 339
Department of Molecular & Human Genetics
Advisor: Huda Zoghbi, M.D. - Department of Pediatrics
Wang, Mengyu ........................................................................................................... 340
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Ido Golding, Ph.D. - Department of Biochemistry & Molecular Biology
Wang, Rui .................................................................................................................. 341
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Wah Chiu, Ph.D. - Department of Biochemistry & Molecular Biology
Wang, Yue ................................................................................................................. 342
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Gad Shaulsky, Ph.D. - Department of Molecular & Human Genetics
Wang, Yumeng .......................................................................................................... 343
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Patrick Barth, Ph.D. - Department of Pharmacology
Wei, Xing ................................................................................................................... 344
Integrative Program in Molecular and Biomedical Sciences
Advisor: Li Xin, Ph.D. - Department of Molecular & Cellular Biology
Wetendorf, Margeaux ................................................................................................ 345
Integrative Program in Molecular and Biomedical Sciences
Advisor: Francesco Demayo, Ph.D. - Department of Molecular & Cellular Biology
White, Joshua James ................................................................................................... 346
Department of Neuroscience
Advisor: Roy Sillitoe, Ph.D. - Department of Pathology & Immunology
Williams, LaTerrica Chemise ..................................................................................... 347
Program in Translational Biology & Molecular Medicine
Advisor: Stephen Gottschalk, M.D. - Department of Pediatrics
Xiao-Tong Song, Ph.D.-Department of Pathology & Immunology
Wu, Chun-Ting .......................................................................................................... 348
Department of Neuroscience
Advisor: Daoyun Ji, Ph.D. - Department of Molecular & Cellular Biology
Xia, Jun ...................................................................................................................... 349
Integrative Program in Molecular and Biomedical Sciences
Advisor: Susan Rosenberg, Ph.D. - Department of Molecular & Human Genetics
Xie, Wei ..................................................................................................................... 350
Integrative Program in Molecular and Biomedical Sciences
Advisor: Huda Zoghbi, M.D. - Department of Pediatrics
Xu, Mingchu .............................................................................................................. 351
Department of Molecular & Human Genetics
Advisor: Rui Chen, Ph.D. - Department of Molecular & Human Genetics
Xu, Xiaowei ............................................................................................................... 352
Department of Biochemistry & Molecular Biology
Advisor: Rachel Schiff, Ph.D. - Department of Medicine
Xu, Yang .................................................................................................................... 353
Department of Pathology & Immunology
Advisor: Gianpietro Dotti, M.D. - Department of Medicine
Xue, Zenghui .............................................................................................................. 354
Department of Biochemistry & Molecular Biology
Advisor: Anna Sokac, Ph.D. - Department of Biochemistry & Molecular Biology
Yang, Eric Yota .......................................................................................................... 355
Clinical Scientist Training Program
Advisor: Christie Ballantyne, M.D. - Department of Medicine
Yang, Liubin .............................................................................................................. 356
Department of Molecular & Human Genetics
Advisor: Margaret Goodell, Ph.D. - Department of Pediatrics
Yatsenko, Dimitri ....................................................................................................... 357
Department of Neuroscience
Advisor: Andreas Tolias, Ph.D. - Department of Neuroscience
Yeh, Szu-Ying ............................................................................................................ 358
Program in Developmental Biology
Advisor: Huda Zoghbi, M.D. - Department of Pediatrics
Yen, Shuo-Ting .......................................................................................................... 359
Program in Developmental Biology
Advisor: Richard Behringer, - Molecular Genetics - M.D. Anderson
Yetman, Michael Joseph ............................................................................................. 360
Department of Neuroscience
Advisor: Joanna Jankowsky, Ph.D. - Department of Neuroscience
Yin, Jiani .................................................................................................................... 361
Department of Molecular & Human Genetics
Advisor: Huda Zoghbi, M.D. - Department of Pediatrics
Yong, Lin-Kin ............................................................................................................ 362
Program in Translational Biology & Molecular Medicine
Advisor: Qizhi Yao, M.D./Ph.D. - Department of Surgery
William Fisher, M.D.-Department of Surgery
Yosef, Nejla ............................................................................................................... 363
Department of Molecular Physiology & Biophysics
Advisor: Mary Dickinson, Ph.D. - Department of Molecular Physiology & Biophysics
You, Ran .................................................................................................................... 364
Department of Pathology & Immunology
Advisor: Farrah Kheradmand, M.D. - Department of Medicine
Young, Melvin Chen................................................................................................... 365
Department of Biochemistry & Molecular Biology
Advisor: Patrick Barth, Ph.D. - Department of Pharmacology
Yuan, Bo .................................................................................................................... 366
Integrative Program in Molecular and Biomedical Sciences
Advisor: James Lupski, M.D./Ph.D. - Department of Molecular & Human Genetics
Yuan, Xiaoyi .............................................................................................................. 367
Department of Pathology & Immunology
Advisor: Farrah Kheradmand, M.D. - Department of Medicine
Yuan, Yuan ................................................................................................................ 368
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Han Liang, Ph.D. - Bio informatics and Computational Biology
Zalewski, Zachary A ................................................................................................... 369
Department of Molecular & Human Genetics
Advisor: David Nelson, Ph.D. - Department of Molecular & Human Genetics
Zaneveld, Jacques Eric Timothy ................................................................................. 370
Department of Molecular & Human Genetics
Advisor: Rui Chen, Ph.D. - Department of Molecular & Human Genetics
Zeng, Huan-Chang ..................................................................................................... 371
Program in Developmental Biology
Advisor: Brendan Lee, M.D./Ph.D. - Department of Molecular & Human Genetics
Zhai, Yijie .................................................................................................................. 372
Department of Molecular & Human Genetics
Advisor: John Belmont, M.D./Ph.D. - Department of Molecular & Human Genetics
Zhang, Jing ................................................................................................................. 373
Department of Biochemistry & Molecular Biology
Advisor: Ido Golding, Ph.D. - Department of Biochemistry & Molecular Biology
Zhang, Xiaotian ......................................................................................................... 374
Department of Molecular & Human Genetics
Advisor: Margaret Goodell, Ph.D. - Department of Pediatrics
Zhao, Li ...................................................................................................................... 375
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Rui Chen, Ph.D. - Department of Molecular & Human Genetics
Zhao, Mingkun ........................................................................................................... 376
Integrative Program in Molecular and Biomedical Sciences
Advisor: Jin Wang, Ph.D. - Department of Pharmacology
Zheng, Liuliu ............................................................................................................. 377
Department of Biochemistry & Molecular Biology
Advisor: Anna Sokac, Ph.D. - Department of Biochemistry & Molecular Biology
Zhou, Quan ................................................................................................................ 378
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Yongtao Guan, Ph.D. - Department of Pediatrics
Zhou, Ying ................................................................................................................. 379
Integrative Program in Molecular and Biomedical Sciences
Advisor: David Moore, Ph.D. - Department of Molecular & Cellular Biology
Karl-Dimiter Bissig, M.D./Ph.D.-Department of Molecular & Cellular Biology
Zhu, Liyuan ................................................................................................................ 380
Department of Molecular Virology & Microbiology
Advisor: Yi Li, Ph.D. - Department of Molecular & Cellular Biology
Zhu, Wenyi ................................................................................................................ 381
Integrative Program in Molecular and Biomedical Sciences
Advisor: Benjamin Deneen, Ph.D. - Department of Neuroscience
Zhu, Ying ................................................................................................................... 382
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Fabrizio Gabbiani, Ph.D. - Department of Neuroscience
Zollinger, Daniel Richard ........................................................................................... 383
Department of Neuroscience
Advisor: Matthew Rasband, Ph.D. - Department of Neuroscience
Alexandra Acevedo-Rodriguez
Department of Neuroscience
Advisor: Shailaja Mani, Ph.D.-Department of Molecular & Cellular Biology
Benjamin Arenkiel, Ph.D.-Department of Molecular & Human Genetics
Anxiety and mood disorders affect 20% of the population and confer large
decrements to the patient’s quality of life. These disorders are frequently associated
with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis which is a system
activated in response to stressors. The sex steroids can modulate HPA axis activity
which may contribute to gender disparities observed in the prevalences of these
disorders. In particular, estrogen receptor β (ERβ) activity has been found to confer
anxiolytic effects and to reduce HPA axis reactivity. This receptor is expressed in the
paraventricular nucleus (PVN) of the hypothalamus which is a key component of the
HPA axis. Within the PVN, ERβ is highly expressed in oxytocin neurons, and in vitro
studies find that ERβ interacts with the oxytocin promoter to regulate oxytocin
transcription. Oxytocin is being researched as a potential therapeutic for numerous
psychiatric disorders, and peripheral oxytocin administration has been found to
decrease HPA axis reactivity and anxiety related behaviors. With ERβ’s expression on
oxytocin neurons and ERβ and oxytocin’s shared anxiolytic behavior, this suggests that
ERβ’s anxiolytic effect may be due to modulating oxytocin signaling. To determine if
oxytocin modulates ERβ mediated anxiolysis, oxytocin null and wild-type animals will
perform a behavioral battery to measure anxiety related behaviors and social aversion.
To determine the locus of oxytocin signaling necessary for ERβ mediated anxiolysis,
oxytocin receptors will be ablated from the PVN, central amygdala, and the median
raphe, regions important in stress circuitry. This will be done by utilizing stereotaxic
injection of an adeno-associated viral (AAV) vector associated with Cre-recombinase,
which will knockdown oxytocin receptor expression in infected cells of OxtrloxP/loxP
animals. In order to determine if ERβ binds to the oxytocin promoter in vivo and if stress
alters the level of this interaction, wild-type animals will be injected with ERβ agonist or
vehicle and then undergo restraint stress or be sacrificed from their home cage. The
PVN will be microdissected out and in vivo chromatin immunoprecipitation will be
performed on this tissue to determine if ERβ interacts with the oxytocin promoter. For all
experiments, male and female animals will be gonadectomized, to prevent endogenous
sex steroid interactions with ERβ, and treated with R-DPN or vehicle. These
experiments will illustrate the interaction between ERβ and oxytocin and how this
interaction relates to physiological stress and anxiety related behaviors.
Contributors: Acevedo-Rodriguez, Alexandra; Oyola, Mario; Malysz, Anna; Kudwa, Andrea;
Carbone, David; Handa, Robert; Mani, Shaila
Carolyn Joy Adamski
Department of Biochemistry & Molecular Biology
Advisor: Timothy Palzkill, Ph.D.-Department of Pharmacology
A comprehensive understanding of the enzymes mediating resistance to the
widely prescribed cephalosporin antibiotics is necessary to address the growing need
for new antimicrobial therapies. The CTX-M-14 β-lactamase enzyme, one of the most
common sources of resistance to cephalosporin antibiotics, was used in this study to
identify residues that are necessary for hydrolysis of cefotaxime, an important
cephalosporin drug. The roles of residues Ser237 and Arg276 in the activity of CTX-M14 β-lactamase were investigated using site-directed mutagenesis, enzyme kinetic
analysis and X-ray crystallography. Ser237 and Arg276 were mutated to alanine and
the catalytic activity of the single and double mutant combinations was evaluated. The
kinetic analysis revealed that, together, Ser237 and Arg276 residues are key
determinants of cefotaxime hydrolysis by the CTX-M-14 β-lactamase. High-resolution
crystal structures of the CTX-M-14 variants alone and in complex with cefotaxime
illustrated the roles of Ser237 and Arg276 in cefotaxime hydrolysis. When both residues
were mutated to alanine several significant changes were seen in the active site
including a compromised hydrogen bonding network, a narrower active site, and a 180°
rotation in the orientation of the aminothiazole ring on cefotaxime. These results identify
Ser237 and Arg276 as key determinants of cefotaxime activity in the CTX-M-14 βlactamase and elucidate their role in catalysis. Furthermore, these studies will help to
inform future drug development studies by highlighting these residues as potential
targets for β-lactamase inhibitor design.
Contributors: Adamski, Carolyn; Cardenas, Ana Maria; Brown, Nicholas; Horton, Lori; Sankaran,
Banumathi; Prasad, B.V.V.; Palzkill, Timothy
Saeed Ahmed
Clinical Scientist Training Program
Advisor: Thomas Giordano, M.D.-Department of Medicine
Elizabeth Chiao, M.D./M.P.H.-Department of Medicine
Background: Early identification and prompt entry into care is critical to reducing
morbidity and mortality in children with HIV. This report describes the impact of the
Tingathe program, which utilizes dedicated community health workers (CHWs) to
improve identification and enrollment into care of HIV-exposed and infected infants and
children at three Ministry of Health centers in Lilongwe, Malawi.
Methods: Three program phases are described. During the first phase,
Mentorship-Only (March 2007 to February 2008), on-site clinical mentorship on pediatric
HIV care was provided. In the second phase, Tingathe-Basic (March 2008 to February
2009), CHWs provided HIV testing and counseling to improve case finding of HIVexposed and infected children. In the final phase, Tingathe-PMTCT (March 2009 to
February 2011), in addition to Tingathe-Basic activities, CHWs were also assigned to
HIV-infected pregnant women to improve mother-infant retention in PMTCT care. We
reviewed routinely collected program data from March 2005 to March 2011.
Results: During the Mentorship-Only phase, 42 children (38 HIV-infected and 4
HIV-exposed) were active in care. During the three years covering the Tingathe-Basic
and Tingathe-PMTCT periods, CHWs conducted 44,388 rapid HIV tests, 7658 (17.3%)
in children 18 months to 15 years of age. Of children tested, 351 (4.6%) tested HIVpositive. During the Tingathe-Basic phase, 238 HIV-infected children were enrolled,
representing a six-fold increase in rate of enrollment from 3.2 to 19.8 per month. The
number of HIV-exposed infants increased from 4 to 118. During the Tingathe-PMTCT
phase, 526 HIV-infected children were enrolled over 24 months, at a rate of 21.9
patients per month. 1667 HIV-exposed infants were enrolled. Following the addition of
PMTCT activities, there was a greater than seven-fold increase in average number of
exposed infants enrolled per month (9.5 to 70 patients per month), resulting in 1667
enrolled with a younger median age at enrollment (5.2 vs 2.5 months; p <.001).
Conclusion: A multipronged approach utilizing CHWs to conduct health center
and home-based HIV testing, link HIV-infected children into clinical care, and provide
adherence support to PMTCT mothers can dramatically improve the identification and
enrollment into care of HIV-exposed and infected children.
Contributors: Saeed Ahmed, MD*1,2§, Maria H. Kim, MD*1,2, Amanda C. Dave, MPH2,
Rachael Sabelli2, Kondwani Kanjelo2, Geoffrey Preidis, MD,PhD1, Thomas P. Giordano, MD3,
Elizabeth Chiao3, MD, Mina Hosseinipour, MD4,5, Peter N. Kazembe, MBChB1,2, Frank
Chimbwandira, MBChB6, and Elaine J. Abrams, MD7.
Patricia Y Akinfenwa
Program in Translational Biology & Molecular Medicine
Advisor: Richard Hurwitz, M.D.-Department of Pediatrics
Donald Parsons, M.D./Ph.D.-Department of Pediatrics
Understanding host pathways regulating the expression of transgenes delivered by
adenoviral and AAV vectors could result in improved gene therapy strategies. Although most
therapies testing systemic administration have been unsuccessful, gene therapy protocols have
been particularly successful in the eye. Our group previously reported the enhancement of AdV
transgene expression (TGE) in the presence of vitreous, the gelatinous material that fills the
posterior eye. Vitreous has no effect on the efficiency of vector internalization but results in
increased transgene mRNA levels. We found that hyaluronan (HA) contributes to the
enhancement of AdV TGE, but another at least one other mediator is involved. The project aim
was to determine other vitreous component(s) and the signaling pathway(s) responsible for
enhancing TGE. Versican, a hyaluronan-binding proteoglycan highly expressed in vitreous, is
proteolyzed into two domains (G1 and G3), both have been shown to have functional effects in
cells. We hypothesize that versican mediates signaling that enhances AdV transgene
Versican is secreted from ACHN cells, a renal adenocarcinoma cell line. Versicanenriched supernatant (VES) was produced by culturing cells for 7 days and concentrating
proteins <300kDa using centrifugal filtration. Recombinant versican G1 and G3 supernatants
were isolated in a similar fashion. Enrichment was verified by silver stain and Western blot
analysis. Y79 or Weri-Rb retinoblastoma cells were transduced with AdV/CMV-Luc and treated
with VES, recombinant G1 or G3 and/or kinase inhibitors and effects on AdV TGE were
measured using a luciferase reporter assay. STAT3/5 phosphorylation was measured by flow
cytometry. The VES and recombinant G1 and G3 resulted in a 2-3-fold enhancement of
luciferase activity, similar to enhancement with vitreous treatment. Vitreous and versican
treatment increased pSTAT3 and not pSTAT5. Small-molecule inhibition of JAK1/2 using 1µM
of Ruxolitinib and STAT3/5 using 10µM C188-9 eliminated the enhancement of TGE mediated
by vitreous or versican, suggesting that enhancement is dependent upon JAK/STAT signaling.
Results support our hypothesis that versican mediates the enhancement of AdV
TGE. This regulation seems to be dependent upon transcriptional activation through JAK/STAT
signaling. In a theoretical model, the G1 or G3 domain of versican binds a cell surface receptor,
CD44 or EGFR, respectively. The signal transduces through the JAK/STAT pathway, mediating
enhanced TGE. These results are the first to implicate versican as an enhancer of AdV TGE, a
finding which could be applied to improve the efficiency of adenoviral-based gene therapy
Contributors: Akinfenwa, Patricia; Bond, Wesley; Hurwitz, Mary; Hurwitz, Richard
Stefanie Alexander
Integrative Program in Molecular and Biomedical Sciences/M.D.-Ph.D. Program
Advisor: Brendan Lee, M.D./Ph.D.-Department of Molecular & Human Genetics
Osteogenesis imperfecta (OI) is a debilitating genetic osteodysplasia that results
in low bone mass, bone deformity, and bone fractures. Most cases of OI are caused by
mutations in the structural protein type I collagen (dominant OI) or in protein complexes
that post-translationally modify type I collagen (recessive OI); both types of mutations
can lead to biochemical overmodification. Type I collagen is important for structural
integrity of bone and extracellular matrix (ECM) cell signaling. It extensively interacts
with ECM components that regulate bioavailability of signaling molecules. Due to the
phenotypic similarities between OI mouse models and TGFβ overexpression models,
we hypothesized that altered TGFβ signaling could contribute to OI pathogenesis in
both recessive (Crtap-/-) and dominant (G610C OI) OI mouse models. In support of this,
we found increased mRNA expression of TGFβ target genes with quantitative PCR and
an increased ratio of phosphorylated Smad2 to total Smad2 with western blot in bone
samples from both OI mouse models suggesting increased TGFβ signaling. In a bone
marrow stromal cell (BMSC) in vitro system, which mimics osteoblast differentiation, we
found that osteoblasts from both OI mouse models demonstrated increased TGFβ
signaling and increased free TGFβ in the media, suggesting altered TGFβ
bioavailability. Treatment of both OI mice with a TGFβ antibody (1D11) significantly
rescued the OI bone phenotype. Together, these experiments demonstrate that
increased TGFβ signaling contributes to the pathogenesis of OI. Small leucine-rich
proteoglycans (SLRPs), ECM components that regulate bioavailability of signaling
molecules, are the only ECM components that can bind both type I collagen and TGFβ.
I hypothesize that changes in type I collagen in OI disturb binding to the SLRPs, which
in turn alters TGFβ availability in the ECM. Surface plasmon resonance (SPR)
demonstrated that the SLRP decorin (Dcn) binds Crtap-/- type I collagen 45% less
effectively than wildtype type I collagen. Altered binding could modulate TGFβ signaling
by preventing proper sequestration to type I collagen. I will use SPR to study binding of
other SLRPs to type I collagen from both OI mouse models. My initial data studying the
interaction between SLRPs and altered TGFβ signaling using the BMSC in vitro system
demonstrates that SLRP mRNA expression increases significantly during osteoblast
differentiation in both OI models compared to WT. This increase correlates with the
increased expression of TGFβ target genes. Further studies will be done to investigate
the relationship between the increased TGFβ signaling, increased SLRP expression,
and the binding of SLRPs to the ECM. Ultimately, establishing the molecular
mechanisms behind altered ECM signaling in OI will lead to more specific treatments for
this disease.
Contributors: Alexander, Stefanie; Grafe, Ingo; Lee, Brendan
Hunter Marshall Allen
Department of Neuroscience
Advisor: Hui-Chen Lu, Ph.D.-Department of Pediatrics
The efficacy of nicotinamide mononucleotide adenylyl transferases (NMNATs) for
neuroprotection in a diverse range of neurodegenerative conditions makes them
promising therapeutic targets. NMNAT2 is the major NMNAT isoform in mammalian
brain and is a key neuronal maintenance factor. Nmnat2 mRNA expression is reduced
in many neurodegenerative diseases. Elevating NMNAT2 in mammals, or Drosophila
NMNAT (dNMNAT) in flies, reduces Tau burden and ameliorates tauopathy model
phenotypes. Molecular chaperones aid in the clearance or refolding of non-native
proteins in neurodegenerative diseases. Here we report a novel chaperone function
requiring a unique C-terminal ATP site that is independent from NMNAT2’s NAD
synthase activity. We found that NMNAT2’s chaperone, but not enzymatic, function is
required to ameliorate Ataxin1-82Q- and Tau-mediated proteinopathies. In the brains of
Alzheimer’s disease (AD) patients, NMNAT2 is greatly reduced at both the mRNA and
protein levels. The residual NMNAT2 in AD brains shifts its solubility and co-localizes
with aggregated Tau. Similarly to heat shock proteins (HSP), NMNAT2 complexes with
Tau (P301L). Thus, these multiple lines of evidence suggest that NMNAT2 acts as a
chaperone to reduce proteotoxic stress and maintain neuronal health.
Contributors: Ali, Yousuf O.; Allen, Hunter M.; Hatcher, Asante; Bjorklund, Nicole; Taglialatela,
Giulio; Lu, Hui-Chen
Samirkumar B Amin
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Lynda Chin, M.D.-Genomic Medicine
The major focus of this study is to uncover emerging functional role of long noncoding RNA (lncRNA) in the development and progression of cancer. Although
structural alterations like mutations in BRAF, NRAS, etc. are found to be driver events in
most of melanoma cases, the cancer genome atlas (TCGA) melanoma working group
have also shown around 15% (n=39) of melanoma samples having none of these driver
events. Given likely absence of structural events in these triple wild type (WT) samples,
epigenetic remodeling may play a dominant role in driving oncogenic transcriptional
program. At the transcription core, such remodeling can take place by differential
interaction patterns of TF with other TFs, co-regulators or availability of TF DNA-binding
site by actions of chromatin remodelers. However, it is unknown whether these TF-coregulator-DNA interactions at known cancer gene sites are coordinated by an
abundance of sequence-specific tethering elements. In this context, our hypothesis
underscores both mechanistic and functional role of lncRNAs in cancer. Based on
recent research, we hypothesize that abundance of lncRNAs harboring sequencespecific motif or structural motif coordinate TF-DNA interactions at promoter regions of
known cancer genes and thus, drive carcinogenesis. Using data from TCGA melanoma
project, we have characterized differential expression of several lncrRNAs in triple WT
melanoma as compare to BRAF/NRAS/NF1 mutant melanoma subtypes. Further, this
subtype specific overexpression of lncRNAs show strong association with normal-like
CpG island methylation (CIMP) phenotype, possibly indicating altered methylation at
promoter regions secondary to lncrna mediated interactions. We also observe
abundance of a few transposable elements from AluYc and LINE-1 families in coding
region of these lncRNAs and promoter regions of expressed consensus cancer genes.
Further, using ChIP-seq data on histone marks, H3K36me3 and H3K27ac on four
melanoma samples, we are integrating epigenetic data with known gene and microrna
expression data to understand potential shift in transcription secondary to expressed
lncrnas in triple WT subtype. Finally, our approach and resulting methods pipeline will
be scalable and extensible in understanding mechanistic and functional role of lncRNA
in other cancers.
Contributors: Amin, Samir B. ; Akdemir, Kadir ; Wu, Chang-Jiun ; Rai, K ; Chin, L.
Viren R Amin
Department of Molecular & Human Genetics
Advisor: Aleksandar Milosavljevic, Ph.D.-Department of Molecular & Human Genetics
Tissue-specific expression of lincRNAs suggests developmental and cell-type
specific functions, yet tissue specificity has been established for only a small fraction of
this abundant gene category. By analyzing 111 reference epigenomes from the NIH
Roadmap Epigenomics project we identify tissue-specific epigenetic regulation for a
total of 3,753 (69% examined) lincRNAs, with 54% active in only one of fourteen
cell/tissue clusters and 15% in up to three. The tissue-specific lincRNAs are strongly
linked to tissue-specific pathways, supporting their important role in development or celltype specific processes. A larger fraction of lincRNA TSSs shows enhancer-like
(H3K4me1) than promoter-like (H3K4me3) tissue-specific activation and a significant
fraction shows a combined pattern. Polycomb-regulated lincRNAs reside in bivalent
state in embryonic stem cells. The exquisitely tissue-specific epigenetic regulation of
lincRNAs and the assignment of a majority of lincRNAs to specific tissue types will
inform future mechanistic and genetic studies of this newly discovered class of genes.
Contributors: Amin, Viren; Harris, Alan; Onuchic, Vitor; Jackson, Andrew; Charnecki, Tim;
Paithankar, Sameer; Subramanian, Sai Lakshmi; Riehle, Kevin; Coarfa, Cristian; Milosavljevic,
Mussie K Araya
Department of Molecular Physiology & Biophysics
Advisor: William Brownell, Ph.D.-Department of Otolaryngology-Head and Neck Surgery
Hearing requires precise detection and coding of the acoustic signal by the inner
ear and equally precise communication of the information through the auditory brain
stem. A membrane based motor in the outer hair cell lateral wall contributes to the
transformation of sound to a precise neural code. Structural, molecular and energetic
similarities between the outer hair cell and auditory brainstem neurons suggest
neuromechanics contributes to hearing. The rapid initiation of action potential by
cooperative activation of voltage gated ion channels enhances neuronal temporal
processing and increases the upper frequency limit for phase locking. We explore the
possibility of electrically evoked mechanical forces mediated by changes in the physical
properties of the lipid bilayer in synchronizing the gating of voltage gated ion channels
for rapid spike generation, thereby introducing the concept of mechanical force in gating
of voltage gated ion channels. The rapid speed of membrane electromechanical
signaling can supports a simple and direct mechanism of ion channel cooperativity.
Using optical tweezers based force bio-sensor; we find that it takes 2-3 times
more pulling force to form membrane tethers from the AIS of hippocampal pyramidal
neurons than from the soma indicating the AIS membrane has a stronger membranecytoskeleton adhesion force. Previous experiments in cultured neurons showed that
channels in soma (which are not anchored by Ank-G) are removed from the membrane
by endocytosis. Our results together with previous experiments suggest that channels
are firmly anchored to the actin–spectrin–AnkG-based cytoskeleton at AIS. Since
cooperative activation is highly dependent on the density of Na+ channels indicating
that the inter-channel coupling is distance dependent, the clustering of Na+ channels at
the AIS by the actin-spectrin-AnkG network can set the stage for cooperative gating
between ion channels. Currently membrane tethers are being pulled under voltage
clamp to a standard length and allowed to relax to an equilibrium force. The
electromechanical coefficient will be measured. The effect of manipulating membrane
material properties on the coefficient and simultaneously measured membrane currents
will be determined. Membrane currents will resolve channel function. A greater
electromechanical coefficient in the AIS would be consistent with a lower threshold for
ion channel gating and support a contribution of electromechanics to ion channel gating.
The manipulations that alter electromechanics also alter ion channel kinetics. Further
support for the membrane’s role in ion channel gating would be for the coefficient and
channel function to vary concomitantly.
Contributors: Mussie K. Araya1 and William E. Brownell1,2
1Molecular Physiology and Biophysics 2Otolaryngology – H&N Surgery Baylor College of
Medicine, Houston, Tx, USA
James Michael Arnold
Department of Biochemistry & Molecular Biology
Advisor: Arun Sreekumar, Ph.D.-Department of Molecular & Cellular Biology
Breast cancer is a significant public health concern and there remain unmet
challenges in the diagnosis and treatment of triple negative breast cancer (TNBC).
Gene expression profiling has revealed that TNBC is composed of a diverse set of
disease states, which serve as an imperfect predictor of response to therapy, thus there
is a need for a more integrated approach to describe phenotypic subtypes within TNBC.
Metabolites are the final products of cell signaling pathways, and the relative levels of
metabolites can be considered a direct readout of the current phenotypic state of the
cell. In a recently published study, our lab profiled 536 metabolites across 67 matchedtumor-normal pairs of breast tissue, all containing matched Affymetrix-derived gene
expression profiles and epidemiological data. Our results suggest altered levels of
specific metabolites within a subset of TNBC, predominantly of African American (AA)
descent. These include elevated levels of unsaturated fatty acids, lipids, and carnitines.
Interestingly, this same group of TNBC tumors is highly enriched for the expression of
FOXM1, an oncogenic transcription factor associated with cell cycle progression known
to be associated with aggressive breast cancer subtypes. Datamining of public datasets
and preliminary siRNA knockdown studies in vitro suggest FOXM1 may regulate key
lipid catabolism genes ACSL3 and HADHB, thus playing a critical role in the regulation
of tumoral lipid profiles and fatty acid beta-oxidation. Furthermore, knockdown of
FOXM1 in multiple TNBC cell lines results in an accumulation of neutral lipids,
observable by lipidtox staining, which further supports the concept that FOXM1 plays an
important role in directing lipid catabolism. These preliminary results suggest FOXM1
may play a significant role in the regulation and maintenance of lipid metabolism in
breast cancer.
Contributors: Arnold, James; Konde, Sai Aparna; Gu, Franklin; Rao, Arundhati; Sreekumar,
Ryan Thomas Ash
Department of Neuroscience/M.D.-Ph.D. Program
Advisor: Stelios Smirnakis, M.D./Ph.D.-Department of Neurology
Methyl-CpG-binding-protein-2 (MECP2) duplication syndrome is a progressive Xlinked disorder of intellectual disability and autism. Interestingly, the mouse model of
MECP2 duplication syndrome exhibits enhanced motor and contextual fear learning in
addition to stereotyped behaviors and social avoidance. We hypothesized that a bias
towards increased synaptic stability could lead to abnormally enhanced memory
consolidation, reminiscent of savant-like behaviors occasionally associated with autism.
Chronic 2-photon microscopy revealed that more dendritic spines are formed and
stabilized during motor learning in apical corticospinal dendritic arbors of MECP2
duplication mice, compared to controls. Learning-associated spines formed clusters
whose number predicted enhanced motor performance in mutants. The Ras-MAPK
signaling pathway was found to be hyperactive specifically after training in MECP2
duplication mouse motor cortex. Pharmacologic inhibition of MAPK signaling normalized
both motor and contextual-fear learning in mutants. We conclude that pathologically
stable learning-associated dendritic spine clusters and hyperactive Ras-MAPK signaling
drive abnormal learning phenotypes in this form of syndromic autism.
Contributors: Ash,Ryan; Buffington, Shelly; Costa-Mattioli, Mauro; Zoghbi, Huda; Smirnakis,
Lisa Michelle Atkins
Department of Molecular Virology & Microbiology
Advisor: Joseph Petrosino, Ph.D.-Department of Molecular Virology & Microbiology
F. tularensis (Ft) is a Gram-negative, facultative intracellular coccobacillus and
the etiological agent of the zoonotic disease tularemia. Subspecies tularensis (type A)
and holarctica (type B) are responsible for 100% of tularemia fatalities in the United
States, with type A infections resulting in significantly higher mortality (24%) compared
to type B (7%). Ft is classified as a Tier One select agent due to its low infectious dose
(<15 CFU), high morbidity, and lack of a licensed vaccine. An attenuated Live Vaccine
Strain (LVS) was empirically derived in the 1950s from repeated passage of a type B
strain. While it was later shown that LVS is less effective against inhalational type A, it is
to date the only vaccine for which formal efficacy data in humans exists. However, LVS
remains unlicensed for public use, and the exact mechanism of attenuation is unknown.
Comparative genomics revealed 51 syntenic block rearrangements in type A
compared to type B genomes, which complicates genetic comparison studies between
LVS and type A strains. Therefore, we aligned the LVS genome to virulent type B
strains and identified 17 genes that are disrupted in LVS, implicating these genes in
LVS attenuation and Ft virulence. These candidate genes are not well described and
their respective roles in pathogenesis are poorly understood. We propose that the
disruption of a subset of candidate genes in virulent type B will result in attenuation as
determined by reduced intracellular replication, increased macrophage activation and
decreased virulence in mice. Preliminary studies to establish attenuation assays in LVS
show that primed macrophages display increased pro-caspase-1 protein levels
compared to uninfected controls and undergo cell death earlier than unprimed
macrophages, effectively removing the replicative niche for Ft. In contrast, virulent type
B replicates to higher numbers in both human and murine macrophages compared to
LVS. Additionally, macrophages infected with virulent type B secrete diminished levels
of Th1 cytokines compared to LVS, thus establishing valid thresholds in order to
evaluate candidate gene disruptions. Characterizing the mechanism of LVS attenuation
may lead to the development of attenuated vaccines, in type A or type B backgrounds,
that are genetically defined and more efficacious against type A.
Contributors: Atkins, Lisa; Ayvaz, Tulin; Petrosino, Joseph
Benu Atri
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Olivier Lichtarge, M.D./Ph.D.-Department of Molecular & Human Genetics
Different substitutions impact the function of a protein in different ways. While
some substitutions cause a large effect, others are neutral or harmless. In light of
widespread exome sequencing, it is critical to distinguish deleterious substitutions from
harmless ones in a clinical setting. We hypothesize that the impact of a substitution on
phenotype depends on two factors: the positional importance of the residue and size of
substitution. To test this, we chose bacterial RecA protein that plays a central role in
homologous recombination and regulates DNA damage repair. RecA is also a key
component of the bacterial SOS response where it controls the expression of many
other DNA damage repair genes. We quantitatively measured the impact of a mutation
on RecA by assaying for DNA damage repair function and recombination function. The
results of these assays reveal a correlation between residue position and the size of
substitution on the functions of RecA. These experimental results complement prior
correlations in retrospective analyses, including the leading performance in independent
evaluations of blind predictions. Together these data shed new light on the relationship
between genotype and phenotype variations.
Contributors: Katsonis, Panagiotis; Adikesavan, K. Anbu; Lichtarge, Olivier
Benjamin Judson Bachman
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Olivier Lichtarge, M.D./Ph.D.-Department of Molecular & Human Genetics
Popular websites such as Netflix and Facebook enhance their users’ experiences
by creating recommendations of items such as “potential friends” and “movies you might
like” to their customers. They do so by using various machine learning tools to predict
interactions. For example, a common method for predicting social interactions is by
looking at the proximity of two users in a network. If they have many mutual friends, it is
likely that they themselves are friends, but simply haven’t connected with each other
online. With Netflix, a common method for predicting a user’s enjoyment of a movie is
to build a model to represent how much each user likes various genres of movies and
how well each movie fits into a particular genre using a method known as collaborative
filtering. Here, it will be shown that these same concepts can be applied to molecular
biology, with a focus on predicting interactions of tumor suppressor p53. Rather than
having a social network or a matrix of user-movie ratings, the information used will be
large-scale biological data, such as protein-protein interaction information and gene coexpression data. In the case of proximity-based predictions, it will be shown that novel
p53 kinases can be found by looking at the set of known p53 kinases and identifying
their potential “friends”, nearby kinases in the network. With the Netflix-style method,
we build a model of the protein-protein interaction network to predict previously
unknown p53 interactions. A meta-algorithm combining these methods is also built to
optimize prediction quality. The predictions made by these machine learning tools are
then tested experimentally to show their prediction accuracy. In addition to predicting
that these interactions exist, it is shown that the model can be used to predict something
about the nature of the interaction. With this study, it will be shown that social network
models can be applied to biological data to successfully predict novel biology, thus
speeding our advancement of knowledge.
Contributors: Adikesavan, Anbu K; Wilkins, Angela D; Lisewski, Andreas; Regenbogen, Sam;
Pickering, Curtis; Donehower, Lawrence; Lichtarge, Olivier
Shawn Samson Badal
Program in Translational Biology & Molecular Medicine
Advisor: Farhad Danesh, M.D.-Department of Medicine
William Mitch, M.D.-Department of Medicine
Despite recent progress in identifying microRNAs (miRNAs) with crucial roles in
the development of diabetic nephropathy (DN), the functional validation and
downstream effectors of these miRNAs in vivo are frequently lacking. Here, we show
that miR-93 plays an important role in the development and progression of DN in vivo.
First, a tissue-specific, tamoxifen-inducible model of miR-93 overexpression was
generated to selectively induce miR-93 overexpression only in podocytes (Pod-miR-93).
At 24 weeks of age, diabetic db/db mice with forced expression of Pod-miR-93 exhibited
improved kidney function as measured by a significant reduction in albuminuria, when
compared to diabetic controls. Additionally, these mice exhibited a significant reduction
in kidney fibrosis compared to controls as measured by Periodic-Acid-Schiff’s (PAS)
staining. Importantly, micrographs of kidney cortices prepared for transmission electron
microscopy revealed reduced podocyte detachment, and improved glomerular
basement membrane thickness in diabetic Pod-miR-93 mice, similar to that of nondiabetic control mice. Second, restoring miR-93 expression via the in vivo delivery of
miR-93 mimic in diabetic mice rescued DN phenotype in a similar fashion. RNASequencing and bioinformatics analysis revealed a potential role for miR-93 as an
epigenetic regulator through a novel target, Msk2. We observed high glucose induced
activation of Msk2 leads to global changes in H3S10 phosphorylation levels, which acts
to alter chromatin states. We propose that miR-93 serves as a relaying link between the
metabolic state and the chromatin remodeling in the diabetic milieu mainly through its
regulatory effect on phosphorylation of histone H3 at serine 10 (H3S10). These findings
uncover a novel role of miRNAs as critical metabolic/epigenetic switches, which has
implications for targeting of miRNAs in DN.
Contributors: Shawn S. Badal, Yin Wang, Jianyin Long, Antony Rodriguez, Benny H. Chang,
and Farhad R. Danesh
David A Bader
Department of Molecular & Cellular Biology/M.D.-Ph.D. Program
Advisor: Sean Mcguire, M.D./Ph.D.-Department of Molecular & Cellular Biology
Androgen receptor (AR)-induced de-novo lipid synthesis enables the growth and
survival of hormone sensitive and castrate-resistant prostate cancer cells, but the
metabolic alterations mediated by AR to support lipogenesis in prostate cancer are
poorly understood. Lipogenesis requires pyruvate to generate lipid precursors (e.g.
citrate) and recent in-vivo imaging strategies have demonstrated that, in contrast to
virtually all other tumors, prostate tumors take up pyruvate more rapidly and reliably
than glucose. Further, a subunit of the mitochondrial pyruvate carrier (MPC), MPC2, is a
direct AR target gene, suggesting a link between AR action pyruvate trafficking, and
lipogenesis. The overall objective of our current project is to determine the mechanism
by which AR alters pyruvate trafficking to fuel lipogenesis in prostate cancer. The
overarching central hypothesis is that AR regulates the MPC to increase pyruvate flux
into mitochondria to enable oncogenic growth by increasing lipogenic capacity of tumor
cells. Characterization of the mechanism underlying increased pyruvate trafficking in
prostate cancer will likely enable the development of strategies to therapeutically
manipulate pyruvate flux. Such treatments could be applied to both hormone sensitive
and treatment refractory castrate-resistant prostate cancer. In order to accomplish the
overall objective, the central hypothesis will be tested by pursuing the following three
specific aims: 1) Determine the impact of altered MPC subunit expression on pyruvate
flux by experimentally modifying MPC subunit composition and measuring a variety of
metabolic endpoints in addition to using mass-spectroscopy-based methods to perform
13C pyruvate tracing. 2) Define MPC-dependent tumor properties using in-vitro
measurements of tumor cell proliferation, invasive capacity, and lipogenic capacity. 3)
Assess the impact of MPC perturbation on prostate tumor growth in-vivo by
transplanting nude mice with prostate cancer cells harboring stable modifications to
MPC subunit expression or treating xenografted mice with the MPC inhibitor UK5099.
Completion of our research plan will define the metabolic and oncogenic consequences
of the altered MPC subunit expression pattern observed in prostate cancer. The
significance of this contribution derives from the expected translational opportunities
made possible by the characterization of lipogenic substrate trafficking in prostate
cancer, disruption of which represents a rational therapeutic strategy in all stages of the
disease. The proposed research is innovative because it focuses on understanding and
disrupting tumor-specific metabolic substrate trafficking rather than enzymatic activities
common to normal tissues and tumor cells alike. It is expected this expansion of focus
to include substrate trafficking will broaden the approach to metabolic targeting in
cancer.Contributors: Bader, David; Coarfa, Cristian; Hartig, Sean; Sreekumar, Arun; O’Malley,
Bert; McGuire, Sean
Dillon Patrick Baete
Department of Neuroscience
Advisor: Mauro Costa-Mattioli, Ph.D.-Department of Neuroscience
Autism spectrum disorder (ASD) is a heterogeneous group of diseases
collectively characterized by impaired social interaction and communication, repetitive
behaviors, and cognitive deficits. Although the etiology of the disease is unknown,
several genes have been identified as mutated in individuals with ASD, including the
phosphatase and tensin homolog gene (PTEN). PTEN encodes for the eponymous
PTEN protein, a tumor suppressor and inhibitor of the PI3K pathway. PTEN directly
antagonizes the actions of PI3K, dephosphorylating PIP3, thereby preventing activation
of the pathway. Mutation of PTEN causes overactivation of the pathway, leading to an
increase in mTOR Complex 1 (mTORC1) activity and also, through an unknown
mechanism, increased mTOR Complex 2 (mTORC2) activity. Pharmacological
reduction of both complexes can rescue many abnormalities associated with PTEN
mutation; however, to date it is unclear which abnormalities are associated with which
complex. We have generated forebrain-specific knockout mice with loss of PTEN and
loss of rictor, a key mTORC2 component. We found that deletion of rictor abrogates
tonic-clonic seizures, anti-social behavior, and deficient learning in memory exhibited by
PTEN knockout mice. In addition, deletion of rictor nearly doubles the lifespan of PTEN
knockout mice. While deletion of rictor ablates mTORC2 activity, mTORC1 activity
remains elevated in PTEN knockout. These results suggest that upregulated mTORC2
activity, and not mTORC1 activity, is responsible for the epileptiform activity and ASDlike phenotypes exhibited by PTEN knockout mice.
Contributors: Baete, Dillon; Huang, Wei; Costa-Mattioli, Mauro
Lakshya Bajaj
Department of Molecular & Human Genetics
Advisor: Marco Sardiello, Ph.D.-Department of Molecular & Human Genetics
Neuronal Ceroid Lipofuscinoses (NCLs) are the most commonly inherited
neurodegenerative disorders of childhood. They are a group of autosomal recessive,
progressive encephalopathies characterized by the accumulation of auto-fluorescent
ceroid lipopigment in various tissues, notably in neurons. Current analyses indicate that
subunit C of the mitochondrial ATP synthase and sphingolipid activator proteins (SAPs)
are the two major aggregates found in the neuronal cells. The clinical course includes
progressive dementia, seizures, progressive visual failure and, death by the second or
third decade of life. CLN6 is an ER transmembrane protein, mutations in which are
known to cause vLINCL. Similar to other NCLs, deposits of ceroid lipopigments are
found in the cells. Neurons are particularly vulnerable to damage caused by
lipopigments and more susceptible to cell death. The progressive death of neurons
contributes to the signs and symptoms of late-infantile NCL. However, it is unclear how
mutations in the CLN6 gene are involved in the buildup of lipopigments and its function
in disease pathogenesis. The ongoing experiments in our lab strongly hinted that CLN8,
another protein causing a very similar form of NCL is acting as a cargo transporter or a
chaperone for various lysosomal hydrolases. As the defects in CLN8 and CLN6 are
majorly lysosomal and these proteins result in two very similar diseases phenotypically,
we postulated that CLN6 might also be acting as cargo transporters or chaperones for
various lysosomal enzymes or maybe is a part of a complex with CLN8 aiding in the
delivery of various lysosomal enzymes. Working towards this hypothesis we performed
a mass-spectrometry analysis on the lysosomal fraction extracted from livers of onemonth-old CLN6 KO mice and we found depletion of various lysosomal hydrolases in
the lysosomal compartment. We further confirmed the depletion of certain lysosomal
enzymes by performing immuno-blot analysis. We investigated the difference in the
enzyme activity between CLN6 KO and WT mice by performing enzyme assay on a
battery of hydrolases. We are also working towards finding the mechanism by which
CLN6 is aiding in the delivery of these lysosomal hydrolases. To check for the same we
performed high throughput direct-interaction analysis between N-terminus YFP tagged
CLN6 acting as bait and the C-terminus YFP tagged various lysosomal hydrolases in a
Bimolecular florescent complementation (BiFC) assay screen using Flow-cytometry.
The candidate interactors are undergoing an unbiased check using CoImmunoprecipitation (CO-IP). Using Bi-FC we have also been able to show that CLN6
homodimerizes and heterodimerizes with CLN8, the latter interaction has been further
confirmed by CO-IP. Taking a lead from these results we are curious to see the
relationship between CLN6 and CLN8 and are currently in a process to generate CLN6/-; CLN8-/- KO mice. Contributors: Bajaj, Lakshya; Di Ronza, Alberto; Palmieri, Michela; Lotfi,
Parisa; Sharma, Jaiprakash; Sardiello, Marco
Miriam Balderas
Department of Molecular Virology & Microbiology
Advisor: Anthony Maresso, Ph.D.-Department of Molecular Virology & Microbiology
Iron is essential trace element for nearly all forms of life and its sequestration
plays an important role in mammalian hosts defense against bacterial infection by
limiting bacterial replication. The mammalian ionic iron level is far too low (10-18) to
sustain normal bacterial growth, further to counteract free iron availability most of the
mammalian iron is compartmentalized in heme, which is further sequestered in
hemoglobin (Hb). Thus, successful bacterial pathogens have evolved mechanisms to
access heme-iron pools to successfully replicate. Bacillus anthracis, a Gram-positive,
spore forming bacteria and the causative agent of anthrax, has evolved mechanisms to
capture sequestered iron mediated by a structural module called the near-iron
transporter (NEAT) domain. In B. anthracis NEAT-containing proteins form a network of
transport proteins that function to import and catabolize heme. In this study, hal (hemeacquisition leucine-rich repeat protein), a gene recently implicated in anthrax disease
progression, was characterized. Hal encodes a single NEAT domain and several
leucine-rich repeats. The purified recombinant NEAT domain of Hal was shown to bind
heme, despite lacking a stabilizing tyrosine common to the NEAT superfamily of
hemoproteins. The NEAT domain also bound Hb and acquired heme from Hb in
solution. Deletion of hal resulted in bacilli unable to grow efficiently on heme or
hemoglobin as an iron source and yielded the most significant phenotype relative to
other putative heme uptake systems, a result that suggests this protein plays a
prominent role in the replication of B. anthracis in hematogenous environments. Finally,
studies using a novel vaccine, composed of the conserved protein NEAT domains of B.
anthracis, suggest the NEAT subunits confer resistance and provide protective
immunity to anthrax disease in a murine model of infection. This project proposes to
move forward the development of NEAT domains as a vaccine for anthrax disease.
These studies advance our understanding of heme acquisition by this dangerous
pathogen and justify efforts to determine the mechanistic function of this novel protein
for vaccine or inhibitor development.
Contributors: Balderas, Miriam; Maresso, Anthony
Anthony Patrick Barrasso
Integrative Program in Molecular and Biomedical Sciences
Advisor: Loning Fu, Ph.D.-Department of Pediatrics
The sympathetic nervous system (SNS) is classically thought to activate the
“fight-or-flight” response as a reaction to external stress. However, the SNS also plays
an important role in maintaining the homeostasis of diverse physiological functions in
vivo. The SNS releases epinephrine and norepinephrine, which bind α- and βadrenergic receptors (AR) to regulate many key cellular processes in peripheral tissues.
There are three sub-types of β-AR (β1, 2, and 3), all of which are known to activate cell
proliferation pathways. Recent studies have linked uncontrolled β-AR intracellular
signaling to the initiation and progression of several cancers in both humans and mouse
models. Especially, β2-AR deregulation, which has been identified as a potential
prognostic marker for cancer in humans. We are specifically interested in the role of βAR signaling in hepatocellular carcinogenesis. We propose to generate a mouse model
lacking all three β-ARs in the liver by first generating mice carrying floxed alleles of βAR1-3 genes via CRISPR/Cas genome editing. These mice will then be crossed with
the transgenic mice expressing cre recombinase under the control of albumin gene
promoter (Alb-cre). We expect to obtain the liver-specific β-less mice within 6 months.
Contributors: Barrasso, Anthony; Fu, Loning
Meagan Amelia Barry
Program in Translational Biology & Molecular Medicine/M.D.-Ph.D. Program
Advisor: Peter Hotez, M.D./Ph.D.-Department of Pediatrics
Laila Woc-Colburn, M.D.-Department of Medicine
Chagas disease is a neglected tropical disease of great importance in the
Americas, with 7-8 million people infected. The causative agent is Trypanosoma cruzi
(T. cruzi), and results in an acute febrile illness that progresses to chronic chagasic
cardiomyopathy in 30% of patients. In endemic areas, Chagas disease is the leading
cause of cardiovascular death between ages 30-50. Current pharmacological
treatments are plagued by significant side effects, poor efficacy, and are contraindicated
in pregnancy. There is an urgent need for new treatment modalities. A therapeutic
vaccine for Chagas disease has potential advantages that include cost savings,
reduced adverse effects, and the potential to be used as a replacement for current
therapies or when paired with chemotherapy. Prior work in mice has identified an
efficacious T. cruzi antigen (Tc24). To elicit a protective cell-mediated immune response
to the Tc24 protein, we have utilized a nanoparticle delivery system in conjunction with
CpG motif-containing oligodeoxynucleotides (ODN) as an immunomodulatory adjuvant.
When tested in a BALB/c mouse model, a dose response study demonstrated a positive
relationship between dose of vaccine and Tc24-specific IFN-γ response. Our
nanoparticle vaccine, comprised of Tc24 and CpG ODN encapsulated in poly(lactic-coglycolic acid) (PLGA) nanoparticles, produced the most robust TH1- mediated CD8+ T
cell immune response. When tested for therapeutic efficacy in T. cruzi infected BALB/c
mice, improved survival was seen in the vaccine group compared to the control groups.
Additionally, there was a significant reduction in the number of parasites in the cardiac
tissue of the vaccine group compared to the PBS sham vaccine group, indicating
protection from parasite-driven cardiac damage. The mice that survived to the end of
the study had almost undetectable numbers of parasites in the cardiac tissue. These
data demonstrate the immunogenicity and efficacy of a Tc24/CpG ODN nanoparticle
vaccine and are convincing evidence for a potential new therapeutic vaccine against
Chagas disease.
Contributors: Barry, Meagan; Wang, Qian; Beaumier, Coreen; Jones, Kathryn; Keegen, Brian;
Bottazzi, Maria Elena; Heffernan, Michael; Hotez, Peter.
Susan Michelle Benton
Integrative Program in Molecular and Biomedical Sciences
Advisor: Jason Heaney, Ph.D.-Department of Molecular & Human Genetics
Colorectal cancer (CRC) is the third most common cancer and the second
highest cause of cancer related deaths in the U.S. Mutations inactivating the gene
Adenomatous Polyposis Coli (Apc) are found in familial adenomatous polyposis and
account for approximately 80% of all sporadic human colorectal cancers. The Apc
multiple intestinal neoplasia (Apcmin/+) mouse model is commonly used to model tumor
initiation and as a tool to discover genetic modifiers of CRC risk.
Cancer requires the up-regulation of oncogenes to grow and metastasize; in fact
key elements of the translational machinery are up-regulated in several cancers.
Several oncogenes (e.g. Cyclin D1, Myc) have been shown to be highly dependent on
efficient translation initiation for their protein expression, and are highly sensitive to
changes in the rate of translation initiation. Our preliminary data show that partial
deficiency for the beta subunit of the translation initiation factor eIF2 (Eif2s2)
significantly reduces both polyp number and size in Apcmin/+ mice, suggesting that
adenoma development is influenced by the rate of translation.
Using both mouse models of intestinal cancer and human colorectal cancer cell
lines we will determine how modulation of translation by eIF2 affects the hallmarks of
tumorigenesis, mechanism by which modulation of eIF2 affects tumorigenesis, and if
drugs that target translation initiation can be used as a colorectal cancer treatment.
Contributors: Maywald, Becky; Heaney, Jason
Grant Daniel Bertolet
Department of Pathology & Immunology
Advisor: Dongfang Liu, M.D./Ph.D.-Department of Pediatrics
T cells are a subtype of white blood cells that can be broadly divided into two
groups based on the expression of one of two different surface proteins: either CD4 or
CD8. CD8+ T cells are also known as “killer T cells”, as their primary function is to kill
infected or malignant cells. CD4+ T cells are known as T helper cells. Their role is more
complex, but they generally serve to direct and augment the immune response through
the secretion of different sets of chemical messenger proteins known as cytokines. For
both subsets, in order to carry out their functions they must first form what is known as
an immunological synapse (IS)– a stable interface between the T cell and another cell in
which biochemical information about the state of the infection is exchanged and the T
cell is accordingly programmed for future action.
PD-1 is a cell surface receptor expressed on both subsets of T cells, and whose
main function is to temper cell activation. PD-1’s expression is known to be upregulated
on virus-specific T cells during chronic viræmia and on tumor infiltrating lymphocytes in
cancer. Blockade of PD-1 signalling has been repeatedly shown to improve the antitumoral and antiviral functions of so-called “exhausted” T cells, which otherwise have
impaired ability to carry out necessary immune function. This demonstrates that PD-1
signalling is an important component of exhaustion. However, it is not completely
understood how exactly PD-1 signalling contributes to exhaustion.
One of the ways that PD-1 appears to influence T cell responses is through
alteration of the IS. PD-1 co-localizes with the T cell receptors (TCRs) in the center of
the IS and attenuates signals transmitted from them. This attenuation comes in the form
of dephosphorylation of important TCR-proximal signalling mediaries such as ZAP-70
and PKCθ. Curiously enough, the ultimate effect of this signal-dampening differs
between CD4+ and CD8+ T cells in regards to their synapse formation. In CD4+ T cells,
PD-1 signalling causes disruption of the synapse, whereas in CD8+ T cells, PD-1
signalling actually strengthens the synapse. It is unknown how PD-1 does this.
It is known, however, that there are innate differences in synapse formation
between CD4+ and CD8+ T cells, independent of PD-1. CD8+ T cells are known to form
stronger, more stable synapses than CD4+ T cells, and this is due to the activity of
PKCθ. PKCθ is known to disrupt synapse formation, and CD4+ T cells recruit more
PKCθ to their synapses than do CD8+ T cells. We thus hypothesize that PD-1
differentially influences synapse formation in CD4+ and CD8+ T cells by acting on the
different levels of PKCθ at the synapse in both subsets.
Contributors: Liu, Dongfang
Onur Birol
Program in Developmental Biology
Advisor: Andrew Groves, Ph.D.-Department of Neuroscience
Foxi3 is a Forkhead transcription factor that is expressed early in chick and
mouse in the preplacodal region. This region gives rise to multiple craniofacial placodes,
such as lens, trigeminal or epibranchial in addition to otic placode, the precursor to the
inner ear. Inner ear induction is triggered by FGF signaling, and competence to induce
otic placode markers in response to FGFs correlates with the expression of preplacodal
markers such as Foxi3. Our lab has knocked out Foix3 in mice and Foxi3 mutants
completely lack all ear structures and also have additional craniofacial defects. Ear
induction appears to fail at the very earliest stages, as we see no markers of the otic
placode, such as Pax2, induced in Foxi3 mutants. Foxi3, like other Forkhead
transcription factors, has a DNA binding domain that resembles H1 and H5 linker
histones and it is believed that due to this structural resemblance it may serve as a
pioneer factor that can bind to condensed chromatin and help to make it
transcriptionally accessible. This in turn may induce competence for the cell to respond
to future inducing signals.
At one extreme, Foxi3 might simply confer competence for the expression of
inner ear genes after preplacodal ectoderm receives FGF signals. Foxi3 might also
provide competence to preplacodal ectoderm by regulating genes (such as FGF
receptors) that to respond to FGF signals. At the other extreme, Foxi3 might be required
to induce preplacodal genes in the first place. We are analyzing the mutant mice to
pinpoint, where exactly the Foxi3 plays role in FGF signaling since the mutant mice lack
Pax2 induction, which is one of the first responsive genes in ear. Our results indicate to
an earlier role of Foxi3 because we have observed that some of the preplacodal
markers are not expressed in mutant mice ectoderm. Moreover, the FGF pathway
genes analyzed to date have revealed no difference in expression between mutants and
wild types. This suggests that the preplacodal tissue is receiving the inducing FGF
signaling; however, due to defects on earlier gene expressions, it is unable to interpret it
to turn on the ear development program.
On the other hand, Foxi3 is also expressed early embryonically in some neural
tissue and later in preplacodal region that is very close to the presumptive neural crest
tissues. We are not sure whether these expressions are biologically relevant. In some
Foxi3 mutant mice, the embryos exhibit exencephaly phenotype. Furthermore, the
branchial arches that are normally filled with neural crest cells fail to form. Considering
all these, we hypothesize that Foxi3 is biologically significant for some neural tissue and
neural crest cells. In order to investigate this part, we plan to perform fate mapping
using Foxi3-CreER mouse line and tissue specific knock out studies.
Vitaliy V Bondar
Department of Molecular & Human Genetics
Advisor: Huda Zoghbi, M.D.-Department of Pediatrics
Spinocerebellar Ataxia type 1 (SCA1) is a dominantly inherited fatal
neurodegenerative disorder for which there is no available treatment. It is caused by the
expansion of a trinucleotide repeat that encodes a polyglutamine (polyQ) tract in
ATAXIN-1 (ATXN1), rendering the protein more stable and resulting in toxic
accumulation of mutant ATXN1. Investigation into molecular mechanisms of SCA1
pathogenesis revealed that mutant ATXN1 exerts deleterious effect through its
interacting partners that include Capicua (CIC), a transcriptional repressor that forms
stable complexes with ATXN1. Previously we showed that in presence of expanded
polyQ ATXN1, CIC significantly increases transcriptional repression of its target genes.
Furthermore, haploinsufficiency of Cic rescues neurodegeneration in SCA1 mouse
model through reduction of aberrant repression. Another interacting partner of ATXN1 is
ATAXIN-1-Like (ATXN1L), which is a paralog of ATXN1 with high amino acid sequence
identity. Yeast-two-hybrid screen revealed that ATXN1 and ATXN1L share many
common interactors such as CIC. Although loss of either Atxn1 or Atxn1L in mice does
not cause lethality, double knock out of Atxn1 and Atxn1L results in perinatal lethality
and thus providing further evidence for their shared function. Interestingly, duplication of
Atxn1L in mice rescues SCA1 by out-competing mutant Atxn1 from its interacting
partners. Given importance of ATXN1L interacting partners in understanding SCA1
pathogenesis we aim to identify interacting network for ATXN1L, in vivo. We propose
that identifying endogenous protein interacting partners of Atxn1L may reveal crucial
players in SCA1 pathogenesis. To accomplish this we engineered an Atxn1L knock-in
mouse (Atxn1L-V mice, V for Venus) with C-terminal tags for which good monoclonal
antibodies are available. Further biochemical studies revealed that epitope tag in Atxn1L
does not interfere with its binding ability. Currently we are performing
immunoprecipitation followed by mass spectrometry to identify Atxn1L interacting
partners in different brain regions.
Contributors: Jefar-Nejad, Paymaan; Tan, Qiumin; Zoghbi, Huda
Meghan Breaux
Integrative Program in Molecular and Biomedical Sciences
Advisor: Nikolaj Timchenko, Ph.D.-Department of Pathology & Immunology
Frederick Pereira, Ph.D.-Department of Molecular & Cellular Biology
Epigenetic control is a key process involved in liver regulation. Recent studies
have shown that several age-related liver dysfunctions are associated with the change
of chromatin structure. These dysfunctions include development of hepatic steatosis
(fatty liver), impaired liver proliferation and age-associated liver cancer. Previous
studies in Dr. Timchenko’s lab revealed that two members of C/EBP family, C/EBP
and C/EBP , are altered by age and are involved in alteration of chromatin structure in
the liver. These observations were obtained in several animal models of aging liver
including C/EBP -S193D knockin mice and recently generated transgenic mice
expressing dominant negative p300, dnp300 mice. We have found that histone
acetyltransferase p300 co-operates with C/EBP proteins in the epigenetic control of liver
functions and that age dramatically changes p300-C/EBP pathways in the liver. The
dnp300 mice have altered expression of C/EBP proteins compared to WT and higher
basal proliferation. Upon challenges to the liver using several methods including Carbon
Tetrachloride treatment and Partial Hepatectomy, we observe that dnp300 mice have
higher rates of proliferation and are more resistant to liver injury. Therefore, the main
goal of my project is to determine the role of p300-dependent epigenetic control in the
key functions of the liver.
Contributors: Breaux, Meghan; Jin, Jingling; Iakova, Polina; Jiang, Yanjun
Alex Jawann Brewer III
Department of Pharmacology
Advisor: Richard De La Garza, Ph.D.-Department of Psychiatry & Behavioral Sciences
AIMS: The dopamine transporter (DAT) has been implicated in the subjective
and reinforcing effects produced by cocaine. Modafinil has shown some efficacy in
reducing these effects. The aim of this study was to determine if the ability of modafinil
to reduce the subjective and reinforcing effects produced by cocaine could be enhanced
by combination with another drug, lisdexamfetamine, that is also known to interact with
the DAT.
METHODS: Non-treatment seeking, cocaine-dependent individuals had a history
of cocaine use via the smoked or intravenous route, were 18-55 years old, were not
dependent on other drugs of abuse excluding nicotine, and did not have unstable
medical conditions or psychiatric diagnoses. Eligible individuals were randomized to one
of four treatments: placebo, modafinil (200 mg, p.o.), lisdexamfetamine (30 mg p.o.), or
modafinil (200 mg, p.o.) + lisdexamfetamine (30 mg, p.o.) for four days. On the fourth
day, individuals participated in two double-blind self-administration sessions for the
opportunity to receive cocaine or saline. Upon receiving a contingent dose of cocaine
(20 mg, iv) or saline, individuals had the opportunity to make 5 subsequent choices for
either another infusion or keep $1 USD. The sessions were counterbalanced so that
some individuals received cocaine in the morning and saline in the afternoon session,
and vice versa. Subjective effects and heart rate and blood pressure were recorded
prior to and throughout each session.
RESULTS: Participants (n = 27) were mostly male (74%), mostly African
American (78%), and non-Hispanic (85%). Expectedly, compared to saline, cocaine 20
mg significantly increased heart rate and systolic blood pressure, and positive
subjective effects of cocaine, including “high”, “like drug”, “good effects”, and
“stimulated” (all p-values < 0.001). Compared to the other treatments, modafinil
attenuated subjective ratings of “high”, “desire”, “any drug effects”, “good effects”, and
“stimulated” , but the combination of modafinil and lisdexamfetamine did not produce
significant attenuations of any subjective or physiological responses induced by cocaine
(all p-values > 0.05).
CONCLUSIONS: These data support previous findings that modafinil is effective
for treating cocaine dependence, yet it does not appear to be more effective when
combined with lisdexamfetamine.
Contributors: Brewer III, Alex J.; Mahoney III, James J.; Newton, Thomas F., De La Garza II,
Amy Elizabeth Brinegar
Department of Molecular & Cellular Biology
Advisor: Thomas Cooper, M.D.-Department of Pathology & Immunology
Alternative splicing increases proteome diversity by producing isoforms that help
define tissue specificity. During development, alternative splicing is highly regulated and
the loss of regulation causes mis-splicing, which can lead to pathogenesis of some
diseases. Postnatal heart development has a well-established global switch in
alternative splicing due to changes in the protein levels of splicing regulators. Skeletal
muscle has a similar switch in alternative splicing regulators during development that
also results in a global switch in alternative splicing. The postnatal period,
encompassing the first four weeks after birth, is a very dynamic time in skeletal muscle
development with many changes at the cellular and molecular level. The skeletal
muscle cells transitions from a highly proliferative population of mononucleated cells to
quiescent terminally differentiated myofibers. Also during this time, innervation of
myofibers matures through neuronal pruning and sarcomeres mature to ensure proper
muscle function. Although some muscle-specific splicing switching has been identified
during postnatal development, very few isoforms have a known biological function
related to muscle physiology. To identify novel transcriptome changes, particularly
alternative splicing events, that affect muscle physiology, I have performed RNA-seq
analysis across different time points of postnatal development. To narrow my scope and
prioritize alternative splicing events, I have also performed GO analysis. From here, I
will determine the biological significance of the splicing events by redirecting particular
splicing events in vivo that will be examined by muscle-related assays. With this, we will
better understand the impact that alternative splicing has on normal muscle biology. If
these splicing events are also mis-regulated in disease, these splicing events can be
targeted to potentially correct muscle function.
Contributors: Brinegar, Amy; Xia, Zheng; Manning, Kathleen; Li, Wei; Cooper, Thomas
Rogers Milton Brown, II
Program in Developmental Biology
Advisor: Andrew Groves, Ph.D.-Department of Neuroscience
The inner ear is a complex and specialized vertebrate organ deeply embedded in
the function of the auditory sensory pathway as well as in the detection of linear and
angular acceleration. The ability of the inner ear to do so is dependent on the six
sensory organs contained within the structure. In general, these sensory organs consist
of apically ciliated sensory epithelial cells called hair cells and the supporting cells that
surround them. There is much that is known about the function of these hair cells, as
well as their morphology, but there is less that is known about the developmental
cascade by which hair cells arise and differentiate.
It is thought that Notch signaling plays a key role in inner ear development at
three stages: first during neurogenesis to generate delaminating neuronal precursors,
later to promote the pro-sensory domains that will give rise to the sensory organs of the
inner ear, and finally to establish the pattern of hair cells and supporting cells during hair
cell differentiation through lateral inhibition. Previous studies performed to analyze the
role of Notch in these processes revolved around removing key ligands or receptors in
the Notch signaling pathway like Jag1 and Jag2, Notch1, and Dll1. The phenotypes
observed in these experiments demonstrated a range of results which suggests that
when some of these elements are removed from the pathway, other receptors or
ligands may be able to compensate in certain situations.
Through the use of a dominant-negative Mastermind-like (dnMAML) and a
conditional knock out of RBPJ-K (RBPJ CKO), we hope to observe the effect of the
complete loss of Notch signaling on the development of the pro-sensory domains that
generate the hair cells and supporting cells of the vestibular sensory organs. By
attacking the “bottleneck” of the pathway we hope to successfully abolish all Notch
signaling in the inner ear. Through the use of paint fillings to understand the threedimensional development of these mutants, and both RNA in situ hybridization and
fluorescent immunochemistry for markers of pro-sensory development, we hope to
obtain a clearer picture of the function the Notch pathway plays in pro-sensory
Contributors: Brown, Rogers II; Basch, Martin; Groves, Andrew
Jason Christopher Burton
Integrative Program in Molecular and Biomedical Sciences
Advisor: Irina Larina, Ph.D.-Department of Molecular Physiology & Biophysics
Investigation of pre-implantation events in the murine oviduct is crucial in
understanding normal reproductive functions; however, detailed analysis of the ciliated
layer of the oviduct has been restricted, due to the complexity of the transport system
and the lack of proper imaging tools. We introduce an innovative approach for threedimensional imaging and tracking of oocytes through the oviduct with optical coherence
tomography (OCT), as well as a novel Functional-OCT imaging modality to analyze the
dynamics of motile cilia in the oviduct. Imaging is performed with a spectral-domain
OCT system operating at 70 kHz A-scan rate with a measured resolution near 5
microns. Oocytes were tracked in vivo from ovulation as they transit through the oviduct
towards the uterus. The movement of the oocyte can be clearly visualized and the
velocity vectors can be quantified based on direct mapping of the trajectories.
Dynamics of cilia beat frequency and location of ciliated cells were extracted from twodimensional OCT images using a Fourier Transform. Extracted frequency information
was mapped back onto OCT images to highlight ciliated epithelial layers. Motile cilia
are responsible for transport of the pre-implantation embryos and a quantitative analysis
of their activity during different stages of pregnancy increase our knowledge of dynamic
events during early pregnancy. To the best of our knowledge, this is the first application
of OCT to the study of dynamic events in the murine oviduct and the first attempt to
develop Functional-OCT techniques that allow for analysis of motile cilia. OCT is
capable of producing detailed structural information of in vivo processes that allows for
comprehensive studies of dynamic pre-implantation events. This study will increase our
understanding of pregnancy events and may lead to further investigations of tubal and
cilia defects and their impact on fertility.
Contributors: Burton, Jason C.; Wang, Shang ; Larina, Irina V.
Tiara T Byrd
Program in Translational Biology & Molecular Medicine
Advisor: Nabil Ahmed, M.D.-Department of Pediatrics
Robert Grossman, M.D.-Department of Neurosurgery
Background: Tumor endothelium marker 8 (TEM8) was discovered by St Croix et
al as one of nine gene products preferentially upregulated in the tumor-associated vs.
normal endothelium. Interestingly, TEM8 has also been identified as a tumor restricted
antigen in triple negative breast cancer (TNBC), a clinical entity associated with a
particularly poor prognosis. Being null for HER2, estrogen and progesterone receptors,
targeted therapies for TNBC are quite limited.
Purpose: To use T cells expressing TEM8-specific chimeric antigen receptors
(CAR) as a novel approach to target both TNBC cells and their tumor-associated
Methods/Results: We used in silico design to construct a novel TEM8-specific
CAR molecule. The antigen recognition exodomain consisted of a single chain variable
fragment based on the TEM8-specific monoclonal antibody, L2. The signaling
endodomain consisted of the costimulatory molecule CD28 and CD3-zeta chain. The
encoding DNA was codon optimized, synthesized and then sequence verified. We used
a retroviral transduction system to express the TEM8 CAR transgene on HEK 293T,
then on primary T cells. Approximately 90% of primary human T cells expressed the
TEM8 CAR, as detected by flow cytometry. The expression of TEM8 was characterized
using flow cytometry and western blot on a battery of TNBC lines, TEM8 transduced
(modest and high expressers) cell lines as well as TEM8 negative cell lines. TEM8 CAR
T cells recognized and killed TEM8 positive target cells in an antigen-dependent fashion
in 51Cr release assays and secreted immunostimulatory cytokines upon encounter of
TEM8 positive cells. There was no reactivity against TEM8 negative cell lines. No
cytotoxicity or cytokine release was exhibited by T cells expressing an irrelevant CAR or
non-transduced T cells from the same blood donor. We are currently testing this
strategy in a vascularized orthotopic breast cancer murine model.
Conclusion: TEM8 specific CAR T cells could serve as a tumor and vascular
targeted immunotherapeutic modality for triple-negative breast cancer.
Contributors: Byrd, Tiara; Fousek, Kristen; Pignata, Antonella; Wakefield, Amanda; St Croix,
Bradley; Fletcher, Bradley; Hegde, Meenakshi and Ahmed, Nabil
Cathryn Rene Cadwell
Department of Neuroscience/M.D.-Ph.D. Program
Advisor: Andreas Tolias, Ph.D.-Department of Neuroscience
The mammalian neocortex gives rise to complex cognitive processes such as
perception and decision-making. Despite tremendous progress in understanding the
physiology and cell biology of individual neurons in the cortex, the process by which
networks of neurons become wired together during development and organize into
functional circuits remains poorly understood. Recent studies suggest that cell lineage
influences the connectivity and functional properties of excitatory pyramidal neurons in
the neocortex, but it remains unclear to what extent cell lineage influences circuit
assembly. We have developed a tamoxifen-inducible Cre/lox system for lineage tracing
in the neocortex that allows us to sparsely label neural progenitors and trace their
daughter cells. When progenitors are labeled at the onset of neurogenesis, they give
rise to columns of clonally related pyramidal neurons spanning all six cortical layers.
Interestingly, we find that sister cells in ontogenetic columns show enhanced
connectivity across cortical layers but not within the same cortical layer. By utilizing 3D
random-access multiphoton (3D-RAMP) imaging, we are also able to study the
functional organization of ontogenetic columns in the visual cortex. These experiments
will shed light on how functional networks are established during development and could
provide a circuit level foundation to study neurodevelopmental disorders.
Contributors: Cadwell, Cathryn; Jiang, Xiaolong; Froudarakis, Emmanouil; Yatsenko, Dimitri;
Tolias, Andreas
Mingbo Cai
Department of Neuroscience
Advisor: David Eagleman, Ph.D.-Department of Neuroscience
A repeated stimulus is perceived as briefer than a novel one. It has been
suggested that this duration illusion is an example of a more general phenomenon,
namely that a more expected stimulus is perceived as briefer than a less expected one.
To test this hypothesis, we manipulated high-level expectation through the probability of
a stimulus sequence, through the regularity of the preceding stimuli in a sequence, or by
violating an overlearned sequence. We found that perceived duration is not distorted by
these manipulations. Repetition of stimuli, on the other hand, consistently reduces
perceived duration across our experiments. Our findings suggest that estimates of subsecond duration are largely the result of low-level processing and independent of highlevel expectation.
Contributors: Cai, Mingbo; Eagleman, David M.; Ma, Wei Ji
Ian Morgan Campbell
Department of Molecular & Human Genetics/M.D.-Ph.D. Program
Advisor: James Lupski, M.D./Ph.D.-Department of Molecular & Human Genetics
Pawel Stankiewicz, M.D./Ph.D.-Department of Molecular & Human Genetics
New human mutations are thought to originate in germ cells, thus making a
recurrence of the same mutation in a sibling exceedingly rare. However, increasing
sensitivity of genomic technologies has anecdotally revealed mosaicism for mutations in
somatic tissues of apparently healthy parents. Such somatically mosaic parents might
also have germline mosaicism that can potentially cause unexpected intergenerational
recurrences. Here, we show that somatic mosaicism for transmitted mutations among
parents of children with simplex genetic disease is more common than currently
appreciated. Using the sensitivity of individual-specific breakpoint PCR, we
prospectively screened 100 families with children affected by genomic disorders due to
rare deletion copy-number variants (CNVs) determined to be de novo by clinical
analysis of parental DNA. Surprisingly, we identified four cases of low-level somatic
mosaicism for the transmitted CNV in DNA isolated from parental blood. Integrated
probabilistic modeling of gametogenesis developed in response to our observations
predicts that mutations in parental blood increase recurrence risk substantially more
than parental mutations confined to the germline. Moreover, despite the fact that
maternally transmitted mutations are the minority of alleles, our model suggests that
sexual dimorphisms in gametogenesis result in a greater proportion of somatically
mosaic transmitting mothers who are thus at increased risk of recurrence. Therefore,
somatic mosaicism together with sexual differences in gametogenesis might explain a
considerable fraction of unexpected recurrences of X-linked recessive disease. Overall,
our results underscore an important role for somatic mosaicism and mitotic replicative
mutational mechanisms in transmission genetics.
Contributors: Campbell Ian; Yuan Bo; Robberecht Caroline; Pfundt Rolph; Szafranski
Przemyslaw; McEntagart Meriel; Nagamani Sandesh; Erez Ayelet; Bartnik Magdalena;
Wisniowiecka-Kowalnik Barbara; Plunkett Katie; Pursley Amber; Kang Sung-Hae; Bi Weimin;
Lalani Seema; Bacino Carlos; Vast Mala; Marks Karen; Patton Michael; Olofsson Peter; Patel
Ankita; Veltman Joris; Cheung Sau Wai; Shaw Chad; Vissers Lisenka; Vermeesch Joris; Lupski
James; Stankiewicz Pawel
James Christopher Campbell
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Choel Kim, Ph.D.-Department of Pharmacology
Membrane bound type II cGMP dependent protein kinase (PKG II) is a central
mediator of the cGMP signaling cascade, which regulates circadian rhythmicity,
intestinal water secretion, bone growth and renal function. PKG II contains a N-terminal
regulatory (R)-domain, and a C-terminal catalytic (C)-domain. The R-domain contains
tandem cyclic nucleotide binding domains (CNB-A and B) each with different affinities
for cGMP, the second messenger that regulates kinase activity of PKG II. Once PKG II
is active by cGMP it phosphorylates pharmaceutically relevant protein target, but due in
part to a lack of structural information drug discovery efforts on have been slow. While
it is known that PKG II needs to be highly selective for cGMP over cAMP to prevent
cross-talk little is known about its cyclic nucleotide selectivity mechanism and the
allosteric activation of PKG II. We seek to understand the cyclic nucleotide selectivity of
PKG II’s CNB domains and their role in the activation of PKG II. In this pursuit, we
characterized the CNB domain’s cyclic nucleotide selectivity. Our findings are that CNBB imparts an almost 500 fold preference for cGMP, while CNB-A only offers a 10 fold
preference. We then solved atomic resolution crystal structures of the two CNB domains
of PKG II, to gain insight into the molecular basis of the selectivity seen. We have
discovered a unique ligand recognition mechanism in CNB-B and tested its role in the
activation of PKG II.
Contributors: Campbell, James; Li, Kevin; Huang, Gilbert; Reger, Albert; Sankaran, Banumathi;
Link, Todd; Ladbury, John; Kim, Choel
Zhijuan Cao
Program in Cardiovascular Sciences
Advisor: Sean Marrelli, Ph.D.-Department of Anesthesiology
Background: Therapeutic hypothermia is a promising strategy for neuroprotection
following stroke. However, current widely available methods are not compatible with
conscious patients and produce unwanted physiological stress. We have recently
demonstrated that pharmacological hypothermia (PH) through transient receptor
potential vanilloid 1 (TRPV1) channel agonism is effective and safe in the conscious
mouse and provides significant neuroprotection following ischemic stroke at 24 hours
reperfusion. We now evaluate whether this method of PH provides sustained
neuroprotection through one month reperfusion.
Methods: Three experimental groups were evaluated: sham operated (Sham),
stroke with normothermia (Stroke/NT), and stroke with PH (Stroke/PH) (n=8, 10, 7).
Stroke was induced by transient occlusion of left distal middle cerebral artery (MCA)
plus left common carotid artery for two hours. For the Stroke/PH group, hypothermia
(32-34ºC) was induced by infusion of TRPV1 agonist (dihydrocapsaicin) for 8 hours;
beginning 90 minutes after reperfusion. We evaluated several behavioral tests from prestroke up to 28 days reperfusion. Total cerebral infarct was then evaluated in Nisslstained brain sections at 30 days reperfusion.
Results: The foot fault test was the most sensitive in demonstrating extended
functional deficit after stroke. With this test, Stroke/NT demonstrated prolonged
deficiency (through 21 days reperfusion) compared to Sham. PH treatment (Stroke/PH)
demonstrated significant improvement in function compared to Stroke/NT. Other tests
demonstrated non-statistically significant performance reduction of Stroke/NT in
swimming time, grip side preference, turning and climbing time. Histological analysis
demonstrated more severe ipsilateral necrosis and hemisphere shrinkage in Stroke/NT
brains compared to Stroke/PH brains.
Conclusions: In summary, we demonstrate long-term neuroprotection with
TRPV1-mediated PH following stroke. These studies support the therapeutic potential of
TRPV1 agonism for promoting hypothermia in the conscious stroke subject.
Supported by NIH R21NS077413 & AHA PRE16900006.
Contributors: Balasubramanian, Adithya; Marrelli, Sean P
Angela N Carter
Department of Neuroscience
Advisor: Anne Anderson, M.D.-Department of Pediatrics
RATIONALE: Research using animal models of epilepsy has implicated
hyperactive signaling of the mammalian target of rapamycin (mTOR) pathway as a
candidate mechanism underlying the pathophysiology of epilepsy and the associated
co-morbidities. In physiological conditions, mTOR signaling regulates protein synthesis
and is necessary for learning and memory. However, how hyperactive mTOR signaling
in epilepsy results in learning and memory deficits is still unclear. In addition, how
seizures affect learning and memory has yet to be fully elucidated.
METHODS: To evaluate the effects of a single seizure on learning and memory
as well as mTOR signaling, we administered either saline (Sham) or the convulsant
pentylenetetrazole (PTZ; 75mg/kg, i.p.) in rats. Our pilot studies show that this dose was
sufficient to induce a generalized seizure. To determine the effects of a single seizure
on learning and memory, animals were subjected to the Fear Conditioning (FC) task. To
evaluate how a single seizure affects mTOR signaling, PTZ was administered and
tissue collected at 3 different time points and homogenized for western blotting. The
time points were: 1-, 3-, and 24- hours post induction. Antibodies against Phospho (P)S6 and P-eEF2 were used as downstream markers of mTOR activation. To determine
the effect of mTOR inhibition on FC as well as P-S6 and P-eEF2, the mTOR inhibitor
rapamycin (Rap) or vehicle was given immediately post seizure. To verify inhibition of
mTOR signaling, tissue was collected for western blotting.
RESULTS: Following a PTZ-induced seizure, neither the Sham nor the PTZ
animals displayed short-term memory deficits in the FC task. When tested for long-term
memory however, the PTZ animals exhibited deficits in contextual and associative
memory (n=7-11, p<0.01) compared to Sham rats. Biochemical analyses reveal that at
1 hour following a PTZ-induced seizure, the levels of P-S6 and P-eEF2 were
significantly increased (n=4-6, p<0.001). At 3-hours post seizure, P-eEF2 remained
elevated while P-S6 levels returned to baseline (n=4-6, p<0.01). Finally, both P-S6 and
P-eEF2 returned to baseline levels 24-hours post seizure. The increased P-S6 levels
(n=3-5, p<0.05) were blocked with Rap treatment however the P-eEF2 levels remained
elevated (n=3-5) and Rap did not rescue the deficits in FC (n=6-11).
CONCLUSIONS: These data confirm that a single PTZ-induced seizure leads to
long-term memory deficits. Additionally, our data show that the deficits observed in FC
are not rescued by inhibition of mTOR signaling. Studies are underway to further
characterize PTZ-induced seizures and to determine other signaling pathways that may
underlie the seizure related memory deficits.
Contributors: Dao, A.T, Muyco, M. A., Lee, W.L., Anderson, A.E.
Lesley S.M. Chaboub
Program in Developmental Biology
Advisor: Benjamin Deneen, Ph.D.-Department of Neuroscience
While very diverse in their origins and presentations, demyelination disorders of
the central nervous system (CNS) share common cellular events. Recruitment of
oligodendrocyte precursor cells (OPCs) at the lesions site, which normally function to
remyelinate axons, is an especially important step in proper remyelination, and has
been shown to be defective in at least two disorders; multiple sclerosis and hypoxic
ischemic encephalopathy (HIE). In order to understand how these cellular events occur
during the disease state, it is imperative to understand how these same processes are
regulated during normal development. Therefore, identification of novel genes involved
in glial cell development might shed light into demyelination disorders, especially white
matter injury (WMI).
In a developmental screen performed in the spinal cord, we recently identified
Arhgef4 as a novel gene expressed in both glial precursor cells and mature astrocytes.
Arhgef4 is a guanine exchange factor for the Rho GTPase family of protein and has
been shown to regulate migration in other systems. During spinal cord development, we
showed that Arhgef4 influences glial precursor cells migration. We hypothesize that
Arhgef4 would also influence migration of precursor cells in disease state. Lysolecithin
injection into the ventral white matter region of the spinal cord is a well-recognized
model of WMI where the kinetics and cellular events of remyelination are well
established. Using this model, we showed that Arhgef4 is expressed in the lesion area,
validating the use of this technique to further our understanding the role of Arhgef4 in
WMI. Lesions performed on Arhgef4 germline knock-out (KO) animals appear to have
delayed remyelination kinetics when compared to heterozygous littermate animals.
Furthermore, Arhgef4 KO lesions present with a significant decrease in the number of
OPCs present inside the lesion area, suggesting that the recruitment of OPCs at lesion
sites is inhibited in the absence of Arhgef4.
We will utilize in vitro OPC cultures to differentiate whether Arhgef4 influences
the migration of OPCs into the lesion site or their proliferation. Ongoing studies will
focus on further characterizing Arhgef4 KO lesions as well as performing misexpression
of Arhgef4 into the lesion to assess whether it can accelerate remyelination. In addition
we will assess the effect of Arhgef4 expression in a mouse WMI hypoxic model that
closely mimics HIE.
This study identified a novel player in OPCs recruitment at the lesion site during
remyelination events. Guanine exchange factors, such as Arhgef4, are potentially good
target for therapies since they affect critical cellular pathways while being tissue
Contributors: Chaboub, Lesley; Lee, Hyun-Kyoung and Deneen, Benjamin.
Chi-Hsuan Chang
Integrative Program in Molecular and Biomedical Sciences
Advisor: Jeffrey Rosen, Ph.D.-Department of Molecular & Cellular Biology
The DNA damage response (DDR) includes activation of cell cycle checkpoints,
apoptosis, and DNA repair. To repair DNA double strand breaks, cells may undergo
homologous recombination (HR) or non-homologous end joining (NHEJ). Adult stem
cells tend to have different DDR mechanisms compared to differentiated cells since they
reside in the tissues for extended periods and continuously ensure tissue homeostasis.
Studies have shown that mouse hair follicle bulge stem cells and hematopoietic stem
cells have higher NHEJ activity and lower apoptosis, resulting in their resistance to
radiation. However, little is known about the DDR in mammary stem cells (MaSCs). In
addition, previous studies from the clinic and mouse models have demonstrated that
tumor initiating cells (TICs) from mammary tumors have an increased resistance to
conventional therapies. However, the mechanisms underlying the DDR to this
therapeutic resistance are still mainly undiscovered. Using a syngeneic p53-null tumor
model, our lab has identified a Lin-CD29HCD24H subpopulation in these tumors with
functional TICs properties, and these TICs are more resistant to radiation. To study the
specific mechanisms that contribute to this resistance, we hypothesized that TICs from
p53-null tumors exhibit increased DDR and elevated NHEJ activity to repair DNA
damage, which might be the similar DDR mechanisms used in MaSCs.
To date, we have shown that MaSCs and basal cells have lower apoptosis as
compared to luminal cells in mammary glands. In cell cycle analysis, a significant
increase of G2/M phase was observed in MaSCs after irradiation, indicating stem cells
have increased G2 checkpoint activation as compared to basal and luminal cells. In
addition, using a high-throughput NHEJ assay, we showed that basal cells, which
contain MaSCs, exhibit higher NHEJ activity as compared to luminal cells. Furthermore,
using Reverse Phase Protein Array (RPPA) analysis, we were able to show that in stem
cells, some stress-responsive proteins were up-regulated, such as p-p38, p-p44, and
conversely that most pro-apoptotic genes were down-regulated, indicating that MaSCs
are intrinsically more resistant to stress and apoptosis. In tumor studies, TICs have
more efficient DNA repair according to γH2AX foci staining and neutral Comet assay.
We also showed that TICs exhibit decreased apoptosis and higher NHEJ activity.
Ongoing studies are designed to identify the “Achilles heel” of these DDR pathways in
TICs, which may act as therapeutic targets.
Contributors: Chang, Chi-Hsuan; Zhang, Mei; Lu, Tao; Coarfa, Cristian; Rosen, Jeffrey
Rich Harrison Chapple
Department of Molecular & Human Genetics
Advisor: Daisuke Nakada, B.A.Sc.-Department of Molecular & Human Genetics
The hematopoietic system is a highly dynamic tissue that exhibits remarkable
plasticity and is characterized by high cell turnover. To circumvent the constant
depletion of these constituents, multipotent hematopoietic stem cells (HSCs) are
infrequently recruited out of quiescence wherein they become proliferative cells. In this
capacity, HSCs are capable of generating new blood cells to promote tissue
homeostasis. However, entry into the cell cycle must be tightly regulated in order to
maintain stem cell integrity and preserve HSC function. One such gene involved in the
regulation of HSC division is B cell-specific Moloney murine leukemia virus integration
site 1 (Bmi1). Bmi1 is a member of the Polycomb repressive complex 1 (PRC1), and is
involved in epigenetic silencing of the Cdkn2a tumor suppressor locus1. Disrupting the
expression of Bmi1 has deleterious consequences on HSC function – overexpression of
Bmi1 is associated with poor prognosis in AML2, and deletion of Bmi1 impairs HSC selfrenewal and long-term reconstitution potential3. Interestingly, we have recently
observed sex differences in the HSC response after Bmi1 deletion. More specifically,
HSC frequency in Bmi1-KO females become progressively depleted compared to that of
male counterparts. We surmised that the female sex hormone estrogen (E2) was
responsible for the observed differences, and subjected male Bmi1-KO animals to an
estrogen regimen. After E2 treatment, a significant reduction in HSC frequency was
observed in Bmi1 deficient animals. The results of these experiments raise the
possibility that Bmi1 deficiency may sensitize HSCs to division-promoting stimuli. We
are now focusing our efforts to determine the molecular underpinnings of E2-mediated
HSC division through NGS applications. The results of these data will not only be used
for further analysis in our Bmi1-KO model, but also will be used for the discovery of
mechanisms that induce HSC division.
1. Sparmann, A. & Van Lohuizen, M. Polycomb silencers control cell fate,
development and cancer. Nat Rev Cancer 6, 846–856 (2006).
2. Chowdhury, M. et al. Expression of Polycomb-group (PcG) protein BMI-1
predicts prognosis in patients with acute myeloid leukemia. Leukemia 21, 1116-1122
3. Park, I. et al. Bmi-1 is required for maintenance of adult self-renewing
haematopoietic stem cells. Nature 423, 302-305 (2003).
Contributors: Chapple, Richard; Takeichi, Makiko; Kitano, Ayumi; Saitoh, Yusuke; Lin,
Angelique; Nakada, Daisuke
Bo Chen
Department of Biochemistry & Molecular Biology
Advisor: Wah Chiu, Ph.D.-Department of Biochemistry & Molecular Biology
Chaperonin is a class of protein that plays an essential role in protein folding for
all cells from bacteria, archaea and eukaryotic cells. Recently it is reported that a type II
chaperonin from archaea Methanococcus Marapaludis (Mm-Cpn) could assist refolding
of human γD-crystallin to its native conformation. Human γD-crystallin is a lens protein
which is associated with the onset of cataract when it partially unfolds. We are
interested in understanding the structural mechanism of how type II chaperonin
recognizes the γD-crystallin.
Here, cryo-electron microscopy (cryo-EM) single particle analysis method was
applied to resolve the structure of Mm-Cpn and human γD-crystallin during their initial
recognition step. A challenge in studying the chaperonin-substrate structure is the
compositional and conformational heterogeneity in the reaction product. We used a
multi-model refinement protocol to sort out particle images according to their structural
uniformity. Our analysis showed three conformations: 33% of the particles (Subset II)
resembled the apo state Mm-Cpn conformation, while 39% of particles (Subset I) had
one-ring less open, one-ring open conformation and 28% of the rest particles (subset III)
did not yield a reliable map. The control cryo-EM map from the Mn-Cpn showed both
ring open which was similar to the map from the subset II of the Mm-Cpn and human
γD-crystallin complex.
Based on the Statistical variance analysis, we conclude that the subset I
corresponds to the Mm-Cpn population with substrate binding. High levels of variances
are observed inside the cis-rings. Subsequent symmetry-free reconstructions of subset I
particle images converged to one-ring less open and one-ring open conformation.
Furthermore, differential conformations of each of the 8 subunits in the less open cisrings were observed to form a tetramer of dimers while both rings in subset II appeared
to have a good 8-fold symmetry. Interestingly the 8-fold symmetries on apical domain
and equatorial domain are largely broken, while intermediate domain still maintain good
8-fold symmetry. It seems that apical domain and equatorial domain work cooperatively
to recognize the unfolded substrate.
In conclusion, our results demonstrate the conformational changes and
symmetry broken features of type II chaperonin upon binding to γD-crystallin.
Contributors: Chen, Bo1; Sergeeva, Oksana2; Goulet, Daniel2; Knee, Kelly2; Joanita, Jakana3; King, Johnathan2;
Chiu, Wah1,3*
1: The Verna and Marrs McLean Department of Biochemisty and Molecular Biology, Baylor College of Medicine,
Houston, Texas, USA 77030
2: Biology Department, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge,
Massachusetts, USA 02139
3: National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular
Biology, Baylor College of Medicine, Houston, Texas 77030
Chun-An Chen
Department of Molecular & Human Genetics
Advisor: Huda Zoghbi, M.D.-Department of Pediatrics
Using whole genome sequencing, our lab identified compound heterozygous
nonsense mutations in a novel gene, TMPRSS9, in a patient with developmental
regression at 2.5 years of age, leading to severe intellectual disability and autism
spectrum disorder. TMPRSS9 encodes for polyserase-1, a transmembrane serine
protease that is poorly studied. To date, the physiological role of TMPRSS9 is unknown
and its endogenous substrates or protein interaction partners have not been identified.
Using qPCR analysis, we showed that TMPRSS9 is expressed in various brain regions,
including cortex, cerebellum, hippocampus, amygdala, brainstem, and hypothalamus.
To test whether loss of function mutations in TMPRSS9 cause developmental
regression leading to intellectual disability and autism spectrum disorder, we generated
a TMPRSS9 knockout mouse model by removing exon 2 using the Cre/loxP
recombination system. Deletion of exon 2 generates a frameshift from exon1 to exon 3,
with a premature stop codon in exon 3. A battery of behavioral tests was used to
evaluate social approach activity, learning and memory, anxiety-like responses, and
motor activity of homozygous knockout mice. The results indicated that knockout mice
display increased body weight, impaired social interaction (three-chamber test), reduced
motor coordination (rotarod), and abnormal sensorimotor gating (prepulse inhibition). To
dissect the physiological function of polyserase-1, we performed immunoprecipitaion
experiments to identify protein-protein interaction partners. The serase-1 domain of
polyserase-1, tagged with HA and FLAG, was expressed in HEK cells. Subsequently,
the purified protein was incubated with mouse brain lysate and subsequently subjected
to tandem affinity purification followed by mass spectrometry. Three putative proteinprotein interactors, ATP1A1, SCAMP3, and NCLN, were furthered validated by coimmunoprecipitation studies and colocalization studies of overexpressed proteins in
HEK cells. Future work will focus on the identification of the endogenous substrates of
Elaine Chen
Department of Molecular & Cellular Biology
Advisor: Lawrence Chan, D.Sc.-Department of Medicine
The endoplasmic reticulum (ER) is an important organelle that performs several
critical functions, including the posttranslational modification, folding, and assembly of
the newly synthesized proteins. Disruption of these physiological functions leads to
accumulation of improperly folded proteins in the ER and the activation of unfolded
protein response (UPR).
Perilipin 2 (Plin2) is a protein found tightly associated to lipid droplets (LD) and
plays an important role in intracellular lipid metabolism. Plin2 is present in multiple
tissues, including the liver and the pancreas. We previously found that when we injected
tunicamycin (TM), an ER stress inducer, into WT and Plin2-/- mice, the liver of Plin2-/mice recovered faster from TM-induced hepatosteatosis compared with the liver of WT
mice. The mechanism whereby Plin2 alleviates the ER stress is not completely
In this study, we first investigated whether the deletion of Plin2 has similar
protective effect in tissues other than livers. An ER stress-induced diabetic mouse
model, Akita mice, was used. Akita mice are heterozygous for a mutation in Ins2 that
results in substitution of tyrosine for cysteine, which prevents normal folding and
secretion of insulin. The Akita mice exhibit markedly elevated glucose levels and a
decline in plasma insulin concentration. Genetic ablation of Plin2 in Akita mice resulted
in ameliorated hyperglycemia and increased β cell mass. Mouse islets with Plin2
ablation showed less UPR activation, indicating that deletion of Plin2 also protects
pancreatic β cell from ER stress. It is believed that autophagy is activated for cell
survival under ER stress. Our data suggest that Plin2 regulates ER stress at least partly
by affecting autophagy activity.
Contributors: Chen, Elaine; Tsai, Luke; Chang, Benny; Chan, Lawrence
Kuang-Yui Michael Chen
Department of Biochemistry & Molecular Biology
Advisor: Patrick Barth, Ph.D.-Department of Pharmacology
G protein-coupled receptors (GPCRs) are one of the largest families of
membrane-embedded receptors that transduce extracellular stimuli into cytoplasmic
responses. Increasing evidence from structural, mutational and spectroscopic studies
on model GPCRs (e.g. rhodopsin, beta-2-adrenergic receptor) indicate that these
receptors signal through long-range conformational changes. However, the atomic-level
sequence/structure/energetic relationships governing such allosteric transitions in this
large receptor family remain poorly understood, thereby hindering the development of
more effective therapeutics (i.e. allosteric regulators) targeting these receptors. We
have developed an integrated homology modeling/multistate design/experimental
approach to reprogram the signaling properties of structurally uncharacterized GPCRs
by redesigning conformational switches in transmembrane (TM) and loop regions. The
method was applied to switch the function of the structurally uncharacterized dopamine
D2 receptor (DRD2) towards the active state. Inactive and active states of DRD2 were
modeled by conformational ensembles using the homology modeling mode of
RosettaMembrane. By manipulating the receptor conformational energy landscape and
rewiring the networks of energetically coupled residues, multistate design of the DRD2
TM region resulted in receptor variants exhibiting up to an eight-fold increase in basal
activity compared to wild type receptor. In agreement with the predictions, biophysical
and pharmacological characterizations indicate that the designed receptor variants
adopt different local conformations exhibiting various levels of basal and ligand-induced
activities. To our knowledge, this is the first computational modeling and design
approach that can reprogram the signaling properties of structurally uncharacterized
GPCRs by rationally designing novel residue conformational switches and energetic
couplings between intra-receptor allosteric regions.
Contributors: Chen, Kuang-Yui Michael; Barth, Patrick
Kuchuan Chen
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M.-Department of Molecular & Human Genetics
Friedreich’s ataxia (FRDA), the most prevalent form of recessive cerebellar
ataxia, is characterized by neurodegeneration and cardiomyopathy. FRDA is caused by
mutations in Frataxin (FXN), which is highly conserved across evolution. It has been
shown that FXN is localized to the mitochondria, and its deficiency leads to impaired
iron-sulfur (Fe-S) cluster biosynthesis and iron accumulation in the mitochondria.
However, evidence to support that either or both defects contribute to the pathogenesis
of FRDA is still a matter of debate. It is generally believed that increased reactive
oxygen species (ROS), which are generated by impaired mitochondria, causes toxicity
and lead to degeneration, However, antioxidant therapy only show limited benefits in
patients. In a forward genetic screen designed to identify mutations in genes that cause
neurodegeneration in Drosophila, we identified the first mutation in frataxin homolog
(fh), a Drosophila homolog of mammalian FXN. fh mutants exhibit an age dependent
neurodegeneration, and the phenotype can be rescued by human FXN, suggesting
human and fly FXN play a conserved role in maintaining neuronal function. Consistent
with previous studies, mitochondrial function is impaired in fh mutants, but ROS levels
are normal compare to control, and overexpression of ROS scavengers cannot rescue
degeneration. Interestingly, both ferrous and ferric irons accumulate in the nervous
system and other tissues in these mutants, and reducing iron levels in the food or
genetically chelating free irons by overexpressing ferritin can suppress and delay
degeneration in fh mutants, suggesting that iron toxicity contributes to
neurodegeneration. Furthermore, we observed an accumulation of lipid droplets in the
glia cells, and feeding myriocin, a drug that inhibits sphingolipid synthesis, delays the
demise of neurons in fh mutants. We argue that accumulated iron triggers impaired
sphingolipid homeostasis and leads to neurodegeneration. We are currently
investigating this pathway in fh mutants.
Contributors: Chen KC; Lin G; Haelterman N; Li Z; Sandoval H; Jaiswal M; Bayat V; Xiong B;
Zhang K; David G; Duraine L; Yamamoto S; Charng WL; Graham B; Bellen HJ
Jinxuan Cheng
Department of Biochemistry & Molecular Biology
Advisor: Kimberley Tolias, Ph.D.-Department of Neuroscience
Neurons undergo a number of highly regulated processes during development
that are required to form a functional nervous system, including migration, axonal and
dendritic outgrowth, and synapse formation and remodeling. The small Rho-family
GTPases Rac1, RhoA and Cdc42 are vital regulators of nervous system development
and perturbations in Rho GTPase signaling are associated with neurological diseases,
such as Intellectual Disabilities, Amyotrophic Lateral Sclerosis and Autism. Like all
GTPases, Rac1 cycles between a GTP-bound activate state and a GDP-bound inactive
state. Active Rac1 binds to effector proteins and stimulates signaling pathways that
induce actin and microtubule remodeling, which promotes directed cell migration, the
growth and branching of dendrites, and the formation and maturation of spines. To
function properly, Rac1 activity needs to be tightly regulated in space and time. Rac1 is
activated by the guanine nucleotide exchanger factor (GEF) Tiam1 and its closely
related homolog Tiam2.
Tiam1 (T-lymphoma invasion and metastasis 1) is a large multi-domain protein,
which has been shown in vitro to regulate different aspects of neuron development.
Tiam2 (STEF, SIF and Tiam1-like exchange factor) is a closely related homolog of
Tiam1. Tiam2 is required for neurite outgrowth and growth cone morphology in cultured
hippocampal neurons. Furthermore, Tiam1 and Tiam2 are both components of the
evolutionary conserved PAR polarity complex (Par3, Par6, aPKC), which is essential for
establishing and maintaining cell polarity during processes such as directed cell
migration, axon initiation, and synapses formation.
The expression levels of Tiam1 and Tiam2 are high in the brain during
development, and remain high in some regions that undergo plasticity. Tiam2, which is
known to regulate cell migration in cell culture, is highly expressed in the cortex during
embryogenesis when neuronal migration happens. This evidence suggests that Tiam1
and Tiam2 may play important roles in nervous system development. However, most of
the studies investigating Tiam1 and Tiam2 have been done in vitro, and little is known
about their roles in vivo.
Here we describe the generation of Tiam1 and Tiam2 conditional knockout
mice and our attempts to use these mice to determine the roles of Tiam1 and Tiam2 in
neuronal migration and synapse development in vivo. Our preliminary data indicate that
Tiam1 and Tiam2 knockout mice show decreased spine density, defects in astrocytes
and interneuron migration. My future work will be using in utero electroporation to study
neuron migration in the Tiam1 and Tiam2 knockout mice, and characterize the role of
Tiam1 and Tiam2 in regulating these processes.
Contributors: Cheng, Jinxuan; Niu, Sanyong; Firozi, Karen; Tolias, Kimberley
Angie Chi An Chiang
Department of Neuroscience
Advisor: Joanna Jankowsky, Ph.D.-Department of Neuroscience
New therapies for Alzheimer’s disease directly target amyloid-β (Aβ), the main
component of pathological plaques. Immunization with an anti-Aβ antibody (clone Ab9)
combined with Aβ suppression significantly reduces amyloid load. However, the
mechanism by which combination therapy works remains unclear. Optimizing the
outcome of immunotherapy requires understanding how antibody treatment lowers
amyloid burden. To address this, we are testing whether microglia, the resident
phagocytes of the brain, are necessary for antibody-mediated plaque clearance. We are
crossing CD11b-HSVTK mice, a mouse model in which microglia can be selectively
ablated from the brain with the administration of ganciclovir, to tet-off APP double
transgenic mice, a mouse model for Alzheimer’s amyloidosis that allows for temporal
control of the transgenic expression of APP. By using the tet-off system to suppress
transgenic APP and Aβ synthesis during administration of an anti-Aβ antibody, microglia
are allowed to work from a static amyloid plaque load. Following treatment with
ganciclovir, microglia cells will induce cell death. Histological assessment of amyloid
burden and microglial ablation will be used for quantification. By understanding the role
of microglia in amyloid clearance, the results of this study may be useful for the
development of better therapies and the avoidance of harmful consequences.
Contributors: Chiang, Angie CA ; Jankowsky, Joanna L.
Sungwoo Choi
Program in Developmental Biology
Advisor: David Moore, Ph.D.-Department of Molecular & Cellular Biology
Type 2 diabetes is a metabolic disease characterized by a high level of blood
glucose due to insulin insensitivity. This disorder is tightly correlated with increased fat
accumulation in the liver. It has been proposed that a vicious cycle of increased fat
accumulation and insulin insensitivity drives the early stages of this disease. In the
forward direction of this cycle serum insulin levels rise in response to obesity. The
elevated insulin results in increased lipogenesis and lipid deposition in the liver, which
drives further whole body insulin resistance. This in turn results in even higher serum
insulin levels and further liver fat accumulation.
Several nuclear receptors are able to reverse this cycle by directly repressing
lipogenesis in the liver. In this case, nuclear receptor activation suppresses liver fat
accumulation and thereby promotes whole body insulin sensitivity. The resulting
decrease in serum insulin feeds forward to further decrease liver fat accumulation, with
the net result of reversing hepatic steatosis and insulin resistance, even in the absence
of effects on overall body weight.
My project focuses on Liver Receptor Homolog-1 (LRH-1), which can be
activated by Dilauroyl Phosphatidylcholine (DLPC). The Moore lab has linked the ability
of this LRH-1 agonist to reverse insulin resistance in mouse models of type 2 diabetes
to decreased expression of the key lipogenic transcription factor SREBP-1. But the
specific mechanisms involved remain unknown. My goal is to understand the
mechasnism of action of the anti-diabetic effects of DLPC.
The transcriptional effects of nuclear receptors are mediated by coactivators and
corepressors, and recent results from the O’Malley laboratory have identified the
coactivator Streroid Receptor Complex-2 (SRC-2) as a key metabolic regulator (PMIDs
21195347, 19039140). Additional preliminary results show that SRC-2 can coactivate
LRH-1 transactivation, and indicate extensive overlap between genome wide LRH-1 and
SRC-2 binding sites in the liver. In vivo, there are also phenotypic similarities between
SRC-2 binding sites in the liver. In vivo, there are also phenotypic similarities between
SRC-2 liver specific knockouts and LRH-1 liver specific knockouts in terms of lipid
metabolism and bile acid metabolism. Thus, my specific hypothesis is that SRC-2 is an
essential mediator of the anti-diabetic and anti-lipogenic effects of DLPC. I am now
trying to determine the impact of both acute and chronic DLPC treatments in SRC-2
liver specific knockout mice and the littermate controls.
Contributors: Choi,Sungwoo; Kim,Mi-Sun; Kim,Seung-Whan; Lee,Jae Man; Moore,David
William Tin-Shing Choi
Program in Developmental Biology/M.D.-Ph.D. Program
Advisor: Mirjana Maletic-Savatic, M.D./Ph.D.-Department of Pediatrics
The fate of the cell, whether it divides, differentiates, enters a transient
(quiescence) or permanent (senescence) growth arrest, or triggers a suicidal
mechanism of death, demands a finely-tuned sequential activation and deactivation of
both biosynthetic and energy generating metabolic pathways. Lipid molecules are
important regulators of stem cell proliferation, including neural stem and progenitor cells
(NPCs). Our previous study of NPCs using proton magnetic resonance spectroscopy
(H-NMR) showed an enrichment of molecule(s) that resonate at 1.28-ppm,
characteristic of –CH2– methylene lipid moiety. The amplitude of the 1.28-ppm signal
correlated with NPC proliferation both in vitro and in vivo, suggesting that the respective
molecule(s) are associated with NPC fate. Further investigations of the chemical
composition of these molecules identified them as a specific group of lipids, some of
which were unique to NPCs as compared to astrocytes. We then asked if these
molecules were functionally important for NPCs, specifically as signaling molecules
regulating NPC cell cycle. Lipid modifiers are known ligands for nuclear receptors,
transcription factors which can be modulated by ligand binding. Transcript analysis of 48
known nuclear receptors found 25 to be expressed in NPCs, 11 of which were orphan
receptors with unknown ligands. Interestingly, 3D-reconstruction and structure
comparison of known ligand—nuclear receptor combinations predicted a high probable
interaction of the 1.28-ppm molecule(s) with the ligand-binding domain of six orphan
nuclear receptors expressed in NPCs. One of those was Nr2e1 (also known as Tlx),
already known to be important for NPC proliferation and self-renewal. Interestingly,
transcriptional activity of Nr2e1 was altered when it was exposed to molecules that
resonate at 1.28-ppm, as shown by mammalian one-hybrid luciferase assay. Altogether,
these data suggest that the NPC fate may involve the interaction of Nr2e1 and the
endogenous lipid molecules enriched in them, thus illuminating a possible mechanism
controlling NPC behavior.
Contributors: Choi, William; Ma Lihua, Thakkar, Aarohi; Gopakuma, Sricharan; Cerda-Smith,
Christian; Young, Damian; MacKenzie, Kevin; Maletić-Savatić, Mirjana
Hsiang-Ching Chung
Integrative Program in Molecular and Biomedical Sciences
Advisor: Thomas Westbrook, Ph.D.-Department of Biochemistry & Molecular Biology
Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer
that is recalcitrant to target therapies. Previous studies in our lab uncovered the codependency of TNBC survival and progression on multiple receptor tyrosine kinases
(RTKs) including MET and PDGFRβ. Notably, combined inhibition of these RTKs with
tyrosine kinase inhibitors (TKis), sunitinib and crizotinib (or S+C) effectively suppresses
TNBC tumor growth across many independent patient-derived xenograft (PDX) models
of TNBC in vivo. However, not all TNBC PDX models are sensitive to S+C.(unpublished
data) To better understand the molecular determinants of S+C sensitivity and
resistance, we performed a genetic (RNAi) screen for modifiers of S+C response in
multiple TNBC models. CUL5 was a prominent candidate from these multiple genetic
screens. We validated that knockdown of CUL5 greatly enhances cell fitness upon S+C
treatment, suggesting CUL5 function is a critical determinant of TNBC drug sensitivity. It
is known that CUL5 functions as a scaffold protein for cullin-based ubiquitin E3 ligases,
however, the role(s) of CUL5 E3 ligases in TNBC pathogenesis and modulating drug
resistance has not been explored (especially resistance to tyrosine kinase inhibitors, the
largest class of anti-cancer targeted therapies). The characterization of how CUL5
controls TNBC drug response represents an unprecedented opportunity to investigate
the role of ubiquitylation in modulating drug response. Identifying genetic or epigenetic
determinants of resistance may enable us to predict resistance mechanisms to S+C in
TNBC patients, and will allow for improved efficacy of target therapies. More broadly,
these discoveries may provide insights or principles governing patient response to other
tyrosine kinase inhibitors (TKIs) used in other cancers.
Contributors: Sun, Tingting; Nair, Amritha; Thomas Westbrook
Andrew Mark Robinson Ciupek
Program in Translational Biology & Molecular Medicine
Advisor: Suzanne Fuqua, Ph.D.-Department of Medicine
Mothaffar Rimawi, M.D.-Department of Medicine
Background: We have previously developed a novel model of hormone therapy
resistance involving shRNA knockdown of Rho GDI(. Although it is known that androgen
receptor (AR) is frequently expressed along with estrogen receptor (ER) ( in breast
tumors, AR’s role in resistance is unknown. Here we explore the role of AR in the
resistance of the Rho GDI( knockdown model.
Materials and Methods: shRNA knock down was used to block expression of Rho
GDI( in the ER(-positive breast cancer cell lines MCF-7 and ZR-75-B. Reverse phase
protein arrays (RPPA) were used to analyze global changes in growth factor pathway
expression. Microarray analysis was used with tamoxifen (Tam) treated cells to explore
pathways promoting Tam resistance. Western blot analysis was used to look at
activation of the AR, ER, and epidermal growth factor receptor (EGFR) pathways. The
effects of AR and EGFR inhibitors and agonists on anchorage independent growth and
proliferation in the presence of Tam were measured in soft agar colony formation
assays and MTT growth assays.
Results: MCF-7 Rho GDI( knockdown cells were resistant to the growth inhibitory
effects of Tam when grown as xenografts in vivo. We discovered that AR was over
expressed in the knockdown cells as confirmed by both western blot and RPPA.
Microarray analysis showed an activation of EGFR and MAPK signaling in Tam treated
ZR-75-B knockdown cells. In addition, western blot analysis showed that rapid activation
of EGFR in these cells could be blocked by both AR and EGFR inhibitors. In MCF-7 and
ZR-75-B knockdown cells, increased basal levels of anchorage independent growth,
proliferation, and Tam stimulated growth were seen which could be blocked by inhibition
of AR with bicalutimide and MDV3100 or the EGFR inhibitors AG1478 and Gefitinib.
Discussion: Since AR was significantly over expressed with the Tam-resistant
phenotype and AR agonists/inhibitors modulated the phenotype, we hypothesize that
AR plays a role in the process. Also, given that EGFR is activated and EGFR inhibitors
block the Tam-resistant phenotype, EGFR also likely contributes to the resistance.
Since an AR inhibitor can block rapid activation of EGFR we hypothesize that AR may
promote Tam-resistance through activation of EGFR signaling. Our results suggest that
AR may represent a new clinical target for hormone therapy resistance of ER( positive
breast cancer.
Contributors: Ciupek A, Brusco L, Covington KR, Weigel NL, Lu Y, Mills G, and Fuqua AW
Sarah Margaret Ciupek
Department of Neuroscience
Advisor: Daoyun Ji, Ph.D.-Department of Molecular & Cellular Biology
Improper aggregation of tau protein occurs in many neurodegenerative
dementias, including Alzheimer’s disease. A major target of degeneration in these
tauopathies is the hippocampus, a classical brain region for memory. In the normal
hippocampus, spatial memory is encoded by place cells, a class of pyramidal neurons
that fire preferentially when the animal is in a specific location in space. Additionally,
proper memory encoding is facilitated by stereotyped oscillations in the local field
potential (LFP) and entrainment of neuronal firing to those oscillations. However, we do
not know which aspects of hippocampal memory processing are altered in tauopathies
and when in disease alterations occur. Such knowledge would bridge our understanding
of the molecular and behavioral level changes that occur in these disease states. Here,
we study this question using the rTg4510 (Tau) mouse line, in which human tau with the
P301L mutation found in patients with frontotemporal dementia is expressed in the
mature forebrain. In this mouse, tau becomes hyperphosphorylated and begins to
aggregate in the hippocampus as young as 2.5 months of age. Neurodegeneration
begins soon after, with 60% of the CA1 layer of the hippocampus already degenerated
by 5.5 months of age. Previous experiments in our laboratory have shown deficits in
place cell stability in older Tau mice (age 7-9 months) that already exhibit profound
neurodegeneration. These older mice also show altered LFP oscillations as well as
impaired firing synchrony during LFP events. However, this previous study only looked
at older animals, when the effects of neurodegeneration cannot be distinguished from
those of tau phosphorylation alone. To test which, if any, of these network level changes
occur prior to neurodegeneration, we have recorded in vivo from the hippocampus of
Tau mice at 2 to 5 months of age as they run through a linear track, explore an open
field, and sleep. Our results show only minor deficits in place cell stability and size
compared to WT mice prior to major degeneration. In contrast, these young mice do
exhibit LFP and synchrony changes. Specifically, there is a decrease in the amplitude of
sharp-wave ripples (high-frequency LFP oscillations associated with memory
consolidation) during slow-wave sleep in Tau mice and a corresponding unusual pattern
of cell firing during ripple events. When graphed by age, the ripple amplitude phenotype
gets progressively worse with age in Tau mice but stays constant in WT mice, with a
starting average ripple amplitude higher than even the youngest Tau mice. These
results allow us an insight into the network level alterations that lead to the debilitating
memory loss symptoms seen in patients with various types of tauopathies, an aspect of
these diseases that is poorly understood.
Contributors: Cheng, Jingheng; Ji, Daoyun
Matthew R Collinson-Pautz
Program in Translational Biology & Molecular Medicine
Advisor: David Spencer, Ph.D.-Department of Pathology & Immunology
Kevin Slawin, M.D.-Department of Urology
The use of dendritic cell vaccines to treat cancer has significant potential,
however that potential has yet to be achieved in treating cancer patients. Fortunately,
pioneering biotechnologies are paving the way for more effective cancer vaccines. Our
lab has developed a drug inducible MyD88/CD40 (iMC) composite adjuvant that
promotes robust cytotoxic T cell priming by iMC activated DCs. Despite the iMC
innovation, its implementation in patient-tailored ex vivo DC vaccines is impractical for
widespread use, due to issues of scalability, distribution, and cost. We have therefore
begun to address the delivery of DC vaccines as “off-the-shelf” therapies using in vivo
electroporation (EP) of plasmid DNA (pDNA) encoding both the iMC adjuvant and tumor
antigen (eVaCIDe). Intradermal EP of pDNA encoding the model antigen LacZ in mice
primed LacZ-specific CD8+ T cell responses, as mice receiving LacZ + EP had
significantly greater portions of antigen responsive, IFNγ secreting T cells than mice that
received that same LacZ vector without EP. Mice vaccinated prophylactically with LacZ
+EP were either protected from establishment of B16/LacZ tumors or demonstrated
significantly slower tumor growth compared to controls. In mice bearing pre-established
B16/LacZ tumors, LacZ-eVaCIDe, but not EP with LacZ antigen alone significantly
reduced tumor burden compared to negative controls. Lastly, eVac encoding the human
Prostate cancer antigen, PSMA, was able to stimulate significant antigen-specific CD8+
T cell and serum antibody responses. Taken together, these data indicate that eVac
with iMC-antigen is an effective “off-the-shelf” cancer vaccine and potentially a platform
for a wide range of disease targets. These findings warrant elucidation of the underlying
immunological mechanisms responsible for the anti-tumor responses observed with
eVac treatment to guide further refinement of the vaccine in a pre-clinical model.
Contributors: Collinson-Pautz, M; Lu, An; Decker, W; Spencer, D; Levitt, J
Zachary Christopher Conley
Department of Biochemistry & Molecular Biology
Advisor: E. Zechiedrich, Ph.D.-Department of Molecular Virology & Microbiology
New antibiotics are needed because resistance has rendered many existing drugs
ineffective. We discovered that the antifungal drug, ciclopirox, prevents growth of problematic,
multidrug-resistant clinical isolates, although the drug target remains elusive (Carlson-Banning
et al. 2013 PLoS One 8:e69646). We showed that both sugar metabolism and the availability of
free iron in the growth medium affect ciclopirox inhibition in of E. coli. Additionally, iron-acquiring
siderophore production in P. aeruginosa was increased in the presence of ciclopirox, implying a
disruption of iron acquisition. To examine which We performed a literature search and identified
103 genes involved in specific iron-utilization and sugar metabolism pathways. To test mightfor
those that are be affected by ciclopirox, we we screened gene deletion strains 103 gene
deletion strains for increased sensitivity to ciclopirox, . Hhoping to identify ciclopirox-specific
targets, . Towe remove filtered out general antibiotic effects, we by comparingred our data to a
study that measured the antibiotic susceptibility of 22 antibiotics forof the entire Keio collection
of E. coli gene deletions and removed from our list any that were affected by any antibiotic (Liu
et. al. 2010 Antimicrob. Agents and Chemother. 54:139310.1128/AAC.00906-09vol, p). To
remove filter out genes involved in general iron chelation affect, as ciclopirox can bind free iron,
we screeneding our 29(how many?) gene sets for increased sensitivity to the known iron
chelator 1, 10-phenanthroline. This stratification resulted in 18 gene deletions with increased
susceptibility to ciclopirox alone. The majority Most (n) of these genes encode proteins identified
are involved with the synthesis of the surface glycolipid enterobacterial common antigen.
Additionally,In support of the idea that ciclopirox affects cell outer membrane, we had already
shown that O-antigen producing bacteriaE. coli grown in sub-inhibitory concentrations of
ciclopirox display less O-antigen and less high molecular weight lipopolysaccharide (CarlsonBanning et al. 2013 PLoS One 8:e69646). We hypothesize that one ciclopirox target exists in
the construction of glycolipids that are attached to the bacterial outer membrane. The next
largest group ofOther stratified genes identified isencode proteins involved in the uptake of the
siderophore enterobactin, a gram-negative-specific siderophore with the highest known iron
binding affinity of any siderophore. We hypothesize that ciclopirox drug targets are not restricted
to one pathway. Here, we identified the construction of glycolipids that are attached to the
bacterial outer membrane as one pathway, and iron uptake through siderophores ais another
pathway affected by ciclopirox. Perhaps the existence of multiple essential targeted pathways
explains why this drug is effective against antibiotic-resistant bacteria. These data are an
important step toward developing ciclopirox or new derivatives of ciclopirox, as drugs against
multidrug-resistant gram-negative pathogens for which few therapeutic options exist currently..
Contributors: Conley, Zachary C.; Carlson-Banning, Kimberly M.; Carter, Ashley; Chou, Andrew;
Hamill, Richard J.; Song, Yongcheng; and Zechiedrich, Lynn
Sean Michael Cullen
Program in Developmental Biology/M.D.-Ph.D. Program
Advisor: Margaret Goodell, Ph.D.-Department of Pediatrics
Our lab recently published the first hematopoietic stem cell (HSC) methylome.
One feature identified was over 1,000 large, unmethylated regions, named “canyons”
(>3.5kb, <10% average methylation, >5 CpG dinucleotides/kb). These canyons were
largely conserved across cell type and species, but were also observed to possess
subtle, dynamic differences between these groups. For example, in mouse embryonic
stem cells (ESCs), 839 canyons were identified, 82% of which were shared with HSCs,
but differed slightly overall in size and methylation levels. As for the purpose of these
canyons, a high number and concentration of HSC-specific TF binding sites were found
within the confines of these canyons. Deletion of DNA methyltransferase 3a (Dnmt3a),
previously shown to be essential for proper HSC differentiation (Challen 2012), was
found to change methylation significantly at canyon borders, but, unexpectedly, both
expanded and contracted the size of many HSC canyons (Jeong 2013). This suggests
Dnmt3a functions to maintain methylation at canyon borders, which leads to our central
hypothesis that direct Dnmt3a binding at canyon edges is essential for canyon stability.
Using an ESC line capable of doxycycline-induced expression of biotinylatedDnmt3a, we performed Chromatin Immunoprecipitation-Sequencing (ChIP-Seq) to
identify the genome-wide binding sites of Dnmt3a. Dnmt3a binding was largely excluded
from ESC canyons, but was found at the edges of these canyons, often at high levels.
The cell line was then used to generate a mouse model capable of examining the
similarities and differences of Dnmt3a DNA-binding in HSCs and ESCs, to determine
how accurately binding differences mimic canyon changes between stem cell
Contributors: Cullen, Sean; Luo, Min; Jeong, Mira; Lin, Xueqiu; Li, Wei; Goodell, Margaret
Hang Dai
Department of Molecular & Human Genetics
Advisor: Suzanne Leal, Ph.D.-Department of Molecular & Human Genetics
A large number of studies on nonsyndromic hearing impairment (NSHI) have
been performed using samples collected from the Indian subcontinent, Middle East and
Europe. There has been limited study of NSHI in African-Americans (AA) and subSaharan Africans. We evaluated the frequency of previously reported “pathogenic”
variants in NSHI genes using data from the NHLBI-Exome Sequencing Project (ESP)
which is a population-based study of AA (N=2203) and European-Americans [EA
(N=4300)]. Out of 201 observed variant sites which are pathogenic according to ClinVar
and/or the Deafness Variation Database, we reclassified 121 (60.2%) variant sites as
likely non-pathogenic based on literature, high allele frequencies in ESP and
bioinformatics tools. In ESP, 80 likely pathogenic variant sites were observed in 5
autosomal dominant (AD) and 14 autosomal recessive (AR) NSHI genes of which 24 of
the variant sites in 7 of these genes cause syndromic hearing impairment (HI), e.g.
MYO7A variants cause both NSHI and Usher syndrome. Of these variant sites, 49 (91
alleles) were found only in EA, 18 (23 alleles) only in AA, and 13 (194 alleles) in both EA
and AA. GJB2 c.35delG was the ARNSHI variant site with the highest allele frequency
in EA [N=89; 1.09% (95%CI: 0.88%, 1.34%)] but with a much lower allele frequency in
AA [N=4; 0.094% (95%CI: 0.03%, 0.2%)]. Additionally OTOF c.2348delG was identified
in 4 AA alleles [0.095% (95%CI: 0.03%, 0.2%)] but is very rare in EA [N=1; 0.01%
(95%CI: 0.0003%, 0.07%)]. For EA, pathogenic variant sites were identified in 11 AR
and 5 AD NSHI genes including variant sites in 7 NSHI genes which cause syndromic
HI, while for AA, pathogenic variant sites were identified in 13 AR and 2 AD NSHI
genes, of which 6 genes have variant sites which cause syndromic HI. MYO7A
c.3764delA which was reported to cause Usher syndrome, is homozygous in 1 AA and
3 EA individuals. The use of EVS in order to exclude nonpathogenic variants must be
done cautiously due to NSHI-causal variants within EVS. Knowledge of the frequency of
NSHI variants in large population samples from different ethnic backgrounds is not only
important to evaluate clinical significance but also aids in evaluating pathogenicity.
Although population-based samples of AA aids in evaluating population-specific
frequency of previously reported pathogenic variants, AA and sub-Saharan Africans
with HI need to be studied to better understand the genes and variants underlying
disease etiology.
Contributors: Dai, Hang; Wang, Gao; Santos-Cortez, Regie; Leal, Suzanne
Michele C. Darrow
Department of Biochemistry & Molecular Biology
Advisor: Wah Chiu, Ph.D.-Department of Biochemistry & Molecular Biology
Huntington’s disease, a neurodegenerative disorder characterized by loss of
striatal neurons, is linked to an expanded and unstable CAG trinucleotide repeat, which
translates as a polyglutamine repeat in the protein product. 40+ trinucleotide repeats
have been described as pathological.
Mutant huntingtin exon 1 with 46 polyglutamine repeats was purified via
thioredoxin tag and incubated with the TRiC chaperonin subunit CCT5 homo-oligomer
complex, markedly slowing mHtt fibril aggregation, as shown using a filter trap assay
and cryo-electron microscopy.
Cryo-electron tomography and averaging was also performed on these samples
with the goal of identifying the structure of CCT5 homo-oligomer in complex with mHtt
fibrils and oligomers. Results indicate that CCT5 homo-oligomer interacts with mHtt at
the tips of the fibrils, and by encapsulating oligomers.
This study provides further information about how a single subunit of TRiC
chaperonin interacts with mHtt fibrils and oligomers to slow aggregation, identifying a
potential target for therapeutics.
Contributors: Sergeeva, Oksana A.; Isas, Jose M.; Galaz-Montoya, Jesus; King, Jonathan A.;
Langen, Ralf; Chiu, Wah
Gabriela Riva David
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M.-Department of Molecular & Human Genetics
The mTOR signaling pathway senses and integrates environmental signals,
cellular nutrition, and energy status to regulate cell growth and metabolism. This
pathway plays a role in many major cellular processes, and its dysregulation has been
implicated in diseases that include neurodegeneration and cancer.
Through a forward genetic screen on the Drosophila X chromosome for essential
genes that play a role in the development, function, and maintenance of the nervous
system, we have isolated a novel component of the mTOR pathway: the Drosophila
homolog of WW domain-containing adapter with coiled-coil region (WAC), which we
have named Wacky.
wacky loss-of-function (LOF) mutant eye clones exhibit functional defects and
morphological degeneration in the adult retina. Looking at the subcellular defects of
wacky LOF mutant clones in the eye and fat body, we observe increase in autophagic
vesicles and lysosomes. Since autophagy is misregulated in wacky mutant cells, we
looked at the activity level of mTOR, which negatively regulates autophagy. We found
that, in wacky mutant larval fat bodies, the phosphorylation levels of downstream mTOR
kinase targets, S6K, 4E-BP, and Akt, are dramatically decreased. Consistent with an
increase in basal autophagy that results from a decrease in mTOR activity, knocking
down WAC in HEK293 cells leads to an increase in the degradation of betaine
homocysteine methyltransferase, a substrate of autophagy.
To determine how loss of Wacky leads to a decrease in mTOR signaling, we
performed immunoprecipitation followed by mass spectrometry to identify protein
interactors of Wacky. From this experiment, we identified four mTOR pathway
components as Wacky physical interactors: mTOR, Vha100-2, Pontin, and Reptin.
Currently, we are focusing on dissecting the interaction of Wacky with these mTOR
pathway components to determine the mechanism by which Wacky positively regulates
this signaling pathway.
Because of its role in the mTOR pathway, Wacky could be a novel target for the
modulation of the mTOR pathway, and further deciphering its functions may lead to the
development of effective therapies for neurodegeneration and cancer.
Contributors: David, Gabriela; Xu, Zhen; Rui, Yan-Ning; Charng, Wu-Lin; Jaiswal, Manish;
Yamamoto, Shinya; Zhang, Ke; Xiong, Bo; Bayat, Vafa; Duraine, Lita; Zhang, Sheng; Bellen,
Shaun Michael Davis
Department of Molecular & Human Genetics
Advisor: Herman Dierick, M.D.-Department of Molecular & Human Genetics
Aggressive behavior is widespread in the animal kingdom. However, its
mechanisms remain poorly understood, and the degree of molecular conservation
between distantly related species is unknown. Here, we used two complementary
genetic approaches to identify the mechanisms that regulate this behavior.
In mice, loss-of-function of the transcriptional repressor Nr2e1 causes extreme
aggression. We used a candidate gene approach and show that knock-down of tailless
(tll), a fly ortholog of Nr2e1, increases aggression in Drosophila melanogaster. Tll
localizes to the adult pars intercerebralis (PI), which is composed of neurosecretory
cells with similarity to the mammalian hypothalamus. Knock-down of tll in the PI is
sufficient to increase aggression and is rescued by co-expressing human NR2E1.
Knock-down of atrophin, which encodes a co-repressor of Tll, also increases aggression
and both proteins physically interact in the PI. The tll-knock-down induced aggression
phenotype is fully suppressed by blocking neuropeptide processing or release from the
PI, showing that aggression is dependent upon a neuropeptide-based mechanism. In
addition, genetically activating PI neurons, which leads to neuropeptide release in flies,
increases aggression and mimics the aggression-inducing effect of hypothalamic
stimulation in mammals. Together our results show that Tll regulates aggression in flies
through a neuropeptide-based mechanism in the neurosecretory cells of the adult PI.
In addition to the candidate gene approach, we developed a forward genetic
screen to identify novel genes that regulate aggressive behavior. One consequence of
excessive aggression is increased physical damage. We found a positive correlation
between number of lunges and percentage of wings with damage. Wild-type male flies
were mutagenized with EMS to perform an X-chromosome forward genetic screen.
Over 1,500 different chromosomes were analyzed; the average percent wing damage
was 7% and only lines with more than 30% damaged wings were kept for further
analysis. Of these 42 lines, 5 showed an increased aggression phenotype. These lines
are now being sequenced to identify the causal mutation. This unbiased approach can
identify novel components and/or pathways that regulate aggression behavior in
Drosophila. Much like the tll-regulated neuropeptide-based mechanism, these results
can provide insight into human aggression.
Contributors: Thomas, Amanda; Nomie, Krystle; Gnerer, Joshua; Huang, Longwen
Emily Packard Dawson
Integrative Program in Molecular and Biomedical Sciences
Advisor: Jason Heaney, Ph.D.-Department of Molecular & Human Genetics
Testicular germ cell tumors (TGCTs) are the most frequent tumor type diagnosed
in young men. TGCTs arise from fetal germ cells whose pluripotent capacities facilitate
tumor differentiation into a variety of somatic cell types. In the 129 inbred mouse model
of human TGCTs, tumors initiate between embryonic days (E)13.5 to 15.5, which
coincides with the mitotic:meiotic switch: germ cells of both sexes lose pluripotent
capacity, female germ cells (oogonia) commit to meiosis, and male germ cells
(gonocytes) enter mitotic arrest. We previously demonstrated that gonocyte
proliferation, retention of pluripotency, and aberrant expression of genes associated
with pre-meiotic oogonia and adult spermatogonia, including cyclin D1 (Ccnd1) and
stimulated by retinoic acid 8 (Stra8), at E15.5 were directly related with increased tumor
risk. Based on the known oncogenic potential of Ccnd1 overexpression, we
hypothesized that aberrant expression of Ccnd1 significantly contributes to TGCT
initiation by inducing a breakdown in the mitotic:meiotic switch, which causes gonocytes
to continue proliferating and retain pluripotency. Using a Ccnd1 knockout on the high
TGCT risk 129-Chr19MOLF/Ei (M19) background, we found that Ccnd1 deficiency
significantly reduced TGCT incidence by 62% (p<0.001) compared to wild-type and
heterozygous animals. Immunohistochemistry experiments suggest that Ccnd1
deficiency reduces the number of proliferating (KI67-positive) and pluripotent (NANOGpositive) gonocytes. Ccnd1 expression in post-migratory embryonic germ cells is
normally restricted to oogonia and is transiently expressed from E12.5 to E15.5, prior to
their entry into meiosis. Expression of Ccnd1 from E12.5 to E15.5 in TGCT-susceptible
gonocytes suggests that a signal normally restricted to the developing ovary is
aberrantly active in the TGCT-susceptible testis. Retinoic acid (RA) signaling normally
precedes Ccnd1 expression in embryonic oogonia, while RA is normally catabolized by
CYP26B1 in the embryonic testis. Curiously, we find an altered expression pattern of
embryonic germ cell sex-specific genes (e.g. Stra8, Ccnd1, Nanos2, and Nodal) in
TGCT-susceptible gonocytes. Therefore, we tested whether gonocyte expression of
Ccnd1 is induced by an ectopic RA signal. Culturing embryonic testes from TGCTresistant or TGCT-susceptible mice in medium containing RA induced CCND1
expression in gonocytes. Furthermore, induction of CCND1 in oogonia and TGCTsusceptible gonocytes deficient for Stra8 demonstrates that RA-induced expression is
independent of Stra8 function. We hypothesize that TGCT-susceptible gonocytes
respond to both an abnormal RA signal and normal gonocyte developmental signals,
which disrupt gonocyte entry into mitotic arrest during the mitotic:meiotic switch and
lead to TGCT initiation.
Contributors: Dawson, Emily; Lanza, Denise; Benton, Susan; Heaney, Jason.
Antonia De Maio
Program in Developmental Biology
Advisor: Huda Zoghbi, M.D.-Department of Pediatrics
Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder
characterized by death of Purkinje cell and brain stem neurons. SCA1 is caused by
expansion of CAG repeats within the coding region of the Ataxin1 gene (Atxn1), leading
to the production of a mutant variant of the cognate protein harboring an expanded
glutamine tract (PolyQ). Previous work in our lab defined the spliceosomal RNA binding
protein, RBM17, as an ATXN1 interactor that preferentially binds upon expansion of the
polyQ tract and requires phosphorylation of the ATXN1 residue S776. These two
aspects of ATXN1 are both critical in the SCA1 pathogenesis suggesting that RBM17
has a key role in the disease mechanism.
To investigate in depth the function of Rbm17 we generated an Rbm17
constitutive knockout and found this protein to have an essential developmental role, as
its constitutive ablation causes early embryonic lethality. To evaluate the effects of
Rbm17 loss-of-function we looked at conditional deletion models lacking Rbm17
exclusively from developing cerebellum or adult Purkinje cells. These mice revealed that
absence of Rbm17 severely impairs development of cerebellum while Purkinje cells
devoid of the protein undergo progressive and fast degeneration.
Having established the critical role of Rbm17 for development and cell viability
we propose that this factor is a key component of the spliceosomal complex and that its
ablation causes aberrant splicing of still unknown RNA targets whose tight regulation is
essential for successful accomplishment of both processes. To test our hypothesis we
will use Immunoprecipitation/mass spectrometry (IP/MS) and Crosslinking and
immunoprecipitation (CLIP) methods to, respectively, identify Rbm17 protein interactors
and its RNA binding pattern throughout the transcriptome. Using MS we analyzed the
composition of Rbm17 immunoprecipitation (IP) samples from wild-type mouse
cerebellum. Our preliminary results confirmed some previously known interactions and
pointed out several putative Rbm17 partners within and beyond the spliceosomal
complex, including splicing factors and RNA metabolism modulators. Currently, we are
optimizing the CLIP protocol for Rbm17 in order to pull down the protein and its
associated RNA targets. RNA-sequencing will eventually reveal the identity of these
targets, shedding light on the essential cellular network regulated by Rbm17. The
integration of these data will permit us to uncover the molecular mechanism underlying
the requirement of Rbm17 for cell survival and will help us establish if and how Rbm17
contributes to SCA1 pathogenesis.
Contributors: De Maio, Antonia; Park, Jeehye; Zoghbi, Huda Y.
George Hilton Denfield
Department of Neuroscience/M.D.-Ph.D. Program
Advisor: Andreas Tolias, Ph.D.-Department of Neuroscience
Neuronal responses to identical visual stimuli are variable, but the source of this
variability is unknown. It is widely accepted that this variability results from neuronal
noise. However, we offer an alternative explanation: this variability is not noise but
reflects, and is due to, computations internal to the brain. Internal signals such as
cortical state or attention interact with sensory information processing in early sensory
areas. However, little research has examined the effect of fluctuations in these signals
on neuronal responses, leaving a number of uncontrolled parameters that may
contribute to neuronal variability. One such variable is attention. We hypothesize that
fluctuations in attentional signals contribute to neuronal response variability and that
controlling for such fluctuations will reduce this variability. We predict attentional effects
on neuronal variability that differ from those predicted by the standard model of
attention, in which attention reduces cortical noise to improve sensory processing,
producing an inverse relationship between degree of attention and neuronal variability.
Our model considers the effects of fluctuations in the attentional signal on recipient
neurons and predicts the greatest degree of neuronal variability at an intermediate level
of attention, corresponding to a state where the focus of attention is most variable. We
control for fluctuations in the attentional signal in a change-detection task, varying
across blocks of trials the degree to which subjects must attend to one of two stimuli
presented in the visual field, while recording macaque primary visual cortex.
Contributors: Denfield, George; Ecker, Alexander; Tolias, Andreas
Avinash Vijay Dharmadhikari
Program in Translational Biology & Molecular Medicine
Advisor: Pawel Stankiewicz, M.D./Ph.D.-Department of Molecular & Human Genetics
Ignatia Van Den Veyver, M.D.-Department of Obstetrics & Gynecology
Alveolar capillary dysplasia with misalignment of pulmonary veins
(ACDMPV) is a rare neonatally-lethal diffuse developmental disorder of the lungs
caused by haploinsufficiency of FOXF1. All affected newborns die in the first month of
life due to severe respiratory distress and pulmonary hypertension. Foxf1 null mice die
by midgestation as a result of defects in mesodermal differentiation and cell adhesion.
Foxf1 heterozygous mice exhibit up to 90% neonatal mortality, depending on genetic
background. For the current study, Foxf1 knockout mice with a deletion of the forkhead
binding domain were generated and are congenic on the C57BL/6J background.
Analysis of RNA from postnatal day 0.5 Foxf1+/- and wildtype lungs using Illumina
mouse WG-6 v2.0 expression bead chip microarray revealed statistically significant
deregulation (p<0.05, fdr<0.05) of several genes, including those involved in pulmonary
vascular development (Sema3C, Dll4, and Ednrb), lung branching morphogenesis
(Fgf10 and Lama1), and the blood pressure regulating renin-angiotensin system (Ren1,
Cma1, and Cpa3). To study the effects of Foxf1 overexpression, we knocked a Creinducible Foxf1 allele into the ROSA26 locus. ROSA26-LSL-Foxf1 mice were mated to
CMV-cre mice to overexpress Foxf1 in all tissues and were mated to Tie2-cre mice to
overexpress Foxf1 specifically in vascular endothelial cells. ROSA26Foxf1; CMV-cre
mice exhibit early embryonic lethality around e12.5 while ROSA26Foxf1; Tie2-cre mice
exhibit perinatal lethality around birth. Transcriptional changes in ROSA26Foxf1; Tie2cre e18.5 lungs (n=3) were compared to e18.5 ROSA26-LSL-Foxf1 (n=3) control lungs
using the Illumina WG-6 v2.0 expression BeadChip kit microarrays. Pathways
associated with granulocyte adhesion and diapedesis were found to be deregulated.
Future studies will be directed towards characterizing lung vascular defects in the
ROSA26Foxf1; Tie2-cre embryos. We are currently mating ROSA26Foxf1; Tie2-cre
mice to Foxf1+/- mice, which we hypothesize, will rescue early postnatal mortality. This
could inform future gene therapy studies in patients with ACDMPV.
Contributors: Dharmadhikari, Avinash; Carofino, Brandi; Zabielska, Joanna; Wronowska,
Weronika; Majewski, Tadeusz; Gambin, Anna; Szafranski, Przemyslaw; Justice, Monica J,
Stankiewicz P.
Turgut Dogruluk
Department of Molecular & Human Genetics
Advisor: Kenneth Scott, Ph.D.-Department of Molecular & Human Genetics
Tumor sequencing projects such as The Cancer Genome Atlas (TCGA) have
revealed the high complexity of cancer genomes that are comprised of both pathogenic
“driver” aberrations and neutral “passenger” events. A major effort in the cancer
research community involves discriminating drivers from passengers with the goal of
prioritizing new therapeutic targets.
To expedite driver discovery, I established a screening platform involving novel
high-throughput mutagenesis and molecular barcoding (HiTMMoB) technology allowing
our laboratory to engineer somatic mutations identified by TCGA into our collection of
>32,000 human open reading frames (ORFs). Wild-type and mutant barcoded ORFs
are subsequently entered into pooled in vitro/vivo genetic screens to identify those that
can drive cancer phenotypes.
Since developing HiTMMoB, I have continued using this technology for my own
screens that include in vitro/vivo driver and pharmacological screens to assess differing
effects of numerous mutations in the PIK3CA oncogene. PIK3CA is frequently mutated
in breast cancer among other tissue types and regulates cancer’s most mutated
pathway, PI3K. However, much of the focus of PIK3CA research is on the hot spot
mutations of the PIK3CA gene and neglects infrequent ‘tail’ mutations. I have created
24 rare mutations of PIK3CA by utilizing HiTMMoB and entered them into few in vitro
and in vivo screens to assess the effects of the mutations on several cancer phenotypes
i.e. tumor formation, soft agar colony formation, growth-factor-independent growth and
drug resistance/sensitivity. Even though there seems to be a general correlation
between PIK3CA tail mutation frequency and ‘phenotype strength’ as expected, these
experiments interestingly revealed that (1) PIK3CA mutations can be classified into two
separate groups in terms of pathway activations, (2) some of the rarest PIK3CA
mutations are still significantly oncogenic. All these data emphasized the need for
‘personalized and mutation specific’ cancer treatment approaches for PIK3CA-positive
In summary, this discovery pipeline revealed that not all the PIK3CA mutations
are the same and thus should be treated differently requiring patient specific cancer
therapies. The HiTTMoB technology together with downstream screening platforms
promise to provide the cancer research community the functional annotation on the
most promising cancer aberrations for drug development and targeting.
Contributors: Dogruluk, Turgut; Dogruluk, Armel; Mills, Gordon B.; Scott, Kenneth L.
Rocio Dominguez-Vidana
Integrative Program in Molecular and Biomedical Sciences
Advisor: Thomas Westbrook, Ph.D.-Department of Biochemistry & Molecular Biology
Background: Targeted therapies exploit the fact that many tumors have genetic
drivers that control tumor progression. Tumors rely on these drivers for their continued
survival; therefore, inhibition of these genes strongly impairs tumor growth with minimal
side effects to normal cells. However, resistance can arise when these drugs are used
as single agents. Clinical and experimental data suggest that there is substantial
heterogeneity in drug-response between patients and even between cells of the same
tumor. However, the mechanisms driving this heterogeneity are poorly understood. The
goal of this project is to identify the genetic networks that govern response to the anticancer drug lapatinib in Her2+ breast cancers.
Experimental design and methods: We performed pooled RNA interferencebased genetic screens in a panel of Her2+ breast cancer cell lines using a short hairpin
RNA (shRNA) library with ~9k unique hairpins targeting ~2k genes. We then assessed
the effects of these shRNAs in untreated cells and in lapatinib-treated cells. We
identified ~300 Lapatinib Sensitivity Regulators (LaSRs); from which we validated ~100
using small interference RNA (siRNA) based assays, and organized in protein networks
using public protein-protein interaction (PPI) databases.
Results: We focused on 12 LaSRs that show a combinatorial effect with lapatinib
in multiple Her2+ models, and we systematically tested whether they shared a PPI and
genetic coregulation. We have identified a potential mechanism in which our LaSRs
WEE1, CSNK2A1, and CDC14B deregulate mitotic entry in conjunction with lapatinib,
causing cell death.
Conclusion: We developed an unbiased functional screen that identified genes
governing sensitivity to lapatinib, and a validated these results using orthogonal
methods. We identified WEE1, CSNK2A1, and CDC14B as a network governing
lapatinib sensitivity from a systematic analysis of genetic coregulation and PPIs.
Contributors: Ronald J Bernardi*1, Rocio Dominguez-Vidana*2,3, Christopher S Bland*2,
Mitchell Rao4, Siddhartha Tyagi2, Kathleen A Scorsone2, Earlene M Schmitt2, Martin J Shea5,
Tamika Mitchell5, Sarmistha Nanda5, Susan G Hilsenbeck5, Joe W Gray6, Carlos L Arteaga7,
Brent N Rexer7, C Kent Osborne5, Chad A Shaw4, Rachel Schiff5, Thomas F
Timothy Lloyd Dosey
Integrative Program in Molecular and Biomedical Sciences
Advisor: Theodore Wensel, Ph.D.-Department of Biochemistry & Molecular Biology
Transient Receptor Potential (TRP) channels are a superfamily of non-selective
cation channels conserved in eukaryotes. In mammals, the Vanilloid (TRPV) sub-family
is involved in sensory processes including nociception and temperature sensing.
However, our understanding of how TRP channel activity is regulated by these diverse
stimuli has been impeded by the lack of structure-function information. Here we report
that TRPV2 as well as a TRPV4 functional fragment can be purified from a yeast
heterologous expression system and we demonstrate that they remain mono-dispersed
in detergent-free buffer when stabilized with amphipoles. Further, we have developed
cell-based and vesicle-based activity assays to investigate TRPV2 and TRPV4 function
and we are utilizing recent advancements in cryo-electron microscopy to achieve high
resolution structures without crystallization. With these structure and function
techniques, we will be able to elucidate the gating mechanisms of these TRPV channels
and gain insights into how other TRP channel families respond to a wide range of
Contributors: Dosey, Timothy; Wang, Zhao; Fan, Guizhen; Zhang, Zhixian; Serysheva, Irina;
Chiu, Wah; Wensel, Theodore
Adetutu Taiwo Egunsola
Department of Molecular & Human Genetics
Advisor: Brendan Lee, M.D./Ph.D.-Department of Molecular & Human Genetics
Shohat type spondyloepimetaphyseal dysplasia (SEMD) is part of a
heterogeneous group of disorders characterized by a combination of vertebral,
epiphyseal and metaphyseal anomalies. Patients with Shohat type SEMD present with
disproportionate short stature, a short neck, a small chest, abdominal distension with
hepatosplenomegaly, lordosis, short limbs, genu varum and joint laxity.
Radiographically, these patients also have a delayed bone age, platyspondyly with
central notches in the vertebral end-plates, radiolucency of the femoral metaphyses as
well as fibular overgrowth. The genetic basis of Shohat type SEMD is unknown. By
exome sequencing of three individuals from two families, we identified a homozygous
donor splice-site mutation in DDRGK1 in all affected individuals, and confirmed it in a
separate family by Sanger sequencing. Patients with this splice-site mutation, a
predicted frameshift mutation, have significantly reduced levels of DDRGK1 compared
to controls. DDRGK1 is an ufmylated ER protein that has a conserved PCI domain, a
domain predicted to participate in protein-protein interactions. To understand the role of
DDRGK1 in skeletal development we knockdown DDRGK1 in zebrafish using
morpholinos. DDRGK1 morphants have abnormal craniofacial features, such as
disfigured cartilages and reduced number of branchial arches. Furthermore, there is a
decreased level of SOX9, a type II collagen transcription activator, when DDRGK1 is
knockdown in differentiated ATDC5, a chondrocyte cell line. Through Co-IP, we found
that DDRGK1 interacts with SOX9. We then performed rescue experiments to
determine whether SOX9 is a downstream target of DDRGK1 and found that
overexpression of SOX9 rescues the craniofacial phenotype seen in DDRGK1
morphants. Therefore, we hypothesize that DDRGK1 regulates the stability of SOX9
and a deficiency in DDRGK1 decreases levels of SOX9. We are currently assessing
whether DDRGK1 regulates the stability of SOX9 by affecting the ubiquitination state of
SOX9. In addition, we are generating Ddrgk1 knockout mice using CRISPR/Cas9
system to future characterize the role of DDRGK1 in skeletal development.
Contributors: Bae, Yangjin; Liu, David; Campeau, Philippe; Lu, James; Cohn, Daniel; Lachman,
Ralph; Swindell, Eric; Shohat, Mordechai; Gibbs, Richard; Lee, Brendan
Joel David Eisenhofer
Department of Neuroscience
Advisor: Richard De La Garza, Ph.D.-Department of Psychiatry & Behavioral Sciences
Individual exercise sessions (usually 10-30 min duration) have been shown to attenuate
cravings to smoke and withdrawal symptoms in humans, though trials lasting several weeks
have not consistently shown reductions in craving or improvements in abstinence. Key
limitations of those trials include lack of sufficiently intense exercise interventions, lack of
exercise adherence measurement, and change in physical activity. In this study, we evaluate
the effects of treadmill exercise on basic fitness measures as well as objective and subjective
measures of nicotine use and craving in individuals with concurrent cocaine and tobacco-use
disorder. Cocaine-dependent cigarette smokers (N=24) were randomized to running or walking
(30 min per session, 3 times per week) or sitting (placebo condition; same amount of time each
day) for 4 consecutive weeks. The intensity of daily exercise for Runners and Walkers was
calculated from each participant’s maximum heart rate (HR) during a treadmill test conducted
while screening. Computerized cognitive behavioral therapy and contingency management were
also given to all participants. Fitness measures included changes in body weight and resting
HR. Objective measures for nicotine use included breath carbon monoxide (CO) and salivary
cotinine. Subjective measures included changes in craving and motivation to quit smoking.
Changes in cocaine use and craving are presented elsewhere (ACNP 2014).
There were no differences in demographic or drug use variables among Runners
(N=10), Walkers (N=7) and Sitters (N=7). On average, participants were Black (71%), male
(80%), 44.7±1.1 (Mean ± S.E.M) years of age, and reported using cocaine for ~20 years.
Participants smoked 12.4±1.8 cigarettes per day (CPD) for 26.4±2.1 years and had FTND
scores of 4.5±0.6. At baseline, participants exhibited moderate scores (scale of 100) for
Motivation to Quit smoking (56.5±6.8) and Confidence to Quit smoking (44.8±5.8).
Across the 4-week study, exercise improved fitness measures including reducing body
weight (-11.3±8.5, -4.0 ±2.9, +2.7±2.3 pounds; p=0.28) and decreasing resting HR (-3.3±4.1,
+5.2±2.6, +9.1±3.0 bpm; p=0.05). Though not statistically significant, exercise reduced
Questionnaire of Smoking Urges scores (-5.2±3.4, -18.4±5.5, -6.1±2.9, p=0.06), current craving
for nicotine (VAS scale of 100)(-11.3±9.5, -20.2±9.9, +1.4±5.5, p=0.25) and CPD (-2.0±2.1, 1.6±2.1, -0.5±1.8, p=0.82). Unexpectedly, exercise reduced Motivation to Quit smoking (33.3±7.0, -3.6±16.9, +7.6±5.1, p=0.03) and reduced CO in Controls to a larger extent than
Runners and Walkers (-1.7±1.5, +2.7±1.8, -5.9±1.9 ppm, p=0.01).
The data show significant improvements in basic fitness measures though significant
reductions in nicotine use and craving were not observed. It is established that cocaine users
are 3-4 times more likely than their non-drug-abusing counterparts to smoke cigarettes, and
smoking cigarettes appears to enhance craving and subsequent consumption of cocaine,
making this a particularly challenging group to treat. Larger sample sizes and distinct exercise
regimens should be evaluated for their potential to reduce smoking in individuals with
concurrent cocaine and tobacco-use disorder.
Contributors: Eisenhofer, Joel D.; Thompson-Lake, Daisy G.Y.; Debrule, Daniel S.; Makanjuola,
Titi; Rodgman, Chris; Newton, Thomas F.; Yoon, Jin H.; De La Garza II, R.
Charlene H Emerson
Department of Molecular & Human Genetics
Advisor: Alison Bertuch, M.D./Ph.D.-Department of Pediatrics
The evolutionarily conserved Ku heterodimer is a DNA end-binding (DEB)
complex involved in both telomere maintenance and non-homologous end-joining
(NHEJ). In Saccharomyces cerevisiae, the Yku70/Yku80 subunits bind DNA ends via a
channel structure. We previously generated DEB defective Ku mutants by mutating
residues in the DNA-binding channel, revealing that DEB is re uired for Ku’s telomeric
functions. Paradoxically, we also found that these mutants demonstrated increased
colony survival in an assay that probes for imprecise NHEJ (Lopez et al. 2011, PLoS
Genet). A subsequent study also generated DEB defective mutants of Ku by deleting
residues to constrict the DNA binding channel (Pfingsten et al. 2013, Cell). These
mutants similarly demonstrated a re uirement for Ku’s DEB activity at telomeres.
However, these mutants were defective for imprecise NHEJ. To reconcile the
differences between the two studies, we generated additional DEB defective Ku
heterodimers and found that they too had increased imprecise NHEJ and defective
telomere function. We also identified a novel region in Yku70 that is required solely for
NHEJ and is predicted to associate with residues deleted in the DEB mutants reported
by Pfingsten et al. We propose that these results reconcile the imprecise NHEJ
differences observed and support our hypothesis that Ku heterodimers solely defective
for DEB can modulate NHEJ off DNA ends. Currently, we are assaying whether the
DEB mutants bind to double strand breaks (DSBs) in vivo. We expect that DEB mutants
will be unable to bind DSBs, suggesting a previously unknown role for Ku in imprecise
Contributors: Emerson, Charlene; Lopez, Christopher; Ribes-Zamora, Albert; Bertuch, Alison
Joseph Mario Fachini
Department of Molecular Physiology & Biophysics
Advisor: Joel Neilson, Ph.D.-Department of Molecular Physiology & Biophysics
In our studies of post-transcriptional gene regulation in human T cells, we
observed a significant upregulation of KRAS protein expression, independent of mRNA
levels, upon antigen receptor stimulation in both primary human T cells and the Jurkat
E6.1 cell line. Through the utilization of polysome fractionation facilitated assays, we
show that KRAS is regulated at the level of translation. Additionally, we have
systematically investigated putative regulatory elements and mechanisms of KRAS
post-transcriptional regulation by the use of luciferase-based KRAS 3’ and 5’
untranslated region (UTR) reporter assays and poly(A) tail length assays. Through
these approaches, we report that the induction of KRAS protein expression upon T cell
activation is 3’ UTR mediated. By 3' UTR mapping via luciferase reporter assays, we
have narrowed the region containing the cis-elements responsible for KRAS posttranscriptional regulation to approximately 1 kb. While several microRNAs have been
implicated in the post-transcriptional regulation of KRAS, we show that the regulation of
KRAS in our system is independent of both let-7 family microRNAs and miR-143.
Through computational analysis of the mapped region, we identified HuR as a regulator
of KRAS. By employing RNA-IP, luciferase reporter knockdown assays as well as
deletional assays, we now demonstrate that HuR both binds to and induces
translational upregulation of KRAS in human T cells post-T cell receptor mediated
cellular activation.
Contributors: Fachini, Joseph; Chaudhury, Arindam; Neilson, Joel
River Uru Faille
Department of Pathology & Immunology
Advisor: Scott Wenderfer, M.D.-Department of Pediatrics
Circulating immune complexes (IC)s deposit in the glomerulus of the kidney in
many autoimmune diseases, including systemic lupus erythematosus (SLE), leading to
proliferative glomerulonephritis and inflammation. IC binding can promote proliferation
or induce apoptosis in human macrovascular EnCs, but the effects on microvascular
EnCs such as those in the glomeruli are unknown. Tumor Necrosis Family (TNF)
members regulate proliferation and apoptosis and are found in endothelial cells. TNF
superfamily member 10 (Tnfsf10/TRAIL) is up-regulated in the kidneys of patients with
IC disease. Membrane bound and soluble forms of TRAIL bind to TRAIL-R2/DR5 to
induce apoptosis in some EnCs. TNF associated protein 3 (Tnfaip3/A20), is antiapoptotic, negatively regulating TRAIL/DR5. Polymorphisms in Tnfaip3 have been
positively associated with disease in SLE. We hypothesize that TRAIL induces
apoptosis in surrounding infiltrating leukocytes and that A20 blocks apoptosis in REnCs.
Using qRT-PCR, we find that mRNA levels of these two TNF family members are upregulated in renal endothelial cells in vitro in the presence of IC binding and in vivo
using a murine aCIC model . Using a Cellular ELISA for Membrane TRAIL, we show a
stepwise increase in expression in REnCs upon treatment with ICs in a dose dependent
manner. Using XTT and CFDA cell proliferation assays, findings suggest increased
proliferation of cells treated with ICs. Future directions include using semi-quantitative
western blots to measure A20 and soluble TRAIL expression levels in REnCs before
and after exposure to IC. We will also study the role of IC binding to REnCs in
regulation of DR5 signaling, caspase activity, and apoptosis, as well as the role of TNF
superfamily members in proliferation and apoptosis. Finally, using co-culture systems,
we will assess the importance of IC induced Trail production by REnCs on the
proliferation or apoptosis of the leukocytes commonly found in inflammatory
Contributors: Suwanichkul, Adisak; Wenderfer, Scott E.
Timothy Farinholt
Department of Biochemistry & Molecular Biology
Advisor: Adam Kuspa, Ph.D.-Department of Biochemistry & Molecular Biology
The social amoeba Dictyostelium discoideum is a model for studying bacteriaamoebae interactions that shape bacterial virulence. These initially solitary amoebae
feed by consuming bacteria, but when starved, they lose their ability to consume
bacteria and enter a 24-hour developmental cycle culminating in a fruiting body. During
the migratory slug stage of development we have discovered a specialized population of
Sentinel (S) cells that release structures similar to neutrophil extracellular traps (ETs)
that ensnare and kill bacteria through an unknown mechanism (Chen et al., Science
317:678-81). ETs are comprised of mitochondrial DNA coated with antimicrobial
proteins. We purified ETs and analyzed them by mass spectrometry, identifying several
proteins, including CadA. Using available polyclonal and monoclonal anti-CadA
antibodies we confirmed the presence of CadA on ETs. Amoeba lacking CadA protein
(cadA-null) displayed variable growth on Klebsiella pneumoniae, which suggests that
CadA is necessary for the killing of bacteria during growth and developmental phases of
the Dictyostelium life cycle. Single wild type amoebae grown on a lawn of bacteria clear
the bacteria in uniformly sized plaques. Single cadA-null amoeba cells form plaques of
widely varying sizes that suggests a deficiency in the first few cell divisions of colony
formation. Our lab’s data suggests Dictyostelium kills bacteria extracellularly before
phagocytosis. Initial data suggest cadA-null amoebae are deficient in extracellular killing
of bacteria. The crystal structure of CadA reveals an immunoglobulin-like and a gammacrystallin-like domains connected by a short linker sequence (Lin, Nat. Struct. Mol. Bio.
13(11):1016-22). Gamma-crystallin domains are found in plant antimicrobial proteins as
well as yeast killer toxin (Huchinson, Protein Eng. 6(3):233-45). I am currently testing
the potential role of CadA in the killing of bacteria by Dictyostelium discoideum. I will
add exogenously purified CadA to bacteria to directly test its antimicrobial function.
Purified CadA will be added to our extracellular killing assay to observe if it rescues the
killing of bacteria in cadA-null amoebae. We hope to garner new insights into the ET
mechanism for killing bacteria in eukaryotes.
Contributors: Farinholt, Timothy; Zhuchenko, Olga
Viktor Feketa
Program in Cardiovascular Sciences
Advisor: Sean Marrelli, Ph.D.-Department of Anesthesiology
Background: Therapeutic hypothermia is an effective and rapidly developing
method of neuroprotection from ischemia. It is being increasingly used for the treatment
of cardiac arrest and explored in stroke and traumatic brain injury. However, current
methods of inducing hypothermia based on physical cooling have serious limitations,
such as shivering, impaired consciousness, and respiratory depression. Novel
approaches to lowering temperature in conscious patients are highly needed. One of
such approaches involves pharmacological manipulation of the molecular
thermosensors, which allows modulating the activity of the thermoregulatory system and
may result in a regulated change in body temperature. In particular, agonists of the
TRPV1 ion channel, also known as the heat and capsaicin receptor, are well known to
induce hypothermia in numerous mammalian species. We aimed to explore if TRPV3
channel, which is closely related to TRPV1, may be targeted pharmacologically to
achieve hypothermia. Similarly to TRPV1, TRPV3 is activated by above-neutral
temperatures, expressed in peripheral tissues and was shown to be involved in
transmitting temperature information to the cortex. These properties suggest that
TRPV3 may also provide afferent input to the thermoregulatory system. However, this
has not been yet conclusively established. We hypothesized that TRPV3-expressing
sensory neurons functionally belong to the skin-warming afferent pathway, and that their
activation by TRPV3 agonists provides spurious skin-warming signal to the
thermoregulatory system, shifts it to less heat-generating and more heat-dissipating
state, and leads to hypothermia in conscious subjects.
Methods: We have determined the effects of systemic administration of TRPV3
agonist carvacrol in conscious mice on core temperature, measured by implanted
telemetric transmitters, and on energy expenditure, measured by indirect calorimetry
using the Comprehensive Laboratory Animal Monitoring System.
Results: Intraperitoneal injection of carvacrol at 31.6 mg/kg b.w. led to ~2.5°C
greater drop in core temperature after 30 min, compared to vehicle injection. This
hypothermic effect was associated with a ~1.6-fold decrease in energy expenditure.
Conclusions: Our findings support the working model, wherein TRPV3expressing neurons provide temperature input to the thermoregulatory system, and
when activated by TRPV3 agonists, modulate the thermoeffector processes, lower
whole-body heat generation, and lead to a decrease in core temperature in conscious
mice. If further confirmed, this model suggests that TRPV3 agonists may be used as
novel agents for induction of therapeutic hypothermia.
Contributors: Marrelli, Sean.
Xiang Feng
Department of Pharmacology
Advisor: Patrick Barth, Ph.D.-Department of Pharmacology
TransMembrane Helical (TMH) domains of integral membrane proteins play
critical roles in diverse physiological functions, such as the signal transduction,
bioenergetics and ion transport, and are thus important drug targets. The lack of
structural information on these proteins, however, hinders our understanding of their
functional regulation and prevents the rational design of selective therapeutics.
Computational modeling techniques represent important alternative approaches but
currently lack the efficiency and accuracy required to consistently predict membrane
protein structures at atomic resolution. Understanding the sequence/structure
determinants controlling the packing of TMHs is a major step in accurately modeling
and designing TMH proteins but such determinants has not yet been characterized for
multiple TMH assemblies.
We conducted a bioinformatics analysis of the sequence/structure relationships
defining the packing of three TM helices. We generated a library of more than 800
closely packed TMH trimer structures from X-ray TM structures. Based on structure
similarity, we found that more than half of the trimers can be classified into only 6 major
clusters with distinct geometrical/topological features. We identified statistically enriched
sequence motifs that correlate with the geometric features of each cluster. The motifs
were used to train an SVM-based predictor of TMH trimer topologies from sequence
that achieved up to 4-fold improvement over random selection, highlighting the
significance and specificity of the motifs. Furthermore, enriched sequence/structure
motifs share similar interaction patterns in diverse protein families, suggesting the
existence of convergent determinants in TMH trimer packing. Analysis of residue
conservation or co-evolution and in silico alanine scanning indicates that most motifs
create energetically important contacts that are under evolutionary pressure. Structural
analysis of atomic contacts mediated by these motifs uncovered novel consensus
physical interactions that are unique to trimer and not found at dimer TMH interfaces.
Our results indicate that a limited number of local sequence/structure motifs can
recapitulate a large fraction of TMH trimer structures. These findings provide important
novel insights into the sequence/structure determinants governing the packing of
multiple TM helices and may guide the prediction and design of complex TMH
Contributors: Feng, Xiang; Barth, Patrick
Lauren Renee Figard
Integrative Program in Molecular and Biomedical Sciences
Advisor: Anna Sokac, Ph.D.-Department of Biochemistry & Molecular Biology
Morphogenesis is driven by cell shape change, which often requires cell surface
growth. Key aspects of this remodeling are still mysterious: What is the source of
membrane for cell surface growth? How is membrane transferred to the site of growth?
How is membrane transfer regulated? To address these questions, we study Drosophila
cellularization, a dramatic tissue-building event that expands the embryo’s surface area
by ~25 fold. We previously showed that microvilli unfold to provide membrane for
cleavage furrow ingression during cellularization. We showed that microvillar
disassembly is controlled by furrow ingression, and that microvillar membrane slides
along the plane of the cell surface into ingressing furrows (Figard et al., 2013). These
results provide the first direct evidence for plasma membrane unfolding, which has long
been suspected as a broadly conserved mechanism of cell surface growth. But how
does furrow ingression drive unfolding and the necessary disassembly of microvillar Factin cores? We propose that furrow pulling increases plasma membrane tension,
antagonizing F-actin polymerization in microvilli and ultimately causing them to unfold.
To validate this proposal, first, we used fluorescence recovery after photobleaching
(FRAP) and drug studies to show that microvillar F-actin is constantly polymerizing and
depolymerizing. Second, we used 3D time-lapse imaging to show that microvillar F-actin
is depleted in sync with furrow ingression. Third, we analyzed a genetic mutant for
furrow ingression and found that furrow ingression controls the depletion of microvillar
F-actin. Fourth, we used physical force assays to show that furrow ingression exerts a
pulling force that is transmitted to far-away microvillar F-actin. In all cells, the surface
folds holding the largest membrane stores will likely be supported by F-actin. Our
results suggest that plasma membrane tension is a critical regulator of unfolding, acting
on F-actin to control the liberation and transfer of membrane during cell surface growth.
Contributors: Figard, Lauren Renee; Sokac, Anna Marie
Olivia Michelle Fitch
Department of Neuroscience
Advisor: Michael Friedlander, Ph.D.-Department of Neuroscience
Mauro Costa-Mattioli, Ph.D.-Department of Neuroscience
In the neocortex, individual cells of like type can undergo heterogeneous
plasticity responses from depression (LTD) to potentiation (LTP), or no change (NC) in
response to a common fixed time delay synaptic conditioning protocol. However it is
not known whether all synapses onto a common cell have the same plasticity outcome.
Nor is it known what role visual experience plays in shaping the distribution of
differential plasticity outcomes. Thus, we evaluated the synaptic plasticity responses of
separable sets of synaptic inputs onto common postsynaptic neurons in primary visual
cortex in response to simultaneous stimulation of distinct sets of afferents in acute brain
slices from visually intact or binocularly deprived mice (deprived from before the natural
time of eye-opening). The two stimulation sites were isolated by occlusion testing
followed by alternative activation of each pathway to evoke a postsynaptic potential
(PSP) every 10 seconds in an interleaved fashion. After a stable ten minute baseline
period, the activation of both pathways was simultaneously paired with direct
postsynaptic activation that preceded the synaptic stimulation by 10 milliseconds
resulting in 3-7 postsynaptic spikes at 0.1 Hz over a 10 min period followed by reversion
to the interleaved stimulation protocol for an additional 30 minutes. The ratio of the
average amplitude of the evoked PSP post/pre conditioning was calculated for each
pathway taking the 5 minutes average peak amplitude over 25-30 minutes postconditioning compared to 5 minutes just before or pre-conditioning. Our results from
132 pathways inputs validate in the mouse cortex our previous findings from other
species demonstrating heterogeneous plasticity outcomes ranging from LTD to LTP for
individual cells with a median post/pre ratio of 0.84 in visually intact mice (both naïve
and anesthesia controls). Visually deprived mice did not show a significant difference in
the change of individual pathways with a mean 0.89 post/pre ratio for 63 pathways
(Rank Sum U-statistic=2027, p=0.424). However, there was relationship between two
pathways onto a common cell differed depending on the level of visual experience.
There was a weak correlation between two pathways in visually deprived mice (median
R=0.49, p=0.005, n=30 cells) and none in binocularly deprived mice (median R =
0.023, p = 0.9, n=31 cells) with a significant difference between correlation coefficients
(t-test, p=0.05) as measured by a linear fit to 10,000 permutations of ordinate and
abscissa designation of post/pre ratios of two pathways onto a common cell for each
treatment group created by Monte Carlo Permutation analysis.
Supported by NIH grant EY-12782 to MJF.
Contributors: Fitch, Olivia M; Friedlander, Michael J
SRC-2 Dynamically Regulates Gluconeogenesis and Glycolysis via Glucose-6phosphatase and Glucokinase
Tiffany C Fleet
Program in Translational Biology & Molecular Medicine/M.D.-Ph.D. Program
Advisor: Bert O'Malley, M.D.-Department of Molecular & Cellular Biology
Clifford Dacso, M.D./M.P.H.-Department of Molecular & Cellular Biology
Steroid Receptor Coactivator 2 (SRC-2) is a well-characterized transcriptional
coregulator of systems-wide energy homeostasis. Ablation of SRC-2 in mice protects
against diet-induced obesity, impairs glucose home¬ostasis, and disrupts efficient
dietary absorption of lipids. Our laboratory has defined SRC-2 as a transcriptional
coactivator for RORα on the Glucose-6-phosphatase (G6pc) promoter, a rate-limiting
enzyme in gluconeogenesis. Loss of SRC-2 results in decreased G6pc expression,
accumulation of liver glycogen, and fasting hypoglycemia. In addition to the fasting
hypoglycemia, SRC-2-/- mice have prandial hyperglycemia after a 24 hour fast. We
determined that expression of Glucokinase (Gck), a rate-limiting step in glycolysis, is
also decreased in SRC-2-/- mice. Therefore, we hypothesize that SRC-2 regulates the
switch between gluconeogenesis and glycolysis by regulating expression of G6pc
during fasting conditions and Gck during feeding conditions. To better understand the
role of SRC-2 in regulating glucose metabolism, we surveyed SRC-2 cistrome
occupancy on the G6pc and Gck promoters. To determine the coregulator complex that
assist in this dynamic regulation, we next made biotinylated PCR fragments of both
G6pc and Gck promoters for a DNA pull-down assay. The biotinylated promoters were
incubated with hepatic nuclear extract containing transcriptional machinery from WT
and SRC-2-/- mice that were either fasted for 24 hours or fasted and refed for 3 hours.
To assess the dynamics of the coregulator complexes formed on the G6pc and Gck
promoter regions, we performed proteomic analysis of the DNA pull-down via mass
spectrometry and immunoblotted selected targets. We found that SRC-2 is involved in
differential recruitment of coregulator complexes in a metabolic and promoter sequence
specific manner.
Contributors: Fleet, Tiffany; Zhang , Bin, Stashi , Erin; Jung , Sung Yun; Rajapakshe, Kimal;
Dean, Adam; Gonzales, Naomi; Foulds, Charles; Coarfaor, Christian; Qin, Jun; York, Brian;
O’Malley, Bert.
Christopher James Foley
Department of Molecular & Cellular Biology
Advisor: Nicholas Mitsiades, M.D./Ph.D.-Department of Medicine
The focus of prostate cancer research and treatment has historically been on
targeting the androgen receptor (AR) signaling axis, as the anticancer activity of AR
inhibition (via chemical castration or anti-androgens) has been well established. Despite
this, if given enough time, prostate cancer will almost always recur as a castrationresistant prostate cancer (CRPC), which can metastasize and grow despite anti-AR
treatments. Although several mechanisms have been proposed to explain this transition
to CRPC, it is clear that targeting only the AR signaling axis is insufficient. The steroid
receptor coactivators (SRCs) are a family of coregulators that play key roles in
transcription. Overexpression of the SRCs has been implicated in numerous cancer
types – including prostate. To understand the role SRCs play in CRPC, we performed
Chromatin Immunoprecipitation-sequencing (ChIP-Seq) for AR, SRC-1, SRC-2 and
SRC-3 in an androgen-dependent prostate cancer cell line and its androgenindependent (CRPC) subclone. We observed a substantial increase in total cellular
protein levels and in the number of chromatin binding sites for both SRC-2 and SRC-3,
as well as a transition away from co-localization with AR chromatin binding, in our
CRPC model compared to its androgen-dependent parental cells. We integrated these
results with gene expression profiles of cells treated with siRNA against SRC-2 and
SRC-3, and identified a set of genes that is regulated by SRCs specifically in our CRPC
model. These SRC-dependent, CRPC-specific genes included the E3 ubiquitin ligase Sphase kinase-associated protein 2 (SKP2), a known oncogene that is overexpressed in
CRPC patient samples. Silencing of SRC-2 or SRC-3 or SKP2 via siRNA exerted
greater anticancer activity against our CRPC model than its androgen-dependent
parental cell line. In agreement with this increased dependency of CRPC cells on SRC2 and SRC-3, we found that a small molecule inhibitor (SMI) of the SRCs, which was
identified via screening of a library of ~360,000 compounds, exerted greater anticancer
activity against CRPC than androgen-dependent parental cells. Both SRC-3 siRNA and
the SRC SMI suppressed SKP2 expression specifically in CRPC cells. Furthermore,
recruitment of SRC-3 to the SKP2 promoter was suppressed by the SRC SMI. Our data
suggest that SRC-2 and SRC-3 contribute to prostate cancer progression to CRPC.
Targeting the SRCs with SMIs is feasible and promises an innovative therapeutic
approach for patients with CRPC.
Contributors: Foley, Christopher; Lanz, Rainer; Fiskus, Warren; Geng, Chuandong; Shou, John;
He, Bin; Shah, Shrijal; Chew, Sue Ann; Coarfa, Cristian; O’Malley, Bert W.; Mitsiades, Nicholas
Andrew Kenji Folick
Program in Developmental Biology/M.D.-Ph.D. Program
Advisor: Meng Wang, Ph.D.-Department of Molecular & Human Genetics
Lipids are known for their roles in energy storage and cellular architecture, but
they also act as signaling molecules involved in the regulation of gene expression and
signal transduction. Although fat storage and metabolism have been associated with
metabolic health and aging, the role that lipid signaling plays in the regulation of
longevity has not been elucidated. Recent work has implicated autophagy and
lysosomal function as involved in the regulation of longevity. Here, we explore the
relationship between lysosomal function and lipid signaling in aging by characterizing
the role of lipl-4, a homolog of human lysosomal acid lipase, as a novel regulator of
longevity in Caenorhabditis elegans, and show that constitutive expression of lipl-4 in
intestinal fat storage tissue decreases fat storage and increases both mean and
maximum lifespan. Increased expression of lipl-4 promotes the expression and
lysosome-to-nuclear translocalization of the lipid chaperone LBP-8. LBP-8 promotes the
activation of transcription by a nuclear hormone receptor complex of NHR-49 and NHR80, and over-expression of LBP-8 is sufficient to promote longevity, dependent on NHR49 and NHR-80.
Using high-throughput metabolomic analysis, we identified several potential lipid
messengers with increased abundance in animals over-expressing lipl-4. Among these,
oleoylethanolamide bound to both LBP-8 and NHR-80 in vitro, and feeding
oleoylethanolamide to worms was sufficient to promote NHR-49/NHR-80 dependent
transcription. Furthermore, feeding oleoyethanolamide was sufficient to increase mean
lifespan, dependent on NHR-80. In summary, we have identified a new pathway by
which lipid catabolism in the lysosome signals to the nucleus to regulate lifespan, and
identified a potential longevity-promoting lipid metabolite.
Contributors: Folick, Andrew; Oakley, Holly; Yu, Yong; Armstrong, Eric H; Kumari, Manju; Sanor, Lucas; Moore, David
D.; Zechner, Rudolf; Ortlund, Eric A.; Wang, Meng C.
Michael David Fountain
Program in Translational Biology & Molecular Medicine
Advisor: Christian Schaaf, M.D./Ph.D.-Department of Molecular & Human Genetics
Daryl Scott, M.D./Ph.D.-Department of Molecular & Human Genetics
The Prader-Willi syndrome (PWS) critical domain is a maternally imprinted, paternally
expressed locus, which contains five protein coding genes and a family of six small nucleolar
RNA (snoRNA) genes or clusters. However, the roles of each of these genes and snoRNAs in
relation to PWS associated phenotypes remain largely unknown. Most recently, four individuals
with major features of PWS were reported to carry truncating mutations in their paternal copy of
MAGEL2, a protein-coding gene in the PWS critical domain. All of these individuals had
previously been diagnosed with autism spectrum disorder (ASD). We hypothesize that loss of
functional MAGEL2 predisposes to ASD, and that mutations in proteins interacting with
MAGEL2 may also lead to neurocognitive and neuropsychiatric phenotypes.
Specific Aim 1: To investigate whether Magel2 null mice display autism-like behaviors.
Previously, Magel2 null mice were generated and evaluated for key behaviors and
characteristics of PWS. However, they have not yet been evaluated for autism-like behaviors.
We propose to assess male and female Magel2 null mice for autism-like behaviors, using a
battery of validated, well-established neurobehavioral tests. Test animals will be compared to
age- and sex-matched wild-type littermates, analyzed in parallel. The behavioral assessment
will include evaluations for communication deficits, social recognition, social interaction, social
dominance, repetitive behaviors and stereotypies, as well as learning and memory.
Specific Aim 2: To identify and investigate the pathogenic significance of mutations in
MAGEL2 interacting proteins. Recently, MAGEL2 was shown to form a functional complex with
TRIM27 and USP7, which then activates the WASH complex (WASH, FAM21, CCDC53, SWIP,
STRUM) in a ubiquitin-dependent fashion. The activation of the WASH complex plays a critical
role in endosomal actin nucleation and protein trafficking. Due to their close physical and
functional relationship, we propose that mutations in MAGEL2 interacting proteins may have
similar phenotypic consequences as MAGEL2 loss-of-function itself. We queried databases of
>100,000 individuals who had clinical array comparative genomic hybridization (aCGH) testing,
and databases of >5,000 individuals who underwent whole exome sequencing at Baylor College
of Medicine, and successfully identified a first set of individuals with loss-of-function mutations in
MAGEL2 associated genes. Primary phenotypes from this query were intellectual disability,
developmental delay, aggressive behavior, seizures, and autism. To investigate the significance
of the respective mutations, lymphoblast and fibroblast cell lines will be established from the
affected patients, which will allow us to assess the functional significance of these mutations on
the MAGEL2-TRIM27-USP7 complex and the WASH complex.
Contributors: Fountain, Michael; Schaaf MD PhD, Christian
Kristen Nicole Fousek
Program in Translational Biology & Molecular Medicine
Advisor: Nabil Ahmed, M.D.-Department of Pediatrics
Helen Heslop, M.D.-Department of Pediatrics
CAR T cells have shown promising results for several cancers in pre-clinical
models as well as in early phase clinical trials. Throughout these studies it has been
observed that first generation CAR T cells demonstrate successful killing of tumor cells
but are unable to proliferate sufficiently while second generation CAR T cells are able to
kill tumor cells as well as proliferate and exhibit improved survival in vitro. Furthermore,
third generation CAR T cells display enhancements in cytotoxicity, proliferation, and cell
survival, however, there has not yet been a comprehensive investigation that
interrogates the correlation of signaling patterns with CAR endodomains and
functionality thereof. Substantial knowledge on TCR signaling exists, and the pathways
downstream of the TCR/CD3 zeta chain as well as those of the CD28, 4-1BB, and
OX40 co-stimulatory molecules on canonical T cells are well understood. In this project I
propose to build upon this T cell signaling knowledge and investigate why distinct CAR
moieties confer enhanced functionalities to T cells by analyzing the activation of
phosphoproteins involved in key signaling pathways on a molecular level. I plan to do
this through conducting the following specific aims:
Aim 1: To construct and validate the phenotype and function of Jurkat and
primary human T cells expressing CARs comprised of permutations of the ζ, CD28, 41BB, and OX40 signaling moieties.
Aim 2: To investigate trends in activation of surrogate nodes involved in distinct
signaling pathways downstream of the co-stimulatory domains in first-, second-, and
third-generation CARs.
I hypothesize that encounter and binding of specific target antigens will activate
CAR signaling molecules in a dose-dependent fashion and generate distinct quantifiable
differences in the activation of surrogate nodes within the signaling pathways,
culminating in correlative changes in the effector functions of CAR T cells.
Contributors: Fousek, Kristen, Byrd, Tiara, Heczey, Andras, Varadarajan, Navin, Heslop, Helen,
Gottschalk, Stephen, Dotti, Gianpietro, Hegde, Meenakshi, Ahmed, Nabil
Pablo Riera Freire
Department of Molecular & Cellular Biology
Advisor: Orla Conneely, Ph.D.-Department of Molecular & Cellular Biology
Hematopoiesis is a dynamic biological process that requires tight coordination
between the differentiation and proliferation of hematopoietic stem cells (HSC) and
progenitors. At the cellular level, these processes are controlled by transcriptional and
epigenetic mechanisms that regulate the correct gene expression program for each
blood cell maturation stage. Our group has previously shown that the members of the
NR4A nuclear receptor family act as tumor suppressors in the hematopoietic system,
and germline deletion of Nr4a1 and Nr4a3 in mice causes death by acute myeloid
leukemia (AML) within 2-4 weeks after birth. However, the cellular and molecular
mechanisms by which NR4As regulate hematopoietic cell development remain poorly
understood. To disclose these mechanisms, we have used a tamoxifen-inducible
Rosa26-Cre-ERT2; Nr4a1fl/fl; Nr4a3-/- mouse model to examine the cellular and
molecular consequences of acute codepletion of Nr4a1 and Nr4a3 on hematopoietic
stem (HSC) and progenitor cell homeostasis in adult mice prior to the development of
AML. We have previously confirmed that temporally restricted depletion of both genes
using this approach leads to development of AML in adult mice within 12 to 15 weeks
after Nr4a1/3 ablation. Expression profiling of NR4A1/3 revealed highest levels of
expression of both genes in the most primitive HSC population with lower levels in the
myeloid progenitors. Consistent with these observations, we find that acute deletion of
NR4A1/3 leads to a rapid increase (within four days) in the frequency of the most
primitive HSC and multipotent progenitor (MPP) populations. Proliferation analysis
revealed that accumulation of HSCs and MMPs was associated with abnormal
activation of cell cycle in both populations. The same analyses were repeated four
weeks after tamoxifen treatment, and we observed a depletion of the HSC compartment
and an accumulation of the granulocyte-monocyte progenitor population. The cell cycle
remained abnormally activated in the HSC and MPP, indicating that the excessive
proliferation caused a reduction of the stem cell pool. We are currently utilizing a
systems approach to identify global NR4A1/3 transcriptional targets by integrating ChIPSeq and RNA-Seq analyses in the rare long-term HSC. We expect these analyses to
uncover new mechanisms controlling normal adult hematopoiesis and neoplasia.
Contributors: Freire, Pablo; Conneely, Orla
Emmanouil Froudarakis
Department of Neuroscience
Advisor: Andreas Tolias, Ph.D.-Department of Neuroscience
Neural codes are believed to have adapted to the statistical properties of the
natural environment. However, the principles that govern the organization of ensemble
activity in the visual cortex during natural visual input are unknown. We recorded
populations of up to 500 neurons in the mouse primary visual cortex and characterized
the structure of their activity, comparing responses to natural movies with those to
control stimuli. We found that higher order correlations in natural scenes induced a
sparser code, in which information is encoded by reliable activation of a smaller set of
neurons and can be read out more easily. This computationally advantageous encoding
for natural scenes was state-dependent and apparent only in anesthetized and active
awake animals, but not during quiet wakefulness. Our results argue for a functional
benefit of sparsification that could be a general principle governing the structure of the
population activity throughout cortical microcircuits.
Contributors: Froudarakis ,Emmanouil; Berrens, Philipp; Cotton, R. James; Ecker, Alexander;
Sinz, Fabian H.; Yatsenko, Dimitri; Saggau, Peter; Bethge, Matthias; Tolias, Andreas S.
Daniel G Fuja
Integrative Program in Molecular and Biomedical Sciences/M.D.-Ph.D. Program
Advisor: Jason Yustein, M.D./Ph.D.-Department of Pediatrics
Osteosarcoma (OS) is the most common form of primary bone cancer. Age of
OS incidence follows a bimodal distribution with a higher incidence in pediatric and
adolescent patients. It is found primarily in long bones such as the femur and humerus,
with metastases most often arising in the lung or other bony sites. Metastatic spread of
OS from the primary tumor to distant metastatic sites drastically decreases survival rate.
Current treatment of metastatic OS still largely parallels that of the non-metastatic
disease, usually consisting of surgical resection of the primary tumor and adjuvant
Treatment of metastatic OS would be facilitated by a better
understanding of factors driving osteosarcoma metastasis. Metastatic progression is
often enabled through altered expression of genes affecting motility, invasive potential,
immune evasion, and other significant functions. We hypothesize that specific genetic
and molecular changes are required for OS cells to metastasize from the primary tumor
and colonize a distant, physiologically distinct site.
Because chemotherapy is begun immediately following diagnosis of OS, there is
limited access to samples of lung metastases which have not already been exposed to
chemotherapeutic regimen and, thus, some selection. In order to investigate untreated
lung metastasis, our lab has developed genetically engineered mouse models (GEMMs)
which phenocopy either metastatic or non-metastatic osteosarcoma. Using highthroughput genome-wide microarray, we screened RNA isolated from GEMM-derived
primary tumors and their corresponding metastatic lesions for genes significantly
altered. These microarrays were subsequently repeated with cell lines derived from
corresponding primary and metastatic lesions. Several factors exhibited higher mRNA
levels in lung metastases than their corresponding primary tumor, both in tissue and cell
line mRNA. Statistical analysis and stringent filtering yielded high-probability candidates
which were subsequently corroborated through multiple-sample quantitative PCR
(qPCR). Some cell line protein levels were also checked through Western Blot to
confirm upregulation. Corresponding data in human-derived samples were analyzed
and confirmed upregulation of Gata6 and TG2. Analysis via Ingenuity Pathway Analysis
(IPA) showed important connections to immune evasion, cell motility, and other prometastatic survival functions for two of the consistently-upregulated factors: Gata6 and
Transglutaminase 2 (TG2). These pro-metastatic mechanisms may be required for OS
dissemination, colonization and metastatic tumor growth. Functional studies indicate
that alterations in Gata6 and TG2 affect cellular phenotypes which might facilitate
metastatic disease through multiple functions. Anticipated in vivo experiments will
demonstrate the extent of metastatic enhancement. Contributors: Fuja, Daniel;
Kurenbekova, Lyazat; Roos, Alison; Gao, Yang; Donehower, Lawrence A.; Yustein, Jason T.
Kenichiro Fujiwara
Integrative Program in Molecular and Biomedical Sciences
Advisor: Lawrence Donehower, Ph.D.-Department of Molecular Virology & Microbiology
A mammalian cell undergoes the DNA Damage Response (DDR) after
encountering various types of genotoxic stresses, including ionizing radiation, which
results in single or double-stranded breaks in the DNA. The sensor kinases ATM and
ATR detect this damage and activate downstream effectors through phosphorylation
cascades to promote various cellular responses, including cell cycle arrest. Once DNA
repair is complete, the expression of Wild-type p53-induced phosphatase 1 (WIP1) is
induced to return the cell to a homeostatic state. WIP1 has been shown to
dephosphorylate and downregulate various DDR mediators and effectors, including p53,
CHK1, CHK2, ATM/ATR, MDM2, and H2AX.
WIP1 is amplified and overexpressed in numerous human cancers. WIP1
overexpression in cancer cells expressing wild-type p53 leads to suppression of p53
activity. WIP1 inhibition, either chemically or through the overexpression of an inhibitory
miRNA, has been shown to suppress tumor growth via induction of cellular senescence
or apoptosis. Thus, understanding the influence of WIP1 on various cellular pathways
involved in cancer is critical, since WIP1 has been suggested to be a potential
therapeutic target.
Currently, our knowledge of the function of WIP1 is mainly limited to its role in the
DDR pathway. Preliminary data from our lab suggests that WIP1 may play a role in cell
cycle regulation. Our findings suggest that WIP1 may target p27Kip1 (also known as
CDKN1B), a known inhibitor of CDK2 and associated cyclins that directly regulate the
cell cycle. Thus, we believe that WIP1 may play a more direct role in controlling the cell
cycle than has previously been suggested.
We hypothesize that WIP1 dephosphorylates p27Kip1 after completion of DDR to
promote cell cycle progression. To investigate the role of p27Kip1 in DDR, we are
examining the ability of WIP1 to target p27Kip1 S140, a site implicated in DDR signaling
by ATM, and testing the ability of WIP1 to promote cell cycle progression. Previous
results indicate that WIP1 dephosphorylates p27Kip1 S140 in vitro, and western blot
analysis suggests that this same site is phosphorylated by ATM in 293 cells. Studies
are currently underway that are designed to observe p27Kip1 S140 dephosphorylation
by WIP1 in cultured cells and to elucidate the mechanism for cell cycle regulation
through WIP1 modulation of p27Kip1. The significance of this study is the potential
discovery of additional cell cycle targets for cancer treatments that involve WIP1
Contributors: Fujiwara, Kenichiro; Donehower, Larry
Paul Thomas Fullerton
Department of Molecular & Human Genetics
Advisor: Martin Matzuk, M.D./Ph.D.-Department of Pathology & Immunology
Pancreatic cancer is fourth-leading cause of cancer death in the United States
with approximately 43,000 cases diagnosed each year. For patients who present with
locally advanced or metastatic disease, average survival times are less than one year
and there are no curative treatments. The transforming growth factor β (TGFβ) signaling
protein SMAD4 is lost in 75% of metastatic pancreatic cancers and has been associated
with poorer prognosis, suggesting that SMAD4 loss is an important event in the
development of aggressive, metastatic disease. However, the mechanisms by which
SMAD4 loss promotes the development of a highly aggressive disease are not well
understood. In our preliminary studies, we discovered that expressing SMAD4 in a
SMAD4-null human pancreatic cancer cell line inhibits proliferation and induces
senescence, suggesting that targeting the TGFβ/SMAD4 signaling pathway could be a
promising therapeutic strategy. We hypothesized that understanding the mechanisms
underlying the anti-proliferative effect of SMAD4 expression would enable the
development of new therapies. To facilitate a more detailed study, we generated an
inducible system for SMAD4 expression in the SMAD4-null human pancreatic cancer
cell line BxPC3. By comparing the expression profiles of this cell line with and without
SMAD4 at a variety of time points after induction of SMAD4 expression, we identified
candidate regulatory partners and targets of SMAD4 in pancreatic cancer. Our future
studies will validate these candidate genes and characterize their connection to the
tumor suppressive role of SMAD4, laying the groundwork for the development of
therapies targeting the TGFβ/SMAD4 signaling pathway.
Contributors: Fullerton, Paul
Robert Steven Fultz
Integrative Program in Molecular and Biomedical Sciences
Advisor: James Versalovic, M.D./Ph.D.-Department of Pathology & Immunology
Chronic intestinal inflammation reduces quality of life and is deleterious to
intestinal tissues. Due to the intimate relationship between the intestinal microbiota and
host intestinal tissues, probiotic therapies are an attractive approach for treating chronic
colitis, yet no such therapy has been proven effective in inducing remission and
maintenance of chronic intestinal inflammation. Currently, therapies with the TNF-α
directed monoclonal antibodies are indicated and effective for IBD refractory to
corticosteroids, but are prohibitively expensive for some patients and require long-term
administration [1]. In vitro, administration of the versatile, biogenic amine, histamine
reduces expression and production of TNF-α in human monocyte derived macrophages
[2]. The probiotic, lactic acid bacterium, Lactobacillus reuteri is a native inhabitant of the
mammalian gut and produces and secretes histamine. Administration of L. reuteri
ATCC 6475 to mice with TNBS-induced colitis ameliorates local and systemic markers
of inflammation in a histamine-dependent manner [†]. Production and secretion of
histamine by L. reuteri ATCC 6475 in the gut is, therefore, hypothesized to reduce colitis
by inhibiting TNF production from intestinal macrophages; however the mechanism by
which this occurs in not fully elucidated. A cell model for studying the effects of L.
reuteri ATCC 6475 on macrophages has been characterized. The mechanism by which
histamine signaling reduces TNF transcription in intestinal macrophages will next be
determined by assaying phosphorylation states of MAP kinase pathway proteins in
macrophages exposed to histamine in vitro, or L. reuteri ATCC 6475 in vivo.
Elucidation of this mechanism is essential for understanding the probiotic functions of
microbial-derived histamine and its clinical applications and limitations.
1. Blonski W, et al. (2011) Inflammatory bowel disease therapy: current state-ofthe-art. Current Opinion in Gastroenterology, 27:346-357.
2. Thomas C, et al. (2012) Histamine Derived from Probiotic Lactobacillus reuteri
Suppresses TNF via Modulation of PKA and ERK Signaling. PLoS ONE, 7(2):e31951.
†. Unpublished data.
Contributors: Fultz, Robert
Upasana Gala
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M.-Department of Molecular & Human Genetics
Autophagy helps deliver sequestered intracellular cargo to lysosomes for
proteolytic degradation and thereby maintains cellular homeostasis by preventing
accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila
designed to identify genes required for neuronal function and maintenance, we
identified multiple cacophony (cac) mutant alleles. They exhibit an age dependent
accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a
degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a
Drosophila voltage gated calcium channel (VGCC) that is required for synaptic vesicle
fusion with the plasma membrane and neurotransmitter release. Here, we show that cac
mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates
suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined
process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes very
similar phenotypes as the loss of cac, indicating that the VGCC is required for AVlysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved
as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to
autophagic defects in mice. Moreover, we find that CACNA1A is localized to the
lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to
a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, increasing
cytosolic calcium levels rescues lysosomal fusion defects in Cacna1a mutant neurons.
We present a model in which the VGCC plays a role in autophagy by regulating the
fusion of AVs with lysosomes through its calcium channel activity and hence functions in
maintaining neuronal homeostasis.
Contributors: Gala, Upasana; Tong, Chao; Tian, Xieujen; Nagarkar, Sonal; Yamamoto, Shinya;
Jaiswal, Manish; Sillitoe, Roy; Bellen, Hugo
Monica Laura Galaz-Montoya
Department of Biochemistry & Molecular Biology
Advisor: Theodore Wensel, Ph.D.-Department of Biochemistry & Molecular Biology
Beta adrenergic receptors are important for cardiovascular regulation and for
physiological responses to the hormones/neurotransmitters adrenaline and
noradrenaline. They are found in the nervous system and throughout the body and are
the targets of numerous widely used drugs. Their canonical signaling pathway involves
activation of adenylate cyclase (AC) and a rise in cyclic AMP (cAMP) levels which leads
to activation of cAMP-dependent protein kinase (PKA). Beta adrenergic receptors have
long been thought to activate distinct signaling pathways from those downstream of Gprotein coupled receptors which activate phospholipase C (PLC) and elevate
intracellular Ca2+. By monitoring of intracellular Ca2+ levels in real time using a
fluorescent indicator dye we found that an endogenous receptor in HEK-293 cells
responds to the adrenergic agonist norepinephrine, by a delayed rise in intracellular
[Ca2+]. The response is blocked by ICI 118,551, a selective antagonist for beta-2
adrenergic receptor (β2-AR), and the relative potency of agonists is isoproterenol >
epinephrine > norepinephrine, consistent with the pharmacological profile of β2-AR.
Treatment with thapsigargin (an inhibitor of the SERCA Ca2+ pump of the endoplasmic
reticulum) and chelation of extracellular Ca2+ revealed that the Ca2+ is released from
intracellular stores. The release is sensitive to inhibition of PLC with U73122 and of
InsP3 receptors with 2-APB. Treatment with cholera toxin, a drug that activates Gαs,
indicated that direct activation of this G protein is not sufficient for Ca2+ release.
Increasing intracellular cyclic AMP levels by treatment with phosphodiesterase inhibitors
IBMX and rolipram, does not mimic the response or potentiate the response of
adrenergic agonists. Additionally, treatment with PKA inhibitors H-89 and KT5720 had
no effect on the Ca2+ signal, and treatment with the cAMP analogue 8-bromo-cAMP,
which selectively activates PKA, did not trigger a Ca2+ response. These results strongly
suggest that AC, cAMP and the cAMP effector PKA are not involved in this signaling
pathway. Together these findings indicate that activation of β2-AR leads to an increase
in cytoplasmic [Ca2+] by a previously unrecognized signaling pathway. The discovery of
a new signaling pathway for β2-AR could have serious implications in the use of beta
agonists and blockers for the treatment of disease.
Contributors: Galaz-Montoya, Monica; Rodriguez, Gustavo; Lichtarge, Olivier and Wensel,
Chunxu Gao
Department of Molecular Virology & Microbiology
Advisor: James Versalovic, M.D./Ph.D.-Department of Pathology & Immunology
Supplementation with probiotic Lactobacillus reuteri strains that naturally colonize
the gut of mammals has been effective at ameliorating intestinal inflammation in rodent
colitis models, but the underlying mechanisms are unknown. Recent pangenomic
studies showed that L. reuteri strains with anti-inflammatory properties contain a
complete hdc gene cluster which is responsible for synthesis and secretion of
histamine, indicating a potential role for histamine in alleviation of inflammation.
L. reuteri strain ATCC PTA 6475 which contains an intact hdc gene cluster was found to
suppress TNF production in activated THP-1 cells through the production of histamine
and activation of histamine receptor 2 (H2R). Targeted mutagenesis of the hdc genes
resulted in diminished anti-TNF activity and loss of histamine production, indicating the
anti-TNF activity of histamine in vitro. Based on these studies, we hypothesize that hdc+
L. reuteri attenuate inflammation in vivo via histamine production and activation of H2R.
Using a trinitrobenzene sulfonic acid (TNBS)-induced mouse model of colitis, we
showed that L. reuteri 6475 administration with a dose of 5×109 CFU once per day for
seven days protected eight-week female BALB/c mice against colitis, as indicated by
significantly decreased weight loss, colonic injury graded by the Wallace score and
serum amyloid A protein concentrations compared to the mice receiving media control.
Positron emission tomography (PET) imaging also showed that L. reuteri 6475
significantly reduced the uptake of [18F]fluorodeoxyglucose ([18F]FDG) in the colon,
indicating attenuation of colonic inflammation by L. reuteri. RT-qPCR experiments
revealed that TNBS instillation induced IL-6 and IL-1β gene expression in the colon
of colitic mice compared to healthy mice while L. reuteri treatment decreased the gene
expression of these proinflammatory cytokines. Further experiments found that
the hdcA mutant of L. reuteri 6475 which failed to produce histamine showed diminished
ability to attenuate colitis compared to the wild type strain. Moreover, H2R was detected
in the mouse colon by immunohistochemistry and blocking H2R with its specific
antagonist ranitidine diminished the anti-inflammatory ability of L. reuteri 6475 while
blocking H1R with its specific antagonist pyrilamine did not affect the anti-inflammatory
activity of this probiotic strain. In addition, feeding mice with a histidine-free diet
diminished L. reuteri’s ability to attenuate colitis compared to the regular diet which
contains 0.4% of histidine. These combined investigations indicate that L. reuteri 6475
attenuates experimental colitis via histamine production, and provide important insights
into understanding the molecular mechanisms underlying probiotic immunomodulation.
Contributors: Gao, Chunxu; Spinler, Jennifer; Major, Angela; Jackson, Vanessa; Lugo, Monica;
Versalovic, James
Courtney Dawn Garcia
Department of Neuroscience
Advisor: Dora Angelaki, Ph.D.-Department of Neuroscience
The vestibular system provides for perception of movement, orientation in space,
and motion related behaviors. In relation to self-motion perception, human yaw rotation
velocity thresholds have been previously measured to be 0.7 deg s-1 at 0.5 Hz (Merfeld
et al. 2008). However, the available data on neuronal direction detection thresholds for
semicircular canal afferents in macaques have been quite disparate. For example,
average neuronal direction detection thresholds for semicircular canal afferents were
reported to be ~3.75 deg s-1 for regular afferents and ~8.25 deg s-1 for irregular
afferents by Sadeghi et al. (2007). In contrast, Yu et al. found average direction
detection thresholds for semicircular canal afferents to be much lower at 2.17 deg s-1 ±
1.32 (n=85) for regular afferents and 1.91 deg s-1 ± 1.49 (n=32) for irregular afferents
(2013 SFN Abstract). Both studies used sinusoidal stimuli without simultaneously
measuring behavioral thresholds. Furthermore, both studies compared neural
thresholds in macaques with human perceptual thresholds. Thus, it is possible that
macaque and human perceptual thresholds are different. Here we set out to
investigate how macaque perceptual yaw rotation velocity detection thresholds
compare with human thresholds. We measured macaque thresholds using transient
smooth trajectories that followed a 1s Gausian velocity profile (peak velocity: 1.3-30 deg
s-1). Two rhesus macaques were trained to perform a two-alternative-forced-choice
task, in which they reported leftward or rightward rotation by making a saccade to one of
two targets presented at motion end. Psychometric functions then determined direction
detection thresholds over time. Mean yaw rotation detection thresholds were initially
8.59 deg s-1 for monkey H and 6.85 deg s-1 for monkey I. However, after continued
perceptual thresholds in both monkeys decreased to 3.31 deg s-1 for monkey H
and 1.48 deg s-1 for monkey I. In order to directly compare neuronal and perceptual
detection thresholds for yaw rotation, we are currently measuring canal afferent
thresholds simultaneously with perceptual detection thresholds in both animals.
This work was supported by NIDCD DC04260.
Contributors: Garcia, Courtney D.; Yu, Xiong-Jie; Liu, S.; Dickman, J. David; Angelaki, Dora E.
Melissa Nolan Garcia
Clinical Scientist Training Program
Advisor: Kristy Murray, Ph.D.-Epidemiology
Chagas disease, infection with the parasite Trypanosoma cruzi, has recently
been identified as an important emerging parasitic disease in the United States. To
describe the cardiac abnormalities among T. cruzi positive blood donors in Southeast
Texas, we performed a pilot study of donors who had screened positive from 20072012. This one-time assessment included: 1) a questionnaire to evaluate source of
infection, cardiac symptoms and health co-morbidities, 2) an electrocardiogram, 3) an
echocardiogram if ECG was abnormal, and 4) measurement of a high-sensitivity
troponin T biomarker. Of those with confirmed infection, 41% (7/17) had an
electrocardiographic abnormality consistent with Chagas cardiomyopathy. In addition,
36% (6/17) were suspected to be locally acquired cases. Biomarker high-sensitivity
troponin T serum levels increased with cardiac severity. Cardiologists should consider
Chagas disease in their differential for patients who may have clinically compatible
electrocardiogram changes or cardiomyopathy, even if the patient has no history of
residing in a Chagas-endemic country
Contributors: Garcia, Melissa; Murray, Kristy; Hotez, Peter; Rossman, Susan; Gorchakov,
Rodion; Woc-Colburn, Laila; Bottazzi, Maria Elena; Rhodes, Charles; Ballantyne, Christie;
Aguilar, David.
Sandy Lynn Garno
Integrative Program in Molecular and Biomedical Sciences
Advisor: Weiwei Dang, Ph.D.-Department of Molecular & Human Genetics
Human mesenchymal stem cells (MSCs) are easily isolated, pluripotent adult
stem cells capable of differentiating into various lineages including (but not limited to)
adipocytes, chondrocytes, and osteoblasts. These characteristics are ideal for stem
cells in regenerative medicine, thus MSCs are widely investigated for their therapeutic
potential. Regeneration is a critical topic when it comes to aging, as aging is defined by
widespread deterioration and dysfunction of normal anatomy and physiology. MSCs
have the potential to regenerate many structures lost or damaged as aging occurs, and
also to treat any condition that requires the replacement of damaged cells. The problem
is that MSCs isolated from older individuals have reduced differentiation potential for all
lineages, and all MSCs are subject to a limited number of expandable divisions in vitro.
This creates difficulty when attempting to collect MSCs for autologous use from aged
individuals, as MSCs can only be safely isolated in small amounts. Thus, it is ideal to
investigate alterations to differentiation potential that occur with aging of human
mesenchymal stem cells in order to maximize their usefulness in regenerative medicine.
It is well known that changes to transcription accompany aging in eukaryotes. We
will perform RNA-Seq analysis on young and old mesenchymal stem cells in order to
determine exactly how transcription is changing with age in these cells. Processes
expected to show significant alteration are intragenic cryptic transcription, non-coding
RNA regulation, and alternative splicing, among others. We will target these processes
in old MSCs via knockdown or overexpression of critical enzymes in order to mimic the
transcription profile of young MSCs. By doing this, we intend to restore the declining
differentiation potential in old MSCs to the robust differentiation potential of young
MSCs. Ultimately this will allow aged individuals access to the full power of
mesenchymal stem cell regenerative medicine that is available to young individuals.
Contributors: Garno, Sandy; McCauley, Brenna; Dang, Weiwei
Leah Ashley Gates
Department of Molecular & Cellular Biology
Advisor: Bert O'Malley, M.D.-Department of Molecular & Cellular Biology
Estrogen receptor alpha (ER ) directs expression of its target genes by binding
particular DNA elements and recruiting different transcriptional coregulators (CoRs) in a
hormone-stimulated fashion. In addition to being regulated by ligands, ER activity is
also modulated through the chromatin environment by particular interactions with
specific CoRs. Our lab is interested in dissecting the mechanisms of transcriptional
regulation, which is essential for deepening our knowledge of basic molecular biology
and may aid in the identification of potential therapeutic targets for hormone-driven
cancers. Epigenetic modifications such as histone marks can promote the recruitment
or dismissal of CoRs that directly influence the transcriptional outcome. H3K4
trimethylation (H3K4me3) is a histone mark found at actively transcribing gene
promoters and is correlated with histone acetylation. The presence of H3K4me3
stimulates acetylation selectively at H3K9 (H3K9Ac) and at several H4 lysines, including
H4K5 (H4K5Ac) on recombinant nucleosomes in vitro with ER . By performing
biotinylated histone peptide pulldowns with HeLa nuclear extract followed by mass
spectrometry and immunoblotting, we now report that the dual modification H3K4me3K9Ac recruits the super elongation complex (SEC) as compared to the H3K4me3
peptide alone. Additionally, H4K5Ac recruits the BET family members Brd2, Brd3, and
Brd4 compared to the unmodified H4 peptide. Based on this data, we propose a model
in which H3K4me3 is required for transcription initiation and promotes acetylation of
H3K9 and H4 lysines through the recruitment of histone acetyltransferases. These
acetylation marks can then recruit elongation machinery via Brd proteins to H4 acetyl
marks and the SEC to H3K9Ac. Next, we will determine the functional relevance of
H3K9Ac and H4K5Ac in ER -mediated transcription by using a combination of
biochemical and cell-based functional assays. Importantly, these studies will advance
our understanding of how ER
and epigenetic marks can regulate transcription at the
level of RNA Pol II elongation in response to hormonal stimulation.
Contributors: Leah A. Gates, Qin Feng, Mark T Bedford, Sung Yun Jung, Jun Qin, Charles E.
Foulds, and Bert W. O’Malley
Violet Gelowani
Department of Molecular & Human Genetics
Advisor: Rui Chen, Ph.D.-Department of Molecular & Human Genetics
A patient family entered the clinic displaying a novel combination of clinical
features consisting of glaucoma, short stature, microphthalmia, bracydactyly, and
choroidal effusions. We were interested in finding the genetic cause for this novel
syndrome. Using whole exome sequencing and pedigree segregation analysis we
identified the patients to be heterozygous for a mutation in LRP6. LRP6 is known to be
involved in the WNT signaling pathway. To test whether the patient mutation disrupts
WNT signaling we performed in vitro luciferase reporter assay and saw that the
mutation reduced WNT signaling. We performed morpholino knockdown and rescue
experiments in zebrafish and found that the mutant LRP6 allele failed to rescue the
skeletal defects and the microphthalmia phenotype of LRP6 knockdown zebrafish. This
observation validated the mutant allele of LRP6 as the genetic cause for the mutant
phenotype in patients. LRP6 heterozygouse null mice were found to have elevated
ocular pressure, optic nerve malformations, and skeletal defects confirming that LRP6 is
haploinsufficient. These results suggest that LRP6 is a novel human disease gene, and
that our newly developed mouse model may serve as a disease model for this novel
syndrome and for glaucoma.
Contributors: Gelowani, Violet; Jacques Zaneveld; Xu Mingchu; Liu, Wei; Wang, Hui; Wang,
Xia; Wang, Feng; Salvo, Jason; Jiang, Lichun; Li, Yumei; Guy, Hughes; Chen, Rui.
Triparna Ghosh-Choudhury
Program in Translational Biology & Molecular Medicine
Advisor: Matthew Anderson, M.D./Ph.D.-Department of Obstetrics & Gynecology
Creighton Edwards, M.D.-Department of Obstetrics & Gynecology
INTRODUCTION: MicroRNAs (miRNAs) are endogenous, non-coding RNA transcripts
that play a critical role in regulating diverse patterns of gene expression. Although altered
miRNA expression has been previously described in ovarian cancer, the role of individual
miRNAs in this disease remains poorly understood.
METHODS: Level 3 data from the Cancer Genome Atlas Consortium (TCGA) were
interrogated by using Kaplan-Meier statistics to test for association between levels of individual
miRNAs and outcome demographics. miRNA mimics were transfected into established ovarian
cancer cell lines (HeyA8, SKOV3ip1, OVCAR8) using Lipofectamine (Invitrogen). Proliferation
and apoptosis were measured using MTS and Caspase Glo 3/7 assays (Promega). Western
blot and quantitative real time PCR were used to validate gene expression. In vivo studies were
done in nude mice injected intraperitoneally with RFP (+) OVCAR8 cells. Statistical significance
was calculated using two-tailed T-tests.
RESULTS: Our analyses indicate that 3 of the 10 miRNA transcripts most robustly
associated with ovarian cancer outcome (miR-148a, miR-148a* and miR-148b) share a
common seed sequence. Transfection of both TP53-null SKOV3ip1 and TP53-mutated
OVCAR8 ovarian cancer cells with mimics for any of these three miRNAs significantly inhibited
proliferation, enhanced apoptosis, inhibited migration and invasion. To identify targets, we
recognized correlations between patterns of gene and miRNA expression using Lasso analyses
with L1-normalization, after which potential miR-148 targets were screened using established
target prediction algorithms (Targetscan, RNA22). These analyses pinpointed MTMR9, a poorly
understood cofactor important for regulating phosphoinositide metabolism, as a key target for
miR-148 in ovarian cancer. Overexpression of miR-148 results in decreased expression of
MTMR9 and decreased catalytic activity of MTMR6 and MTMR8 contributing to apoptosis of
cells. Direct knockdown of MTMR9 using siRNAs reduces proliferation, enhances apoptosis and
decreases rates of migration and invasion in OVCAR8 and HeyA8 cells. Treatment with
siMTMR9 liposomes in vivo reduces ovarian cancer growth and metastasis. Control mice were
treated with siNT and had significant metastasis throughout abdomen and diaphragm. KI67
stainings of cross sections of dissected tumors showed a large number of control cells to be
proliferative where as significantly lower amounts were seen in siMTMR9 mouse cross sections.
CONCLUSION: Our findings indicate that the frequent loss of miR-148 miRNAs
promotes ovarian cancer growth and metastasis by enhancing MTMR9 expression. These
findings not only validate the unique approach we have adopted for screening high throughput
data sets for relevant miRNA targets, but also implicate novel aspects of phosphoinositide
metabolism in ovarian cancer biology.
Contributors: Ghosh-Choudhury, Triparna; Wan, Ying-Wooi; Liu, Zhandong; Anderson, Matthew
Mary Elizabeth Girard
Department of Molecular Virology & Microbiology
Advisor: Christophe Herman, Ph.D.-Department of Molecular & Human Genetics
Bacterial infections resistant to antibiotics are an alarming threat as the incidence
numbers continue to rise, leaving us in need of new antimicrobial methods. We propose
to engineer an antimicrobial bacterium, or rather, a bacterium able to kill other bacteria.
With this novel antimicrobial approach, we highjack bacterial conjugation as a platform
to deliver toxic elements to bacteria. Conjugation is the horizontal transfer of genes
between bacteria via a conjugative plasmid through a sex pilus from the donor to the
recipient bacterium. This process is a very efficient mode of gene transfer and is quite
promiscuous, theoretically allowing us to target a variety of bacteria. Additionally, with
the current methodologies of bacterial genetic engineering, we are able to construct a
variety of conjugative plasmids encoding toxic genes of choice with ease. For the toxic
component of our antimicrobial platform, we envision utilizing either toxins from bacterial
toxin-antitoxin systems or the Cas endonuclease from bacterial CRISPR/Cas systems.
Both options are highly advantageous, untapped resources for potential antimicrobials.
The TA system toxins are known to be quite toxic to their bacterial hosts in absence of
the cognate antitoxin, and have thus often been suggested as putative alternatives to
antibiotics. Here, the engineered bacterium carrying the toxin-encoding conjugative
plasmid mates with a recipient, transferring and expressing the toxin, killing the recipient
bacterium. For our other choice toxic element, the CRISPR/Cas systems are also
known to be lethal to the host bacterium if targeted to cleave bacterial host DNA. The
CRISPR system is very exciting as a putative antimicrobial as the Cas nuclease can be
targeted to cleave very specific SNPs of DNA, and this specificity allows us to target its
lethal activity to bacterial pathogen SNPs. In this case, the engineered bacterium carries
a conjugative plasmid encoding the Cas nuclease targeted to a desired pathogen SNP.
Upon mating, the Cas nuclease specifically cleaves the pathogen DNA, thereby only
killing the desired pathogen.
Taken together, this antimicrobial platform combines the toxic elements of TA
systems and CRISPR/Cas systems with conjugation as a delivery mechanism,
transferred by a harmless bacterial carrier. We are initially testing this antimicrobial
strategy by performing mating assays between our engineered strains against nonpathogenic lab strains of E. coli, scoring for recipient cell viability. Preliminary data
shows this method is effective against E. coli, and next, we will be performing mating
and viability assays against a variety of bacterial pathogens. In the future, this work may
lead to the development of this system for application in biotherapeutics or
Contributors: Girard, Mary; Herman, Christophe
Guilherme Godoy
Clinical Scientist Training Program
Advisor: Seth Lerner, M.D.-Department of Urology
Introduction and Objective: We have used the prostatic urethra biopsy as part of
our preoperative management and clinical staging prior to radical cystoprostatectomy
(RC). Our hypothesis is that a negative preoperative transurethral resection (TUR)
biopsy of the prostatic urethra accurately predicts a final negative apical urethral margin,
safely replacing an intraoperative frozen section for urethral preservation and orthotopic
neobladder utilization.
Methods: TUR biopsies of the prostatic urethra adjacent to the verumontanum at
5 and 7 o’clock were performed on 272 male patients that underwent RC at our
institution between 1987 and 2013. Pathology results of biopsy were correlated with
those of final apical urethral margin status of the RC specimen, and whether or not the
surgeon had done an intraoperative frozen section of the urethral margin and performed
a primary or secondary urethrectomy. Whole mount step section of the entire prostate
was performed and examined microscopically in the majority of cases.
Results: Of the 272 patients, 200 (74%) had a negative and 72 (26%) had a
positive biopsy. The overall final positive apical urethral margin rate in the RC specimen
in this series was 2.2% (6/272 ). Five of those patients underwent a primary or
secondary urethrectomy. In one patient with a positive final margin, a urethrectomy was
not performed as the frozen section of apical urethra had been negative despite the
finding of an invasive urothelial carcinoma on the preoperative TUR biopsy. The
negative predictive value of a TUR biopsy was very high (99.5%) with a very low
positive predictive value (PPV) for the final pathological margin status.
Conclusion: The apical urethral positive margin rate is very low in patients
undergoing RC. A positive TUR biopsy has a low PPV for a positive final apical margin
(7%). A negative preoperative TUR biopsy of the prostatic urethra can reliably predict a
negative final margin and may obviate the need for intraoperative frozen section
evaluation. This information facilitates preoperative counseling patients about the
feasibility for orthotopic neobladder reconstruction of the bladder after RC.
Contributors: von Rundstedt, Friedrich-Carl; Godoy, Guilherme; Shen, Steven; Lerner, Seth P.
Ganga Gokulakrishnan
Clinical Scientist Training Program
Advisor: Steven Abrams, M.D.-Department of Pediatrics
BACKGROUND: Antenatal GLC exposure is known to accelerate the maturation
of several tissues in the fetus, but whether it is also true for the skeletal muscle is
unknown. It has been shown that in utero GLC exposure results in decreased skeletal
muscle mass and decreased protein content independent of its effect on maternal food
intake. Satellite cells are mononuclear myogenic cells responsible for postnatal skeletal
muscle growth and mass. However the effects of in utero GLC exposure on satellite
cells in the perinatal period are unknown.
OBJECTIVE: To test the hypothesis that the decrease in skeletal muscle mass
following in utero GLC exposure is a result of a decrease in satellite cell population.
The aims were to determine if fetal exposure to GLC alters 1) myofiber cross sectional
area (CSA) 2) myonuclear accretion 3) Satellite cell population.
DESIGN/METHODS: Three groups (n=7/group) of timed-pregnant SpragueDawley rats were studied: Control (CON): ad libitum food intake; Dexamethasone
(DEX): ad libitum food intake; dexamethasone (1 mg/L drinking water ad lib from
embryonic day (ED) 13 to 21). Pair-fed (PF): pair-fed to DEX group from ED 13 to 21;
no DEX. Three injections of 5-bromo-2′-deoxyuridine (BrdU) were given 12 hours apart,
36 hours prior to retrieval. On ED 22 (term), fetuses were retrieved, dissected and
muscles collected. Quadriceps muscles were analyzed for the following: Muscle fiber
cross sectional area (CSA), myonuclear number, myonuclear proliferation and satellite
cell number using immunofluorescence techniques.
RESULTS: The mean CSA was different among all 3 groups with DEX group
being the smallest (P < 0.01). The myonuclei/fiber ratio was 0.79, 0.55 and 0.68 in the
CON, DEX and PF groups respectively with DEX significantly different from CON (P =
0.014). BrdU labeling of myonuclei was similar among all groups. Satellite cell/myofiber
ratio was 0.13, 0.07, 0.16 in CON, DEX and PF groups respectively with all 3 groups
being significantly different from each other (P< 0.001). The percentage of satellite cells
noted within the myofiber was similar among all groups.
CONCLUSIONS: In utero GLC exposure resulted in a decrease in fiber size as
evidenced by cross sectional area. The decrease in myonuclear accretion noted in the
DEX group can be explained by the decrease in satellite cell population secondary to
impaired proliferation. No impairment in satellite cell fusion was noted. Apoptosis of
myonuclei could also have contributed to decrease in myonuclear accretion. These
findings suggest a possible permanent reduction in skeletal muscle mass in adulthood.
Contributors: Gokulakrishnan, Ganga; Chang, Xiaoyan; Fiorotto, Marta L.
Maria Monica Gramatges
Clinical Scientist Training Program
Advisor: Alison Bertuch, M.D./Ph.D.-Department of Pediatrics
Introduction: Constitutional mutations in telomere maintenance genes are associated with
short telomeres and a spectrum of disorders including dyskeratosis congenita (DC). DC confers a
90% lifetime risk for bone marrow failure, a 200-fold risk for acute myeloid leukemia (AML), a 2500fold risk for myelodysplastic syndrome (MDS), and is associated with chemosensitivity in affected
individuals. Constitutional defects in telomere maintenance genes are also enriched in patient
cohorts with various hematologic malignancies, including AML. Given this association, we
investigated telomere length and the incidence of constitutional telomerase variants in de novo
pediatric AML cases and their role in therapy-related adverse events (AE’s). We hypothesized that
variants would be (1) more frequent in AML cases compared with controls, (2) associated with
characteristics of telomere biology disorders, and (3) in addition to telomere length, would further
characterize AML cases with specific AE’s.
Methods: We sequenced the exons and flanking intronic regions of the telomerase-related
genes TERT, DKC1, hTR, and TINF2, in a local pediatric AML/MDS cohort (n=104), a Children’s
Oncology Group (COG) AML cohort (n=115), and local controls racially and ethnically matched to
our AML/MDS cohort (n=254). We blindly reviewed medical records in the local AML cohort for six
characteristics suggestive of DC. For the COG cohort, we compared the number of variants and
remission telomere content (TC), measured by qPCR, in subjects with expected blood count
recovery after chemotherapy (n=62) to those with significantly prolonged recovery times (n=53). A
relationship between variants, TC, and specific grade 3 or 4 AE’s was also explored.
Results: In the local AML/MDS cohort, the number of novel or rare telomerase variants
significantly exceeded what was observed in both population databases and local controls
(p<0.0001). Retrospective medical record review demonstrated a significant association between the
number of DC features present and presence of a novel or rare variant (p=0.04). Within the COG
AML cohort, a significant association was observed between less TC at the end of initial
chemotherapy and delays in time to blood count recovery in later cycles of chemotherapy (p=0.03).
Conclusions: Novel and rare constitutional telomerase variants are enriched in pediatric
AML/MDS cases compared with controls. These variants are significantly associated with clinical
features of telomere biology disorders. In addition, less TC at end of AML induction is associated
with significant delays in time to blood count recovery in later cycles of AML chemotherapy,
suggesting that short telomeres are indicative of an underlying impairment in capacity for marrow
stem cell reconstitution.
Contributors: Gramatges, MM; Sasa, G; Gerbing R; Alonzo T; Gamis A; Meshinchi S; Plon, SE; Bertuch, AA.
Joshua Daniel Graves
Integrative Program in Molecular and Biomedical Sciences
Advisor: Weei-Chin Lin, M.D./Ph.D.-Department of Medicine
The E2F family of transcription factors consists of crucial proteins in higher
eukaryotes with functions including cell proliferation, apoptosis, senescence, and
metabolism. How a handful of proteins, and in some cases the same family member
(e.g. E2F1), can carry out such opposing functions is an unresolved paradigm. Thus far,
specifying and directing activity has been largely attributed to the numerous posttranslational modifications found on E2Fs. I have demonstrated that the small ubiquitinrelated modifier 2 (SUMO2) modifies E2F1-5. Due to the high frequency of
perturbations in the Rb/E2F1 pathway in all cancer types, E2F1 is the main focus of
further study. More than 95% of SUMO2 conjugation on E2F1 requires two lysines of
E2F1. Overexpressing Ubc9, the sole E2 conjugating enzyme in the SUMO pathway,
greatly induces modification dependent on these two lysines. When using an E2F1specific luciferase reporter assay, Ubc9 overexpression reduced E2F1 transcriptional
activity by up to 60%. Additionally, E2F1 point mutants for sumoylated lysines
significantly rescued this Ubc9-mediated transcriptional repression. This suggests that
sumoylation specifically on E2F1 is responsible for the observed decrease in activity.
Furthermore, reduction of transcriptional activity by Ubc9 has been recapitulated at
endogenous E2F1 target genes by qRT-PCR. I hypothesize that SUMO2 modifying
E2F1 serves as an additional means to inhibit specific E2F1 transcriptional activity,
resulting in changes to cell cycle progression, apoptosis, senescence, or metabolism.
E2Fs1-3 have essential roles in promoting cell growth, as is exemplified by the fact that
a triple knockout mouse is embryonic lethal. Consequently, the pathways that inhibit
their activity are highly mutated in all cancer types. By further understanding how
SUMO2 addition to E2F1 inhibits its transcriptional activity, as well as how this
mechanism is perturbed in cancer, new opportunities could be presented as
pharmacologic targets to inhibit E2F1 dependent cell growth. Moreover, since SUMO2
has also been observed on E2Fs2-3, the findings of this and future work might yield a
means to shut down all E2F dependent growth in cancer.
Contributors: Graves, Joshua; Lin, Weei-Chin
Deric Maurice Griffin
Program in Translational Biology & Molecular Medicine
Advisor: Robia Pautler, Ph.D.-Department of Molecular Physiology & Biophysics
Stephanie Abrams, M.D.-Department of Pediatrics
Non-alcoholic fatty liver disease (NAFLD) is a spectrum of disorders,
encompassing hepatosteatosis (fat in the liver), non-alcoholic steatohepatitis (NASH),
and cirrhosis. NAFLD’s incidence has been increasing alongside obesity in the U.S. and
is becoming a leading cause for liver transplant in the United States. Oxidative stress
has been heavily implicated in disease pathology, but its full role along with an effective
antioxidant therapy is still unclear. Here we use a methionine-choline deficient diet
(MCDD) model of NAFLD, which captures the liver phenotype of the disease and leads
to decreased production of the antioxidant glutathione (GSH).
We used the MCDD model to define the effect of overexpressing the
endogenous antioxidant, superoxide dismutase 2 (SOD2), which breaks down the
reactive oxygen species (ROS) known as superoxide radical. We also sought to
evaluate the efficacy of pegylated-hydrophilic carbon clusters (PEG-HCCs), a potent
bioengineered nano-antioxidant, on NAFLD progression. In the context of MCDD, mice
overexpressing SOD2 showed increases in oxidative stress markers and no
improvement in markers of liver damage, while PEG-HCC treated mice had significant
decreases in oxidative stress markers in addition to displaying trends towards less liver
damage. We predict that these observed differences are partially due to an increase in
hydrogen peroxide, which is a ROS by-product of SOD2 activity. We suspect that this
increase in hydrogen peroxide due to increased SOD2 activity, along with the restriction
of GSH production, led to increased oxidative stress in MCDD fed SOD2
overexpression mice. This effect was not observed in the PEG-HCC treated group and
we believe this to be due to the PEG-HCCs ability to scavenge oxygen radicals without
the producing ROS byproducts, thus highlighting their efficacy as a potential
Contributors: Griffin, Deric M.; Criss II, Zachary K.; Tour, James; Pautler, Robia
Caitlin Grzeskowiak
Department of Molecular & Human Genetics
Advisor: Kenneth Scott, Ph.D.-Department of Molecular & Human Genetics
Discoveries in cancer genomics have revolutionized advancements in
personalized medicine. Through large-scale sequencing efforts, many critical genetic
alterations have been identified to drive tumor initiation and metastasis. Several
consortiums have been established to analyze biopsied patient tumors to identify
potential oncogenic mutations. However, these mutations often still require extensive
validation efforts as they are usually diluted in these studies with passenger mutations
in late stages of disease progression. Alternatively, large scale in vitro screening efforts
using cell culture assays do not faithfully recapitulate hallmarks of spontaneous tumors
presented in the clinic. To combat these challenges, in vivo models are often used to
validate oncogenic drivers. We have developed an in vivo screening platform to identify
oncogenic drivers of tumor initiation and metastasis in a Genetically-Engineered-Mouse
(GEM) lung cancer model. In this approach, GEM models provide the proper
microenvironment, yet drivers are readily identified with previously generated barcoding
technology developed in our lab. Cre recombinase is activated in LSL-KRASG12D mice
while simultaneously expressing barcoded potential drivers. Using a pool of candidate
oncogenes, we are able to readily identify the genetic drivers of metastasis and tumor
initiation by sequencing for barcode enrichment. To demonstrate the power of this
method, dominant negative forms of p53 were expressed with Cre recombinase in the
lung. Six months after intubation, mice were sacrificed and tumor analysis showed
obvious primary tumors with metastases, confirming this approach as powerful way of
identifying not only drivers of primary tumors, but also metastatic determinants.
Therefore, we are able to reproducibly activate oncogenic KRAS with proper genetic
context in addition to expressing candidate oncogenic drivers exclusively in the lung.
Once established, these models can be used directly as a preclinical model for testing
therapeutics targeted to specific mutations that arise in individual patient tumors.
Contributors: Wu, Ping, Scott, Kenneth
Nele Haelterman
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M.-Department of Molecular & Human Genetics
Proper muscle physiology is central to allow an organism to move and is thus
essential for every organism’s survival. Multiple fatal human diseases, such as Spinal
Muscular Dystrophy, are caused by muscle dysfunction. To identify novel genes
required for normal muscle development and function, our lab performed a forward
genetic mutagenesis screen on the Drosophila X-chromosome. From this screen, we
identified several mutations in ariadne-1 (ari-1), which encodes a conserved Ring-In
Between-Ring (RBR) E3 ubiquitin ligase and hence plays a role in maintaining protein
homeostasis. Under normal circumstances, nuclei are evenly spaced within the
multinucleated muscle fiber and this positioning is required to maintain proper muscle
function. For instance, abnormal myonuclear positioning is seen in human patients with
Central Nuclear Myopathies. We found that loss of ari-1 leads to nuclear clustering
within larval body wall muscles, a phenotype that is very rarely observed in wild type
It is well established that the cytoskeleton plays a critical role in nuclear
positioning, but less is known about the proteins that connect the nucleus to the
cytoskeleton. Recently, the KASH-domain containing proteins Msp-300 and Klarsicht
were found to form a complex that links the outer nuclear membrane to both the
microtubular and the actin cytoskeleton. Interestingly, the subcellular localization of
Msp-300 is severely affected in ari-1 mutants. In addition, the microtubular network
appears fragmented upon loss of ari-1, whereas the actin cytoskeleton remains intact.
One way to interpret our data is that Ari-1 directly regulates microtubule dynamics, the
malfunction of which affects nuclear migration. However, a similar microtubular
phenotype was observed in Msp-300 mutants that lack the KASH-domain. It is therefore
possible that, once properly placed, Msp-300 is required to maintain the astral
microtubular network that surrounds nuclei. Ari-1 would then control the localization,
and hence function, of Msp-300, possibly by mono-ubiquitinating the protein. If our
hypothesis holds true, Ari-1 would be the first known protein to fine-tune Msp-300’s
function in controlling nuclear positioning and sustaining muscle function.
Contributors: Haelterman, Nele*; Tan, Kai Li*; Jaiswal-Nagarkar, Sonal; Lee, Pei-Tseng; Bellen,
Katherine Haines
Department of Molecular & Human Genetics
Advisor: Sharon Plon, M.D./Ph.D.-Department of Pediatrics
Introduction: Hepatoblastoma, a pediatric cancer of the liver, has few recurrent
genomic abnormalities. Known alterations include Wnt pathway activation through
somatic mutations of CTNNB1 or rare germline APC mutations, an unmapped recurrent
t(1,4) translocation, and gains or losses of whole chromosomes; however, these
abnormalities do not fully explain the molecular basis of this tumor. We hypothesize that
gene fusion events may present a mechanism for oncogenesis in hepatoblastoma.
Methods: We have used RNA sequencing (RNA-seq) to survey a cohort of 24
hepatoblastomas for chimeric transcripts. High quality RNA (RIN: 6.6-9.7) was extracted
from fresh-frozen tissue and strand-specific, poly-A+ RNA-seq libraries were prepared
for Illumina sequencing. Approximately 85 million paired-end reads (42.5 million
fragments) of 2 x 100 bp length were generated per sample. Fusion transcripts were
detected using deFuse (v.0.6.1) and/or TopHat-Fusion on FASTQ files. Candidate
fusions were verified using BLAST and validated using RT-PCR.
Results: A total of 143 in-frame chimeric transcripts were detected. Of all
chimeric transcripts 60% represented likely read through events. These include one
transcript, WDR81-SERPINF2, which was detected in 9 of 24 hepatoblastomas. This
transcript results from a read through of the coding regions from two adjacent genes on
chromosome 17. Additional filtering yielded 109 fusions across the tumor set that
contain at least one gene partner from the COSMIC Cancer Gene Census with a known
role in cancer.
Conclusions: We have established an RNA-seq pipeline for identification of
fusions in pediatric solid tumors. From this initial cohort of hepatoblastomas, 100% of
samples displayed at least one chimeric transcript, alterations which would not have
been observed with DNA sequencing or standard clinical assays. This pipeline has
identified a novel read through, WDR81-SERPINF2 which is also detected in matched
normal liver but not lymphoblastoid cell lines and therefore may be a liver-specific
transcript of unknown significance.
Contributors: Katherine Haines1, Angshumoy Roy2,3, Linghua Wang4, Pavel Sumazin2, Kyle
R. Covington4, Donna M. Muzny1,4, Vijetha Kumar3, Dolores Lopez-Terrada2,3, David A.
Wheeler1,4, Sharon E. Plon1,2,4 and D. Williams Parsons1,2,4
Departments of 1 Molecular and Human Genetics, 2Pediatrics, 3Pathology & Immunology,
and 4Human Genome Sequencing Center, Baylor College of Medicine and Texas Children’s
Hospital, Houston, TX 77030
Meade Haller
Department of Molecular & Cellular Biology
Advisor: Dolores Lamb, Ph.D.-Department of Urology
Genitourinary (GU) birth defects comprise some of the most common yet least
studied congenital malformations and range in severity from conditions such as
undescended testes (cryptorchidism) and ventrally misplaced urethral meatus
(hypospadias) to highly complex malformations such as bladder exstrophy epispadias
complex (BEEC) and ambiguous genitalia. Congenital anomalies of the kidney & urinary
tract (CAKUT) are also common and include phenotypes such as duplicated tract
components, kidney agenesis, congenital hydronephrosis, horseshoe kidney, and cystic
kidneys. Genomic aberrations such as copy number gains and losses can result in
congenital malformations of the GU tract, among other organ systems. Genome wide
array comparative genomic hybridization (aCGH) together with an extensive literature
review allowed identification of genomic hotspots of GU development and delineated the
smallest CNV regions of maximum overlap. CNV mapping revealed over 30 patients
with GU defects harboring duplications or deletions in the syndromic genomic region,
16p11.2 – the most common known pathogenic gene dosage region in humans. The
only gene covered collectively by all the mapped CNVs in patients with GU defects was
MYC-associated zinc finger (MAZ). MAZ encodes a transcription factor with a similar
consensus sequence to that of WT1, and is implicated in WNT signaling. In situ
hybridization on mouse embryos and isolated mouse GU tracts defined the expression
profile of Maz during development. In situ experiments showed robust staining of the
GU tract including the kidneys, ureters, testes, bladder and genital tubercle.
Immunohistochemistry confirmed this expression pattern at the protein level. Analysis of
the lab’s cohort of genomic DNA from GU-abnormal patients and GU-normal fertile
controls via CNV qPCR determined the frequency of MAZ CNVs in these groups.
Validated by two independent probe sets, 6% of our GU abnormal cohort displayed
CNVs in MAZ (n=258) compared to 0% in controls (n=57). As predicted based on its
consensus sequence, knockdown or overexpression of MAZ in human embryonic
kidney (HEK293) cells results in differential expression of several WNT pathway genes
including DKK1/2, LRP5, FZD9/10, FRZB, DACT1, SFRP5, WNT2, WNT4, and WNT11.
MAZ, once thought to be simply a housekeeping gene, encodes a dosage sensitive
transcription factor that may play a key role in urogenital development and contribute to
the congenital malformations of the 16p11.2 phenotype.
Contributors: Haller, Meade; Lamb, Dolores J.
Mark Patrick Hamilton
Department of Molecular & Cellular Biology/M.D.-Ph.D. Program
Advisor: Sean Mcguire, M.D./Ph.D.-Department of Molecular & Cellular Biology
MicroRNAs are a large class of noncoding RNA that suppress mRNA expression.
MicroRNAs bind through their seven nucleotide “seed” region in complex with the
Argonaute protein, typically to the 3’ untranslated region (3’UTR) of target mRNAs. A
given microRNA may have hundreds of targets, and individual targets may only exist in
specific cellular contexts. Due to this, the microRNA target spectrum in any given cell
includes tens or hundreds of thousands of context specific interactions. Traditional
methods of microRNA target interaction include analysis of conserved sequence in the
3’UTR and computation of biochemical binding properties between a microRNA’s seed
and its putative target. While these methods are effective, they are often unable to
distinguish non-canonical sites outside the, are only inferences of possible microRNA
binding, and are not context specific.
Argonaute crosslinking immunoprecipitation (AGO-CLIP) is a method to directly
query global microRNA binding in the cell by sequencing the mRNA targets associated
with the Argonaute-microRNA complex. AGO-CLIP technology is underutilized, with
data existing in only 9 human cell lines and 2 mouse tissues due to the complexity of
the AGO-CLIP method, which requires cDNA library preparation following Argonaute
protein pull-down and radiolabeling of Argonaute-associated RNAs. Library preparation
requires multiple gel isolations and takes several weeks to perform. Further, cDNA
library preparations reported in current AGO-CLIP protocols require in-house-generated
reagents inaccessible to most researchers. Finally, the RNA yield from Argonaut pulldown is low, requiring multiple rounds of PCR amplification that reduce the unique
sequence composition of the AGO-CLIP library leading to inefficient sequencing results.
Here we report the development of the AGO-CLIP protocol using the Illumina
TruSeq Small RNA sample preparation kit. TruSeq library preparation removes the
need to isolate multiple gels during library preparation saving time and preventing RNA
loss. The TruSeq kit is readily accessible and requires no in-house generated reagents.
Finally the TruSeq kit is rapid and contains multiplex compatible sequencing primers.
We are able to rapidly generate successful AGO-CLIP libraries using the TruSeq RNA
kit using less than 15 cycles of PCR amplification that exhibit improvements in percent
of unique sequence and thus overall gains in sequencing efficiency. Use of the TruSeq
kit promises to increase the speed and quality of AGO-CLIP library generation allowing
an unprecedented view of microRNA binding in cells.
Contributors: Hamilton, Mark P.; Rajapakshe, Kimal; Hartig, Sean M.; Coarfa, Cristian;
McGuire, Sean E.
Lindsay Jeanette Harrigal
Integrative Program in Molecular and Biomedical Sciences
Advisor: Janet Butel, Ph.D.-Department of Molecular Virology & Microbiology
SV40 induces tumors, including lymphomas, in the Syrian golden hamster model
and has been found in peripheral blood mononuclear cells (PBMCs) of
immunocompromised monkeys. SV40 has been detected in human PBMCs, in human
tonsils, and in non-Hodgkin lymphoma (NHL), suggesting that SV40 may be
lymphotropic in humans. Other polyomaviruses have also been detected in lymphoid
tissues and cells, including JCV, BKV, and MCV. We hypothesize that SV40
establishes a persistent infection in B cells that is rarely productive. Preliminary results
using SV40-infected DG75 cells (human B cell line) suggest that viral genomes are
maintained at low copy numbers and that SV40 early protein T-antigen (T-ag) is
expressed. One goal is to determine the mechanism by which the SV40 genome is
maintained in human B cells. We speculate cellular mitotic protein(s) could function in
tethering T-ag-bound viral DNA to the chromosomes during cell division, allowing for
proper segregation to each daughter cell. Genome tethering or a “piggyback”
mechanism has been observed in other virus systems that establish persistent
infections, including human papillomavirus (HPV) and Epstein–Barr virus (EBV).
Currently, a mechanism for SV40 genome maintenance in human cell lines is
unknown. T-ag binds to the origin of replication in the SV40 genome and is predicted to
function in a tethering mechanism, allowing segregation of the viral genome during cell
division. Transient DNA transfections were used to examine SV40 genome tethering in
human B cell lines. The pEGFP-N1 vector was used to represent the SV40 genome in
transient transfections with the human B cell line DG75. Cells were transfected with
pEGFP-N1 and pCMV-CPC-T (encoding SV40 T-ag). The collected cells were
analyzed by flow cytometry for EGFP expression. Through 72 hours post transfection,
there were greater numbers of EGFP+ cells in the co-transfected (EGFP+CPC-T) DG75
cells compared to the vector control (EGFP only). The presence of SV40 T-ag led to an
increase in EGFP expression detected by flow cytometry, which suggests T-ag
promoted the retention of SV40-like DNA during B cell division; the observed “T-ag
effect” was significant. To further investigate the genome tethering ability of T-ag, sitedirected mutagenesis was used to disrupt the binding site on T-ag for the SV40 origin of
replication or binding sites on T-ag for cellular proteins that might act as linkers. These
could theoretically block the tethering of SV40 DNA to the chromosome. Transient
transfections will be performed with the SV40 T-ag mutants to evaluate changes in the
“T-ag effect” on human B cells.
Contributors: Harrigal, Lindsay J.; McNees, Adrienne L.; Butel, Janet S.
Anne M Hause
Program in Translational Biology & Molecular Medicine
Advisor: Pedro Piedra, M.D.-Department of Molecular Virology & Microbiology
Robert Atmar, M.D.-Department of Medicine
Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract
infections (LRTI) among infants and young children. The fusion (F) protein is a
promising vaccine target because it is relatively conserved among RSV-A and RSV-B
subgroups and induces neutralizing antibodies. Antigenic site II of the F protein is the
targeted site of palivizumab, a monoclonal antibody that is used prophylactically for
high-risk infants. Palivizumab is approximately 50% effective in preventing RSV related
hospitalization and a small number of patients have palivizumab resistant viruses after
therapy. Candidate vaccines, which are based on the historical GA1 genotype of RSVA, might provide variable levels of protection dependent on the subgroup and genotype
of the infecting virus. Our objective is to develop a bivalent RSV-F vaccine that is
composed of a consensus F protein for the RSV-A genotypes (Fa) and consensus F
protein for the RSV-B genotypes (Fb).
The G and F genes of RSV isolates (n=118) stored at BCM were sequenced.
The F and G sequences of 118 isolates from GenBank were obtained. Together, these
sequences were used to generate phylogenetic trees based on the G gene. The F
sequences were then used to create a consensus sequence for each of the 7 RSV-A
genotypes and 4 RSV-B genotypes identified in the phylogenetic trees. The genotype
consensus sequences were then used to generate an overall consensus sequence of F
protein for RSV-A (Fa) and RSV-B (Fb).
We identified 169 RSV-A sequences (GA1=10, GA2=94, GA5=36, GA7=5,
NA1=10, SAA1=2, ON=12) and 67 RSV-B sequences (BA=41, GB1=3, GB3=10,
GB4=13). Among the genotype consensus sequences of each subclass, there is
approximately 98% nucleotide conservation. However, this number decreases 5% when
comparing contemporary to historical genotypes. There is approximately 90% amino
acid and 80% nucleotide conservation between Fa and Fb. Amino acid variation is
greatest in the signal peptide (64%), no annotation domain 2 (23%), antigenic site ø
(17%), transmembrane domain (12%), and cytoplasmic domain (12%).
Although the F protein is generally well conserved between the genotypes of
each subclass, there is variability in domains that contribute to the overall structure of
the protein. Differences in protein domains could contribute to changes in quaternary
structure or accessibility of the antigenic sites. We hypothesize that a bivalent vaccine
(Fa & Fb) based on consensus of the F proteins will provide broader protection among
all circulating genotypes when compared to a monovalent RSV-F (Fa) vaccine derived
from historical virus (GA1 genotype).
Contributors: Piedra, Pedro A. ; Avadhanula, Vasanthi
Zongxiao He
Department of Molecular & Human Genetics
Advisor: Suzanne Leal, Ph.D.-Department of Molecular & Human Genetics
Autism spectrum disorder (ASD), a heterogeneous disorder with substantial
heritability, is defined by impaired social communication, deficits in language
development, and the presence of restricted interests and/or stereotyped repetitive
behaviors. Genome-wide association, de novo mutation and copy number variant
studies have reported more than 100 different genes and genomic regions to be
associated with this complex trait, but for at least 70% of autism cases the underlying
genetic component remains unexplained. Under the hypothesis that rare variants may
be involved in the etiology of ASD, we applied the rare variant extension of transmission
disequilibrium test (RV-TDT; He et al. 2014) on the exomes of 2,377 parent-child trios
exhibiting sporadic ASD. TDT tests whether or not the frequency of transmitted alleles is
the same as the alleles not transmitted to an affected child, and an excess of an allele
of one type transmitted to the affected offspring indicates a disease-susceptibility locus
for the trait that is both linked and associated with the marker. To enrich the association
signal from rare variants in sequence data, RV-TDT incorporates four commonly used
rare variant association methods: CMC (Li & Leal, 2008), WSS (Madsen & Browning
2009), BRV (Morris & Zeggini, 2010) and VT (Price et al. 2010). After a stringent quality
control, genotypes were accurately phased by BEAGLE (Browning & Browning, 2009).
The gene-based RV-TDT analysis was performed on rare and low frequency [minor
allele frequency (MAF) < 5%] nonsynonymous and putative splices site variants. Strong
associations were observed for a burden of rare variants within gene IFT20 on genomic
region 17q11.2 (p value = 4.8x10-4) and gene CPNE2 on genomic region 16q13 (p
value = 9.6x10-4). Our results show that RV-TDT analysis of trio-based sequence data
is a powerful tool to identify ASD genes and suggest that rare variants are highly
involved the etiology of ASD.
Contributors: Zong-Xiao He, Suzanne M. Leal
Corey W Hecksel
Department of Molecular Virology & Microbiology
Advisor: Wah Chiu, Ph.D.-Department of Biochemistry & Molecular Biology
Latent Herpes Simplex Virus type 1 (HSV-1) infections are a special public health
concern as asymptomatic viral shedding is believed to be responsible for over 70% of
primary infections and has been shown to increase the rate of HIV transmission in
patients with concurrent infections. HSV shares a common evolutionary origin with
tailed bacteriophages, which contain a DNA packaging motor, or portal, at one vertex of
the icosahedral particle. The bacteriophage tail is a complex structure that assembles at
the portal vertex and provides the mechanism for delivering the viral genome into the
infected cell. Using electron cryo-tomography (cryo-ET), we have identified a previously
unsuspected tail-like structure at the portal vertex (termed PVAT) in the HSV-1 virion.
The location of this tail-like structure suggests that it is important for virion architecture
and may play a role in herpesvirus infection.
Contributors: Hecksel, Corey; Schmid, Michael; Rochat, Ryan, Bhella, David; Chiu, Wah; Rixon, Frazer
Sarah Marie Hein
Department of Molecular & Cellular Biology
Advisor: Yi Li, Ph.D.-Department of Molecular & Cellular Biology
An early aged pregnancy leads to a reduced proliferative rate in the parous
breast and to an overall reduction in the lifetime risk of breast cancer, but a late aged
pregnancy leads to increased long term breast cancer risk. As increasing numbers of
women choose to delay childbirth, it is vitally important to understand how a pregnancy
may promote or inhibit breast cancer so that effective prevention strategies may be
developed. We have reported that pregnancy can instigate preexisting early lesions in
their progression to cancer. However, it is not clear whether pregnancy causes the
breast epithelium to become more or less susceptible to oncogenic mutations that will
be gained later in life.
We utilized the RCAS-TVA viral vector system to introduce the oncogene ErbB2
into similar numbers of mammary epithelial cells in fully-involuted and age-matched
nulliparous mice. We performed a tumor latency study and compared the fully
developed tumors. We find that a prior pregnancy accelerates tumorigenesis of
oncogene activated cells. Tumor phenotype does not appear to be affected by parity.
We subsequently evaluated precancerous lesions at multiple stages to determine how
tumor initiation is being accelerated. We found that early lesions are initiated at similar
rates in both cohorts of mice. However, lesions in the parous mice advance more
rapidly than those in the nulliparous group, and lesion advancement is associated with
reduced rates of apoptosis.
Collectively, these data suggest that parity reprograms the mammary epithelium
to lower the apoptotic anticancer barrier to oncogenic mutations and to become more
susceptible to tumorigenesis initiated by an oncogenic mutation. These findings stand in
direct contrast to all known mechanisms of parity acceleration of tumorigenesis, which
assume that the initiating insult must occur prior to pregnancy. Therefore, these findings
uncover a novel mechanism by which pregnancy regulates breast cancer risk.
Contributors: Hein, Sarah; Haricharan, Svasti; Huang, Shixia; Li, Yi
Amanda Y. Hendrix
Program in Translational Biology & Molecular Medicine
Advisor: Farrah Kheradmand, M.D.-Department of Medicine
Anita Sabichi, M.D.-Department of Medicine
The number one cause of cancer death for men and women in the US is lung
cancer. The poor 16.8% 5-year relative survival rate is attributed to the late-stage
diagnosis of lung cancer; 57% of lung cancer patients are initially diagnosed with
metastatic disease. The MAGE-A protein family of cancer/testis antigens is expressed
in 59-69% of non-small cell lung cancers and is associated with decreased overall
survival. In addition, expression increases proportionally as the disease progresses,
interfering with the p53 tumor suppression transcriptional program.
Advances in bicistronic adenoviral (Adv) vector-mediated activation of in vivo
dendritic cells (DCs) provide a promising platform for a novel adjuvant therapy against
metastatic lung cancer. This system is a departure from recent clinical trials involving
expensive and lengthy ex vivo DC-maturation protocols that have produced suboptimal
clinical outcomes; in these trials, DCs express low levels of co-stimulatory molecules
and TH1-polarizing cytokines, show poor migration to lymphoid tissues, insufficient
longevity, and potentially tolerogenic outcomes. In contrast, in vivo bicistronic delivery of
our drug-inducible composite activation receptor with a tumor-specific antigen activates
DCs and has been shown to successfully enhance potent CD8+ T-cell responses
against antigen-specific tumor growth.
Coupled with the widely expressed lung tumor antigen MAGE-A, we hypothesize
that our bicistronic Adv will target DCs for potent activation of specific anti-tumor T cell
responses against Mage-a+ lung tumors in vivo.
Mage-a3 and Mage-a4 murine mRNA have been found to be expressed in more
than one lung tumor model. We have designed and produced the bicistronic adenoviral
vector iMC-Mage-a, combining the drug-inducible composite activation receptor MyD88
and CD40, “iMC,” with the tumor-specific antigen Mage-a. The murine Mage-a3 and -a4
proteins are fused using a glycine flexible linker. Initial functional studies show protein
expression of the iMC activation receptor and the Mage-a3 and Mage-a4 antigens, and
the construct is able to induce nucleofactor-kappa B signaling critical to the adjuvant
function and anti-tumor response. A high titer of the vaccine is currently being produced
by a third party.
Future work will evaluate murine in vitro and in vivo T cell tumoricidal function
following activation of iMC-Magea bicistronic Adv-transduced DCs. Then we will
evaluate in vivo anti-tumor function and efficacy of the iMC-Magea bicistronic Adv using
murine lung tumor models expressing murine Mage-a.
Contributors: Hendrix, AY; Spencer, D; DeMayo, F; Levitt, J; Kheradmand, F in collaboration
with the Helis Foundation.
Alexander Michael Herman
Program in Developmental Biology
Advisor: Benjamin Arenkiel, Ph.D.-Department of Molecular & Human Genetics
Acetylcholine has long been proposed to influence feeding behavior. However,
this idea has been largely based on anecdotal evidence associated with tobacco users
who attest to the appetite-suppressing effect that tobacco has on the body. This link
between acetylcholine and tobacco is important to consider because nicotine, the
addictive component in tobacco that is believed to act as an appetite suppressant, acts
on the same receptors in the body as acetylcholine.
Therefore, endogenous
acetylcholine may act in a similar way to curb appetite under normal physiological
conditions that nicotine does in tobacco users. Thus far, however, a source site of
acetylcholine in the brain that might play a homeostatic role in suppressing appetite is
unknown. Using conditional genetic approaches to impair cholinergic signaling from the
diagonal band of Broca (DBB) – the major component constituting the cholinergic basal
forebrain (CBF) – we show that adult mice lacking cholinergic DBB neurons display
increased food intake and develop severe obesity. Furthermore, conditional removal of
cholinergic neurotransmission from the DBB by targeted knockout of the choline
acetyltransferase (Chat) gene recapitulates these findings. In addition, viral-mediated
mapping strategies uncovered presynaptic inputs into the cholinergic DBB originating
predominantly from the hypothalamus, the master-regulatory site of feeding behavior in
the brain. In turn, feeding-associated postsynaptic projections from the DBB converged
in the median eminence, another site important in regulating food intake. Taken
together, these data reveal a novel role of cholinergic CBF neurons in modulating
satiety in order to promote normal feeding behavior.
Contributors: Herman, Alexander; Ortiz-Guzman, Joshua; Garcia, Isabella; Arenkiel, Benjamin
Jose A. Herrera
Program in Translational Biology & Molecular Medicine
Advisor: Jeffrey Neul, M.D./Ph.D.-Department of Pediatrics
Jeffrey Kim, M.D.-Department of Pediatrics
Background: Rett Syndrome (RTT) is an X-linked dominant neurodevelopmental
disorder primarily caused by mutations in Methyl-CpG-binding protein 2 (MECP2), a
transcriptional regulator. Twenty-five percent of all deaths in RTT are sudden and
unexpected, and autonomic nervous system (ANS) dysfunction is hypothesized to be
the cause of sudden deaths in RTT. However, the role of MeCP2 in cardiac autonomic
function has not been investigated in depth.
Design/Methods: Male (2 months) and female (9 months) Mecp2 deficient and
wildtype mice were implanted with ETA-F10 telemeters and 24h recordings were taken
to calculate heart rate, heart rate variability, and incidence of arrhythmic events. Effects
of pharmacological stimulants or inhibitors on heart rate were normalized to saline
injection response.
Results: Male Mecp2 deficient mice (NULL) have a decreased heart rate (WT =
634 ±3, NULL = 518 ±8, p<0.001), while female deficient mice (NULL/+) do not present
with a decreased heart rate. Interestingly, NULL and NULL/+ mice both have a high
incidence of sinus pauses and AV block (WT 1/5, NULL 6/6, NULL /+ 4/4, p<0.05).
Additionally, both NULL and NULL/+ mice have increased bradycardia events defined
as a heart rate below 200 bpm, (WT = 17 ±6, NULL = 307 ±94, NULL/+ = 198 ±67,
p<0.05) and an increased heart rate variability. The normalized heart rate response to
atropine, a parasympathetic blocker, of NULL mice was above wildtype levels (p<0.05)
Chemical denervation by combined antagonism of parasympathetic (atropine) and
sympathetic (propranolol) resulted in NULL mice having an increased normalized heart
rate response compared to wildtype (p<0.05). Finally, the incidence of AVB/sinus
pauses counts per 30 minutes post-acute treatment with atropine and chemical
denervation (NULL saline 49 ±10, NULL atropine 8 ±5, NULL atropine/propranolol 0.5
±0.5 p<0.05) decreased the severity of the cardiac phenotype in NULL mice.
Conclusions: In summary, Mecp2 is required for a normal heart rhythm. Loss of
Mecp2 causes bradycardia, sinus pauses, AV block, and increased heart rate variability
that may be attributed by aberrant innervation. Acute atropine and chemical denervation
was therapeutic and ameliorated the cardiac phenotype observed in Mecp2 deficient
Contributors: Herrera J, Ward, CS, Neul, JLN
Tyler John Hilton
Integrative Program in Molecular and Biomedical Sciences
Advisor: Theodore Wensel, Ph.D.-Department of Biochemistry & Molecular Biology
John Wilson, Ph.D.-Department of Biochemistry & Molecular Biology
Retinitis pigmentosa (RP) is a neurodegenerative disease that causes
progressive loss of vision due to defective retinal cell function. RP is a very
heterogeneous disease, with over 60 genes associated with the disease phenotype. Of
these genes, rhodopsin is linked mostly to Autosomal Dominant RP (ADRP), and
accounts for about 10% of all cases of RP. Mutations in this gene alter rhodopsin
function or localization, resulting in progressive degeneration of the rod photoreceptors
in the retina. Past experiments in the lab have used zinc-finger nucleases to cause a
double strand break in the last exon of the rhodopsin gene, which was found to be
repaired predominantly by non-homologous end-joining (NHEJ) and produce an
insertion or deletion. If such a frameshift site were inserted upstream of the mutations in
rhodopsin associated with ADRP, it could potentially attenuate the effect of the toxic
protein products. Combined with a functional copy of the rhodopsin gene resistant to the
double-strand break agent, this “kill and replace” gene therapy strategy may be used to
treat the ADRP phenotype from the associated rhodopsin mutations. Thus far, we are
focusing on using TALENs and CRISPR/Cas9 systems to target sites in the first and
second exons of the rhodopsin gene, to introduce a double-strand break that will be
repaired by NHEJ in the rod photoreceptors. These double-strand break agents will be
tested in HEK 293 cells to determine activity before they will be packaged into rAAV and
used in murine models of ADRP.
Contributors: Hilton, Tyler; Wilson, John H.; Wensel, Theodore G.
Antentor Othrell Hinton
Integrative Program in Molecular and Biomedical Sciences
Advisor: Yong Xu, Ph.D.-Department of Pediatrics
Background: Hypertension is the leading cause of cardiovascular disease worldwide.
The etiology of essential hypertension is unknown. Psychological stress contributes to
development of hypertension in humans. The effects are mediated at least partly in the
amygdala which is part of the brain limbic system that mediates emotional and hormonal
responses to stress. In humans, mice and rats, neural activities in the medial amygdala (MeA)
are positively associated with levels of blood pressure (BP) during stress and estrogens
attenuate stress-induced c-fos expression in the MeA. Estrogen receptor-α (ERα) is highly
expressed in the MeA. Thus, we hypothesize that ERα expressed by MeA neurons mediate the
anti-hypertensive effects of estrogen during stress. Methods: Experiment 1: SIM1-Cre mice
were subjected to bilaterally stereotaxic injections with AAV-hM3Dq-mCherry into the MeA and
after two weeks for the virus to replicate, mice were inserted with telemetry probes. After seven
days recovery, mice received ip injections of CNO or saline and blood pressure was recorded
for 3 hours to establish baseline and then mice were subjected to 1 hour stress restraint.
Experiment 2: C57BL/6 female mice were subjected to bilateral ovariectomy (OVX) and given a
vehicle (V) or 17-β-Estradiol pellet (E). One week later, female OVX + V and E mice received
either no stress or 1 hr stress and were perfused and stained for c-fos. Experiment 3: We used
the Cre-LoxP system to remove ERα from specific MeA neurons in mice (ERαlox/lox/SIM1-Cre).
Female ERαlox/lox/SIM1-Cre mice and controls (ERαlox/lox) were subjected to bilateral
ovariectomy (OVX), given a vehicle (V) or 17-β-Estradiol pellet (E), and inserted with telemetry
probes on day one. After seven days recovery, blood pressure was recorded for 3 hours to
establish baseline and then mice were subjected to 1 hour stress restraint. Experiment 4:
C57BL/6 Female mice received OVX surgery and were given V and E therapy. 1 week later,
mice received either no stress or 1 hr stress and then amygdala tissue was harvested for
western blot analysis of glutamate receptors. Results: We found that the basal BP and HR
(before stress started) were significantly elevated by CNO compared to saline injections. In
addition, all mice showed increased BP/HR upon restraint stress, and CNO-treated mice
displayed significantly potentiated stress-induced responses compared to saline-treated mice.
C57BL/6 OVX + V and OVX + E mice receiving no stress had comparable c-fos activity.
However, stress-restraint C57BL/6 OVX + V mice showed increased c-fos activity in the MeA
whereas stress-restraint C57BL/6 OVX + E mice had attenuated c-fos in the MeA. ERαlox/lox
OVX + V mice showed increased mean arterial blood pressure (MAP) and systolic arterial blood
pressure (SAP) in response to stress. Conversely, ERαlox/lox OVX+ E mice had blunted stressinduced hypertension (MAP and SAP). Interestingly, ERαlox/lox/SIM1-Cre OVX + E and OVX +
V group both showed increased MAP, SAP, and RPP in response to stress. AMPA Receptor
Subunits GluR1 (GluA1) and GluR2 (GluA2) increase after stress in OVX+V group, responses
which appeared to be blunted in OVX+E group. OVX+E group shows higher membrane
expression of GluA1 and GluA2 levels in basal condition compared to OVX+V group.
Conclusions: We demonstrated that selective activation of SIM1 neurons in the MeA increases
basal BP and potentiates hypertensive responses provoked by psychological stress (restraint)
in conscious mice. We also demonstrated that estrogens protect against increased neural
activity under stress. Also, ERα in the MeA is required to mediate estrogenic actions to prevent
stress-induced hypertension. Our results also indicate that estrogen increases glutamatergic
membrane receptors, which may mediate the anti-hypertensive effects of estrogen during
Contributors: Hinton,Jr., Antentor; Xia, Yan; Henderson, Alexander; Xu, Pingwen; Saito, Kenji;
Cao, Xuehong; Yan, Xiaofeng; Reynolds, Corey; Xu, Yong
Hsing-I Ho
Department of Molecular & Human Genetics
Advisor: Gad Shaulsky, Ph.D.-Department of Molecular & Human Genetics
The evolution of sociality and altruism is enigmatic because cooperators are
constantly threatened by cheaters who benefit from cooperation without incurring its full
cost. Kin recognition is the ability to recognize and cooperate with genetically close
relatives. It has also been proposed as a potential mechanism that limits cheating, but
there has been no direct experimental support for that possibility. Here we show that kin
recognition protects cooperators against cheaters. The social amoebae Dictyostelium
discoideum cooperate by forming multicellular aggregates that develop into fruiting
bodies of viable spores and dead stalk cells. Cheaters preferentially differentiate into
spores while their victims die as stalk cells in chimeric aggregates. We found that the
victims escaped exploitation by different types of non-kin cheaters. This protection
depends on kin-recognition-mediated segregation, because it is compromised when we
disrupt strain segregation. The genetic variability in this recognition system is puzzling
because cells expressing incompatible cues are expected to be eliminated during
development. We show that rare recognition variants are excluded at first, but rejoin the
cooperative aggregate and produce spores due to subsequent suppression of kin
recognition. Cheating through several strategies is also reduced at late development,
thus limiting the threat posed by chimerism. These findings provide direct evidence for
the role of kin recognition in cheater control, and suggest a mechanism for the
maintenance of stable cooperative systems.
Contributors: Ho, Hsing-I; Hirose, Shigenori; Kuspa, Adam; Shaulsky, Gad
Szu-Yu Ho
Program in Developmental Biology
Advisor: Matthew Rasband, Ph.D.-Department of Neuroscience
The scaffolding protein ankyrinG is required for Na+ channel clustering at axon
initial segments. It is also considered essential for Na+ channel clustering at nodes of
Ranvier to facilitate fast and efficient action potential propagation. However, in contrast
to these widely accepted roles, we show here that ankyrinG is dispensable for nodal
Na+ channel clustering in vivo. Surprisingly, without ankyrinG, erythrocyte ankyrin
(ankyrinR) and its binding partner βI spectrin substitute and rescue nodal Na+ channel
clustering. In addition, channel clustering is also rescued after loss of nodal βIV spectrin
by βI spectrin and ankyrinR. Thus, ankyrinR/βI spectrin protein complexes function as
secondary reserve Na+ channel clustering machinery. In mice lacking both ankyrinG
and ankyrinR, Na+ channels fail to cluster at nodes. Thus, two independent
ankyrin/spectrin protein complexes exist in myelinated axons to cluster Na+ channels at
nodes of Ranvier.
Contributors: HO, Tammy Szu-Yu; Zollinger, Daniel R.; Chang, Kae-Jiun; Xu, Mingxuan;
Cooper, Edward C.; Stankewich, Michael C.; Bennett, Vann; Rasband, Matthew N.
Xue B Holdman
Integrative Program in Molecular and Biomedical Sciences
Advisor: Jeffrey Rosen, Ph.D.-Department of Molecular & Cellular Biology
Background: Tumor dormancy appears to play a critical role in the late
recurrence of luminal breast cancers. Fibroblast growth factor receptor 1 (FGFR1),
amplified in more than 20% of luminal B breast cancers, is correlated with endocrine
resistance, poor prognosis and increased recurrence. However, the molecular basis of
the recurrence remains elusive.
Experimental Design and Methods: We have employed a transplantable luminal
B breast cancer genetically engineered FGFR1/Wnt1-induced mouse mammary tumor
model to study the mechanisms involved in dormancy, resistance and tumor recurrence.
Tumor bearing mice treated with a pan-FGFR specific inhibitor BGJ398 resulted in rapid
tumor regression due to both inhibition of proliferation as well as increased apoptosis.
Results: Following 10 days of treatment with BGJ398 the tumors disappeared,
and treatment was stopped. After approximately 60 days of dormancy, the tumors
recurred. The recurrent tumor cells were still sensitive to BGJ398 treatment, and again
could be eliminated following treatment, but again recurred when treatment was
stopped. Interestingly, the second recurrence occurred sooner (25 days) than the first
recurrence. Western blot analysis shows that the recurrent tumors had enhanced
phosphorylation of the mTOR kinase and 4EBP1 as compared to the primary tumors,
suggesting that there are escape pathways activated to circumvent the effect of
Conclusion: In summary, we have demonstrated that mTOR kinase can be
phosphorylated through FGFR1 signaling and that the increased activation of the
protein translation pathway is correlated with recurrence. Future studies are focused on
the molecular characterization of the dormant cells after BGJ398 treatment. Supported
by NIH grant CA16303.
Contributors: Holdman, Xue; Rosen, Jeffrey
Matthew Valle Holt
Department of Biochemistry & Molecular Biology
Advisor: Jun Qin, Ph.D.-Department of Biochemistry & Molecular Biology
Recent advances in stem cell research have shown intricate and robust
mechanisms which are responsible for the establishment and maintenance of
pluripotency: the ability to generate any embryonic germ layer. The addition of only four
transcription factors is capable of reprogramming somatic cells into a pluripotent state.
Embryonic stem cells lack the capability of significantly contributing to extra-embryonic
lineages such as the trophectoderm, and thus are different than totipotent cells which
can contribute to both. However, the addition of two inhibitors (CHIR99021 and PD
0325901) has been shown to cause embryonic stem cells to express a trophectoderm
marker in conjunction with pluripotency markers and actively contributing both
embryonic and extra-embryonic lineages. The primary objective of this project is to
understand the key players involved in the response to this inhibitor treatment and
which are the key transcription factors involved in totipotency. Transcription factor DNA
binding activity and abundance can be determined at the protein level by using
transcription factor response elements as an affinity reagent to pull-down DNA binding
proteins (catTFRE pull-down). These proteins can be subsequently analyzed by liquid
chromatography and mass spectrometry, and through our database software package
(iSPEC and Align!) to determine which transcription factors and associated proteins are
activated in a totipotent state. We have identified 436 DNA binding transcription factors
in pluripotent and totipotent stem cells. Furthermore, distinct transcription factor patterns
arise from each individual inhibitor treatment potentially underscoring the transcription
factors directly involved in the transition from pluripotency to totipotency.
Contributors: Holt, Matthew; Yucer, Nur; Choi, Jong Min; Jain, Antrix; Kim, Beom-Jun; Chan,
Doug; Malovannaya, Anna; Wang, Yi; Qin, Jun
Chuan Hong
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Wah Chiu, Ph.D.-Department of Biochemistry & Molecular Biology
The functional and structural knowledge of assembly principles of viruses, often
dsDNA viruses composed of only proteins and nucleic acids, have extended our
understanding of viral capsid maturation and genome packaging processes. Viruses
with lipids possess an additional complexity when exploring the mechanistic and
structural properties of such fundamental functions.
PRD1 phage, which is known to infect Salmonella typhimurium, is a good
candidate for a possible antibacterial agent. PRD1 is an icosahedral dsDNA bacterial
virus with an inner membrane (Tectiviridae family). Based on previous X-ray
crystallographic results of PRD1, its major capsid protein has similar fold as those of
several other viruses such as adenovirus, PBCV-1 and STIV. However, the structure of
the non-icosahedrally arranged portal complex anchored in the inner membrane
remains elusive. Biochemical and immuno-electron microscopic studies have identified
four proteins in the portal complex: the packaging ATPase P9, the packaging efficiency
factor P6, and the integral membrane proteins P20 and P22. The goal of our study is to
reveal the structures of the portal machinery of PRD1.
We used single-particle electron cryo-microscopy (cryo-EM) to study the mature
virion and three procapsid mutants of PRD1 using symmetry-free reconstructions. Their
density maps allow us to conclude the locations and features of the four portal proteins
at a unique vertex. The P20 and P22 form a hexamer of dimers embedded in the viral
membrane and function as a conduit for the DNA packaging. The P20 or P22 cannot
exist alone without the other. The P6 and P9 form a 12-mer of a portal complex with
ATPase activity similar to other phage portal protein complex. This is the first structural
evidence of the PRD1 packaging complex operating at a specific vertex, and shows the
connection between the membrane and the capsid shell providing a conduit for DNA
translocation in an ATPase-driven reaction.
Contributors: Hong, Chuan; Liu, Xiangan; Jakana, Joanita; Oksanen, Hanna; Bamford, Dennis;
Chiu, Wah
Stanley Eugene Hooker
Department of Molecular & Human Genetics
Advisor: Suzanne Leal, Ph.D.-Department of Molecular & Human Genetics
With the advent of next generation sequencing there is great interest in studying
the involvement of rare variants in complex trait etiology. For many complex traits
sequence data is being generated on DNA samples from African Americans and
Hispanics to elucidate rare variant associations. Analyses of admixed populations
present special challenges due to spurious associations which can occur because of
confounding. However using information on admixture and local ancestry can also be
highly beneficial and increase the power to detect associations in these populations.
Here a local haplotype sharing (LHS) method (Xu and Guan 2014) was extended to test
for rare variant (RV) associations in admixed populations. Previously the Weighted
Haplotype and Imputation-based Test (WHAIT) (Li et al. 2010) was proposed to test for
rare variant associations using haplotype data. The RV-LHS method unlike WHAIT,
does not require reconstruction of haplotypes which can be both computationally
intensive and error prone. Additionally the RV-LHS uses information on local ancestry
which is particularly advantageous when analyzing admixed populations. Results will be
shown from simulation studies performed for rare variant data from an admixed
population. Both Type I and II errors are evaluated for the RV-LHS method. Additionally
the power of the RV-LHS method is compared to WHAIT as well as several other nonhaplotype-based rare variant association methods including the combined multivariate
collapsing (CMC) (Li and Leal, 2008), Variable Threshold (VT) (Price et al. 2010) and
Sequence Kernel Association Test (SKAT) (Wu et al. 2010). Several heart, lung and
blood phenotypes were analyzed using sequence data on African-Americans from the
NHLBI-Exome Sequencing Project to better evaluate the performance of the RV-LHS
compared to other rare variant association methods.
Contributors: Hooker, Stanley; Wang, Gao T; Li, Biao; Guan, Yongtao; Leal, Suzanne M
Benjamin Daniel Hornstein
Department of Molecular Virology & Microbiology
Advisor: E. Zechiedrich, Ph.D.-Department of Molecular Virology & Microbiology
Gene therapy requires the delivery of nucleic acid to replace, regulate, or correct
genes to treat diseases. Viral vectors deliver DNA efficiently to cells, but are often
unsuitable for therapeutic applications because their integration into the host genome
can disrupt or dysregulate essential genes or proto-oncogenes. Nonviral vectors do not
have these issues, but many human cell lines are refractory to transfection with plasmid.
Our laboratory developed minimized nonviral vectors that can be as small as 250 bp,
called minivectors. Minivectors are devoid of bacterial sequences, and are unlikely to be
silenced. Published and preliminary data show that minivectors transfect hard-totransfect cell types, including primary cells and cells in suspension, and cause no
During transfection, a DNA vector needs to enter the cell, translocate to the
nucleus, and be expressed to mediate an effect. Whereas minivectors get into cells
extremely well, the observed minivector-mediated knockdown was less efficient than
plasmid when the same number of molecules were transfected. I hypothesized that
smaller vectors get expressed less efficiently than larger ones. To test how vector
length affects transfection, I created DNA vectors expressing shRNA against GFP. The
lengths were 383, 735, 1,026, 1,869, 2,844, 3,913, 4,265, and 4,556 base pairs (bp). I
tested transfection efficiency by measuring GFP knockdown in HeLa cells stably
expressing GFP. By using HeLa cells, which are easily transfected, we can test how
vector length affects nuclear localization and expression without including cell entry as a
variable. Flow cytometry and fluorescence microscopy were used to quantify the data.
Indeed, the smallest vector showed the least knockdown, and the largest three vectors
showed similar knockdown. To test directly whether these differences in GFP
knockdown are a consequence of inhibition of RNA polymerase, I am quantitatively
measuring in vitro transcription. Because each clinical application is different,
understanding the effect of vector size on transfection is necessary for calculating the
amount of DNA vector needed to elicit the appropriate expression level for any given
application. The ultimate goal of this project is to find the optimal minivector size for
transfection so we can design a nonviral gene therapy vector that maximizes expression
while minimizing toxicity from transfection vehicle and vector.
Contributors: Arevalo-Soliz, Lirio Milenka; Szafran, Adam; Mancini, Michael; Zechiedrich, Lynn
Jason K Hou
Clinical Scientist Training Program
Advisor: Hashem El-Serag, M.D./M.P.H.-Department of Medicine
Purpose: Patients with inflammatory bowel disease (IBD) perceive that diet
impacts their gastrointestinal symptoms. There are conflicting data regarding the effect
of dietary modifications on IBD activity; however, patterns and patient perceptions of
food aversions among IBD patients are not well defined. Unnecessary food aversion
may lead to macro- and micro- nutrient deficiencies. The aim ofthis study was to
evaluate the frequency and patient perceptions of food aversions among patients with
Methods: A case-control study of 104 patients with an established diagnosis of
IBD and 101 healthy controls was performed. Patients with known diagnosis of
Ulcerative Colitis (UC) and Crohn’s disease (CD) were recruited in consecutive fashion
from an IBD referral center. Controls were healthy volunteers without IBD, family
history of IBD, or a diagnosis of irritable bowel syndrome. A 38 question selfadministered food frequency questionnaire was administered to patients and controls.
Chart review of all cases was performed to confirm diagnosis of IBD and obtain clinical
characteristics using a standardized abstraction form. Comparisons of patterns of use
of vitamins, supplements, and food aversions were performed using Chi square and
Fisher-exact test. Multivariate logistic regression was performed to adjust for age, sex,
and ethnicity.
Results: A total of 104 IBD cases (50 UC, 54 CD) and 101 healthy controls were
included. After adjusting for age, sex, and ethnicity, IBD patients were more likely to
take vitamin/mineral supplements (OR 5.30, 95% CI 2.41-11.66) and use liquid meal
replacements (OR 4.66, 95% CI 1.76-12.37). IBD patients were also more likely to
have food aversions overall (OR 3.49, 95% CI 1.47-8.30), and specifically to fruit/juice
(OR8.24, 95% CI 2.98-22.81), vegetables (OR 6.40, 95% CI 2.72-15.05),
pork/beef/poultry (OR 2.83, 95% CI 1.26-6.34), and peanuts/nuts (OR 11.79, 95% CI
4.00-34.80) compared to healthy controls. Exacerbation of IBD symptoms was the most
commonly patient-reported reason for food avoidance amongst IBD patients.
Conclusions: Avoidance of fruit, vegetables, meat, and nuts are more common
among patients with IBD than healthy controls. Patients perceive that these food
groups exacerbate their IBD symptoms. Dietary restrictions may increase risk of
nutritional compromise in these vulnerable patients and increase unnecessary stress
among IBD patients. Further studies on the perceptions of dietary intake on IBD
symptoms may identify adjunct therapy such as elimination or exclusion diets to control
symptoms, prolong remission, and involve patients actively in their care.
Contributors: Chen, Tien-Chun; Cruz, Guillermina; Sellin, Joseph; Hou, Jason
Corey F. Hryc
Program in Structural and Computational Biology an Molecular Biophysics
Advisor: Wah Chiu, Ph.D.-Department of Biochemistry & Molecular Biology
Structural biology plays a key role in the development of novel biological
inhibitors and drug delivery methods. Recently, structures of large macromolecular
complexes ranging from 400 kDa to tens of MDa have been solved by single particle
electron cryo-microscopy, resulting in near-atomic resolution structures, however
lacking validation. This absence of validation devalues the derived models and their
usefulness in drug discovery. Large datasets exist for these structures in various
databases, including the Protein Data Bank (PDB) and Electron Microscopy Data Bank
(EMDB), but this data must be validated to annotate the level of structural uncertainty
prior to their use in translational medicine. By understanding this structural variability,
such as atom locations and interactions between molecules, pharmaceutical chemist
can target specific domains and interfaces for drug design with confidence.
We have repurposed existing crystallographic software for our near-atomic
resolution structures determined without crystallography. In an attempt to understand
the model on a per-atom basis, we developed a number of model building and
optimization protocols to interrogate the expansive data set that comprises cryo-EM
density maps. These protocols are currently being tested with a known virus, in addition
to various unknown structures. We can then apply rigorous validation test, as in
crystallography, for structures commonly used in drug design. Using this approach, we
can analyze and improve model validity for current and future structures. Our early
results provide a statistical assessment that can be used in conjunction with fit-todensity and stereochemistry scores to assess model uncertainty.
This research was funded by a training fellowship from the Keck Center of the
Gulf Coast Consortia, on the Training Program in Biomedical Informatics, National
Library of Medicine (NLM) T15LM007093-21, PI - G. Anthony Gorry.
Contributors: Hryc, Corey; Chen, Dong-hua; Wang, Zhao; Afonine, Pavel; Baker, Matthew; Adams, Paul; Chiu, Wah
Hsiang-Ting Hsu
Department of Pathology & Immunology
Advisor: Jordan Orange, M.D.-Department of Pediatrics
Natural killer (NK) cells are cytolytic immune lymphocytes that survey the host
environment for stressed or diseased cells and are required for maintenance of human
health. NK cell activation triggers a stepwise series of events leading to degranulation of
specialized lysosomal-related organelles called lytic granules. Prior to degranulation,
preformed lytic granules rapidly converge from dispersed cytoplasmic locations to the
microtubule-organizing center (MTOC), and then polarize to the immunological synapse
(IS), which is defined as the contact site with the target cell. Once polarized, lytic
granules fuse with NK cell membrane and release their contents onto the target cell to
trigger target death. Our lab has previously identified that granule convergence requires
LFA-1 signaling and Src family kinase activities, is dependent on dynein motor function
but not on actin reorganization, and does not require commitment to NK cell cytotoxicity.
Despite these advances the purpose of lytic granule convergence in NK cell cytotoxicity
is unknown. We hypothesize that granule convergence can improve efficiency of
targeted lytic granule secretion and prevent collateral damage to neighboring tissue. By
using a Drosophila S2 cell-based expression system which, unlike mammalian cells,
does not express ligands for adhesion and activation receptors of human NK cells we
aim to control signal inputs from a target cell to precisely control granule convergence
and degranulation. Specifically, engagement of LFA-1 induces convergence of granules,
but not degranulation, whereas ligation of CD16 triggers undirected degranulation.
Thus, we propose to test our hypotheses using S2 cells expressing only intercellular
adhesion molecule (ICAM)-1 as ligand of LFA-1, S2-specific IgG as ligand for the Fc
receptor CD16, or ICAM-1+IgG. By mixing differentially labeled IgG-loaded and IgGunloaded target cells we determined the efficiency and specificity of individual NK cells
activated through LFA-1, CD16 alone, or LFA-1+CD16. Indeed, we showed that NK
cells activated by anti-S2 IgG had diffuse subcellular granule localization and undirected
lytic granule release causing non-specific killing of neighboring cells. In contrast, NK
cells triggered by S2-ICAM1 cells resulted in granule convergence but not cytotoxicity,
whereas NK cells receiving both signals killed targets more precisely and efficiently. By
understanding the purpose of granule convergence, we believe we will be able to
harness a powerful regulatory step in NK cell cytotoxicity that can be exploited to tailor
immunological therapies.
Contributors: Hsu, Hsiang-Ting and Orange, Jordan S.
Teng-Kuei Hsu
Department of Biochemistry & Molecular Biology
Advisor: Olivier Lichtarge, M.D./Ph.D.-Department of Molecular & Human Genetics
Whole genome sequencing uncovers a plethora of genetic alterations that
are often with unknown functional and clinical impact. A major challenge is to estimate
the consequences of these alterations. As a complement to traditional computational
tools that follow machine learning, statistical and biophysical modeling approaches and
trained with numerous available data, we sought to assess the impact of novel
mutations from a phylogenomic perspective, namely by relating specific genotype
variations to specific speciation events. In our retrospective data analysis, this approach
i) correlated with the loss of protein function in viral, prokaryotic and eukaryotic proteins,
ii) separated the disease-associated mutations from the benign, iii) matched the
morbidity of monogenic disorders. When this approach was tested by independent
judges at the two most recent international CAGI contests, it also consistently achieved
top ranking. In order to assess its practical value in a complex disease, we asked
whether it could prognosticate for poor outcomes in head and neck squamous
carcinoma (HNSCC), which often bear TP53 mutations. We found in a small cohort but
at a statistically significant level that cancer patients can be further stratified into
subgroups by the impact of mutations in TP53 determined by our method, such that
patients with greater deleterious impact in TP53 mutations associate with the poorest
clinical outcomes. Together, these results may open a novel and complementary
approach to assess genetic variations at the molecular level and may eventually play a
role in the stratification of cancer patients for precise treatment selection.
Contributors: Katsonis, Panagiotis; Lichtarge, Olivier
Tiffany Hsu
Integrative Program in Molecular and Biomedical Sciences/M.D.-Ph.D. Program
Advisor: Thomas Westbrook, Ph.D.-Department of Biochemistry & Molecular Biology
c-MYC (MYC) overexpression or hyperactivation is one of the most common
drivers of human cancer. Despite intensive study, the MYC oncogene remains
recalcitrant to therapeutic inhibition. Like other classic oncogenes, hyperactivation of
MYC leads to collateral stresses onto cancer cells, suggesting that tumors harbor
unique vulnerabilities arising from oncogenic activation of MYC. Herein, we discover the
spliceosome as a new target of oncogenic stress in MYC-driven cancers. We identify
BUD31 as a MYC-synthetic lethal gene, and demonstrate that BUD31 is associated with
the core components of the human spliceosome. These BUD31-associated core
spliceosomal factors (SF3B1, U2AF1, and others) are also required to tolerate
oncogenic MYC. Notably, MYC hyperactivation induces an increase in total pre-mRNA
synthesis, suggesting an increased burden on the core spliceosome to process premRNA. In contrast to normal cells, partial inhibition of the spliceosome in MYChyperactivated cells leads to global intron retention, widespread defects in pre-mRNA
maturation, and deregulation of many essential cell processes. Importantly, genetic or
pharmacologic inhibition of the spliceosome in vivo impairs survival, tumorigenicity, and
metastatic proclivity of MYC-dependent breast cancers. Collectively, these data suggest
that oncogenic MYC confers a collateral stress on splicing and that components of the
spliceosome may be therapeutic entry points for aggressive MYC-driven cancers.
Contributors: Hsu, Tiffany Y.-T.; Simon, Lukas M.; Neill, Nicholas; Marcotte, Richard; Sayad,
Azin; Bland, Christopher S.; Sun, Tingting; Dominguez-Vidana, Rocio; Kurley, Sarah J.; Tyagi,
Siddhartha; Karlin, Kristen L.; Hartman, Jessica D.; Renwick, Alexander; Bernardi, Ronald J.;
Skinner, Samuel O.; Jain, Antrix; Orellana, Mayra; Lagisetti, Chandraiah; Golding, Ido; Jung,
Sung Y.; Neilson, Joel R.; Zhang, Xiang; Webb, Thomas R.; Neel, Benjamin G.; Shaw, Chad A.;
Westbrook, Thomas F.
Gilbert Huang
Department of Biochemistry & Molecular Biology
Advisor: Choel Kim, Ph.D.-Department of Pharmacology
Cyclic guanosine monophosphate (cGMP) is a key secondary messenger that is
produced in response to nitric oxide. One of the key mediators of cGMP signaling,
cGMP-dependent protein kinase (PKG), is activated upon binding to cGMP and
phosphorylates downstream substrates in a process required for important physiological
processes such as vasodilation, nociception, and memory formation. PKGs are also
known to mediate most effect of drugs that increase cellular cGMP levels, including
nitric oxide-releasing agents and phosphodiesterase inhibitors, which are used for the
treatment of angina pectoris and erectile dysfunction, respectively. We have
investigated the mechanism of cyclic nucleotide selectivity by PKG by determining
crystal structures of the cGMP-selective carboxyl-terminal cyclic nucleotide-binding
domain (CNBD-B) of human PKG I bound to cGMP and in the apo form. Our crystal
structure of CNBD-B with bound cGMP reveals that cGMP adopts the syn configuration
in the binding pocket and is coordinated by a previously unidentified arginine residue.
Furthermore, comparison of the cGMP-bound crystal structure of the apo structure
suggests a role for a C-terminal tyrosine residue in capping the nucleotide into the
binding pocket. The interaction of this tyrosine residue with cGMP appears to result in
conformational rearrangement of the C-terminal helix, suggesting a mechanism for
kinase activation by cGMP.
Contributors: Huang, Gilbert; Kim, Jeong Joo; Reger, Albert; Casteel, Darren; Bertinetti,
Daniela; Lorenz, Robin; Zhao, Chi; Moon, Eui-Whan; Melacini, Giuseppe; Herberg, Friedrich;
Kim, Choel
Longwen Huang
Department of Neuroscience
Advisor: Benjamin Arenkiel, Ph.D.-Department of Molecular & Human Genetics
The exquisite balance between excitation and inhibition determines information
processing in neural circuits, where inhibitory neurons play significant roles in sculpting
principle neuron output and maintaining proper brain function. In prominent models of
olfactory bulb circuitry, mitral cells, the principle neurons, receive inhibitory input from
granule cells, which are considered to mediate lateral inhibition, facilitate information
processing, and/or control the gain of mitral cell activity. Additionally, we recently
reported a novel population of GABAergic interneurons located in the external plexiform
layer (EPL) of the olfactory bulb, which make reciprocal connectivity with mitral cells.
However, the connectivity map between EPL interneurons/granule cells and mitral cells,
and how these interneurons contribute to odor processing remain unknown. Using
optogenetics, electrophysiology, and fast scanning microscopy, we show that EPL
interneurons make spatially broader connectivity with mitral cells, and their activity have
subtractive effects on mitral cell’s tuning to odorants; while granule cell-mitral cell
circuitry show distinct connectivity patterns, which is much narrower and specific.
Furthermore, we are currently implementing light-assisted circuit mapping techniques
and behavioral studies to investigate the inhibitory circuit plasticity of EPL
interneurons/granule cells; these studies will provide more insight onto the principle
underlying the organization and dynamics of inhibitory circuitry in the brain.
Contributors: Huang, Longwen; Garcia Isabella; Saggau, Peter; Arenkiel Benjamin.
Teng-Wei Huang
Program in Developmental Biology
Advisor: Jeffrey Neul, M.D./Ph.D.-Department of Pediatrics
Rett syndrome (RTT) is a dominant X-linked neurodevelopmental disorder
caused by MeCP2 mutations. RTT associated with loss of communication and
purposeful hand skills as well as several autonomic deficits including abnormal
temperature regulation, decreased heart rate variability, and respiratory deficits. These
autonomic disorders may contribute to the sudden death observed in a fraction of
people with RTT. We found that MeCP2 is both necessary and sufficient within the
brainstem and spinal cord for normal lifespan, control of heart rate, and respiratory
response to hypoxia. These results suggest MeCP2 is critical within autonomic and
respiratory control centers for survival, and suggest a possible correlation between
abnormal cardiorespiratory regulation and premature lethality.
To study MeCP2 functions in specific circuits in the brainstem, HoxA4-Cre line
was generated to target caudal medulla and spinal cord. HoxA4-Cre line 1 conditional
knockout (CKO) mice have deficient motor functions, increased ventilation hypoxic
response, and early lethality. Re-expression of MeCP2 in HoxA4 domain rescued basic
motor coordination, forelimb grapping strength, and survival. HoxA4 domain including
important component of respiratory network: nucleus of the solitary tract (NTS), PreBötzinger Complex (preBötC) and ventral respiratory column (VRC). Interestingly,
although previous study indicates that losing MeCP2 function in the pontine respiratory
group results in increased baseline breathing rate, the development of this phenotype in
HoxA4 rescue animals is much slower than that in non-rescue animals. In addition,
HoxA4 rescue animals had apnea at 7 weeks old but the level of apnea was
ameliorated with age. These findings suggest that MeCP2 re-expression in the
downstream component of the respiratory network allowed it to adopt and/or resist the
mis-regulated inspiratory/expiratory transition signal and maintain a relative normal
pacing rhythm. MeCP2 expression in HoxA4 domain partially rescued the decreased
heart rate. ECG recording by implanted probe is now performed to determine the detail
of cardiac phenotype.
Contributors: Huang, Teng-Wei; Ward, Christopher; Neul, Jeffrey Lorenz
Yu-Mei Huang
Department of Neuroscience
Advisor: Matthew Rasband, Ph.D.-Department of Neuroscience
Spectrins are a family of cytoskeletal proteins, which provide structural support
of cells, link membrane-associated proteins to actin and serve as platforms for cell
signaling. Spectrins are classified into α and β subunits, forming heterotetramers to
function as a complex. Among the spectrins, αII-spectrin is highly expressed in both
neurons and myelinating glia. It is also implicated in the neurological disorder, West
syndrome, which is the infantile spasm with symptoms including cerebral
hypomyelination, epilepsy and brain atrophy. Moreover, the importance of αII-spectrin in
the nervous system is emphasized by embryonic lethality of constitutive knockout mice
with nervous system malformation.
Owing to the fact that αII-spectrin constitutive knockout mice die in utero
whereas myelination and node formation do not take place until birth, there is no mouse
model to study αII-spectrin in myelination and node-related structure formation.
Therefore, I generated αII-spectrin conditional knockout (cko) mice to bypass the
embryonic lethality and to identify the roles of neuronal and glial αII-spectrin.
When αII-spectrin is deleted in the CNS (Nestin-cre), mutant animals have
profound phenotypes and die perinatally. Nestin-cre cko mice are smaller and weaker
and they have motor function deficits. Besides, immunohistochemistry shows that
neurodegeneration occurs throughout the brain and cko mice have defects in neuronal
migration in the cerebral cortex while Purkinje cells are dramatically reduced in the
cerebellum. Furthermore, axon initial segments are fragmented and remarkably
reduced. However, the role of αII-spectrin in CNS myelin and node formation remains
unknown due to the early perinatal lethality.
To eliminate αII-spectrin exclusively in myelinating glia, αII-spectrin floxed
mice were crossed with CNP-cre mice. Surprisingly, myelin and the nodes of Ranvier
subdomains can still form. In addition, βII-spectrin remains at bands of Cajal in
Schwann cells despite loss of αII-spectrin, suggesting that the spectrin network may
work in a different fashion in myelinating glia.
On the other hand, when neuronal αII-spectrin is knocked out only in the
pheriphery sensory nervous system (advillin-cre), animals have hind leg clasping and
significantly worse motor function. Immunohistochemistry shows that paranodes are
extensively disrupted and even invade into nodal regions. Moreover, potassium channel
clustering is aberrant.
This study will reveal the function of αII-spectrin and provide insights into
the different roles of spectrin networks in neurons and in myelinating glia.
Contributors: Huang, Yu-Mei; Rasband, Matthew
Redwan Huq
Department of Molecular Physiology & Biophysics
Advisor: Christine Beeton, Ph.D.-Department of Molecular Physiology & Biophysics
Autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis, are
mediated by a type of white blood cell—T lymphocytes. Current treatments for these
diseases are broad-spectrum immunosuppressants associated with life-threatening
side-effects, necessitating the development of new therapeutic strategies. The
inflammatory microenvironment in these diseases generates large quantities of harmful
reactive oxygen species (ROS). However, low levels of intracellular ROS act as
signaling molecules necessary for T lymphocyte activation. Therefore, intracellular ROS
represent attractive targets for modulating T lymphocyte activity and for treating
autoimmune diseases.
Carbon nanoparticles can scavenge ROS with higher efficacy than dietary and
endogenous antioxidants. The affinity of carbon nanoparticles for specific cell types
represents an emerging tactic for targeted therapy. Here, we show that nontoxic
poly(ethylene)-glycol-functionalized hydrophilic carbon clusters (PEG-HCCs) are the
first carbon nanoparticles to be preferentially internalized by T lymphocytes over other
splenic immune cells. We use this selectivity to attenuate T lymphocyte function in vitro
without affecting major functions of macrophages, immune cells crucial for the
physiological activation of T lymphocytes. We demonstrate the effectiveness of PEGHCCs in reducing T lymphocyte-mediated inflammation in delayed-type hypersensitivity
and in ameliorating experimental autoimmune encephalomyelitis and pristane-induced
arthritis, animal models of multiple sclerosis and rheumatoid arthritis, respectively. Our
results suggest that the remarkable selectivity of PEG-HCCs for T lymphocytes is a
novel and promising route for treating T lymphocyte-mediated autoimmune diseases
without inducing broad-spectrum immunosuppression.
Contributors: Huq, Redwan; Samuel, Errol L.G.; Lee, Thomas; Tanner, Mark R.; Khan, Fatima S; Tajhya, Rajeev B;
Inoue, Taeko; Pautler, Robia; Tour, James M.; Beeton, Christine
Amy Marie Hurwitz
Program in Translational Biology & Molecular Medicine
Advisor: Timothy Palzkill, Ph.D.-Department of Pharmacology
Robert Atmar, M.D.-Department of Medicine
Norovirus (NoV) infections cause over 96% of non-bacterial gastroenteritis and
lead to about 21 million new cases annually in the United States alone. Existing
diagnostics have significant limitations in feasibility for point-of-care applications, so
there is a clear need for more reliable, rapid, and simple-to-use diagnostic tools to
prevent epidemic outbreaks and to inform appropriate actions. In this study, phage
display technology was used to screen libraries of phages displaying random 12-mer
peptides for those that bind to NoV virus-like particles (VLPs). Since NoV strains
classified in genogroups I and II cause the majority of human outbreaks, we focused
initially on the prototypical Norwalk strain within genotype GI.1. After five rounds of
library selection against GI.1, we amplified the phage DNA from phage populations
eluted from each round for high throughput sequencing. Using an in-house
computational algorithm for analysis, a consensus motif was identified from thousands
of sequences for binding GI.1. Peptides containing this consensus motif were then
validated for their ability to bind GI.1 VLPs in multiple formats. Overall, the work here
provides a proof-of-concept for the use of high-throughput phage display sequence
analysis for the identification of peptide motifs with specific binding affinity for a target
protein of interest.
Contributors: Hurwitz, Amy; Huang, Wanzhi; Atmar, Robert; Palzkill, Timothy
Diane Smith Hutchinson
Program in Translational Biology & Molecular Medicine
Advisor: Joseph Petrosino, Ph.D.-Department of Molecular Virology & Microbiology
Stephen Pflugfelder, M.D.-Department of Ophthalmology
Background: Norovirus (NoV) pathogenesis is not fully understood, and the
associations and potential role of the gut microbiota in NoV infection have not been
described thoroughly. While the gut microbiome contributes to the development of gut
immunity, it has also been demonstrated to enhance enteric viral replication and
systemic pathogenesis. We assessed the interaction between NoV, the intestinal
microbiota, and the human host in fecal samples collected from the Norwalk virus (NV)
challenge study performed at Baylor College of Medicine.
Methods: The study population consisted of 55 individuals who participated in an
experimental challenge with NV. Fecal samples collected from all subjects at 6
timepoints (days -7, 2, 4, 8, 21, and 56) were analyzed to assess changes in the fecal
microbiota related to NV infection. Of the 55 subjects included in our study, 35 were
uninfected (including 9 non-secretors and 6 placebo) while 20 subjects were infected.
16S rDNA sequencing was performed on the V4 hypervariable region of 328 samples
using the Illumina MiSeq platform.
Results: Pre-challenge fecal microbiome composition was not associated with
susceptibility to NV infection. Following challenge with NV, infection did not induce
changes in the structure of the fecal microbiome when compared to uninfected controls.
Among infected individuals, the microbiome of long shedders was more similar to the
pre-challenge microbiome composition than that of short shedders. Additionally, long
shedders exhibited increased abundance of Subdoligranulum, a butyrate producer
generally thought to improve intestinal health, compared to short shedders. During
infection, the presence of gastroenteritis or specific symptoms was not associated with
microbiome composition. Unrelated to NV infection, secretor positive subjects exhibited
either high or low abundance of Prevotella, while secretor negative subjects only
exhibited low abundance of Prevotella.
Conclusions: These results indicate that NV infection does not alter the
composition of the fecal microbiome with the exception of short shedders. This suggests
that long shedders are able to maintain a healthy microbiome throughout infection.
Currently, we are performing metagenomic sequencing to examine gene content in long
and short shedders. Further study of the host genetics underlying shedding duration
and its association with microbiome stability may lead to better intervention/prevention
strategies for NoV infection.
Contributors: Hutchinson, Diane S; Ajami, Nadim J; Finkbeiner, Stacy R; Neil, Frederick H;
Opekun, Antone R; Metcalf, Ginger A; Muzny, Donna M; Gibbs, Richard A; Graham, David Y;
Atmar, Robert L; Estes, Mary K; Petrosino, Joseph F
Regis Aaron James
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Chad Shaw, Ph.D.-Department of Molecular & Human Genetics
Genome wide genotype data are increasingly important in the molecular
diagnosis of human disease. However, interpretation of these genome wide data is
challenged by the large number of variants observed in individual patients. We
hypothesize that efficient integration of available clinical data characterizing patient
indications for genetic testing can improve the diagnostic workflows, physician
communication with the diagnostic lab and, ultimately, prioritization of variants. We
have developed scalable quantitative analysis tools for mining indication content.
Fundamentally, our approach exploits semantic similarity computed on patient
phenotypes to comprehensibly represent patient features in simple composite scores.
Our tool can help to elucidate cohort substructure by segmenting large, heterogeneous
groups of subjects into subgroups based on shared multidimensional phenotypes.
Identification of this structure can help to identify and establish cohorts for associative or
experimental study, accelerating the investigation of genetic variation. Identification of
cohorts can also lead to discovery of novel functional variants that drive complex
phenotypes. We have also developed a prototype graphical interactive web-based
viewer exploiting our computational toolkit. This viewer can aid decision support
through visual query and analysis of structured input data. This tool can help simplify
identification of clinically relevant variants by facilitating indirect matching of patient
phenotypes to cataloged variants already known to be associated with semantically
similar patients or reported OMIM diseases. We use our tools, in the context of the
Human Phenotype Ontology representation of patient indications, to analyze a pilot
cohort assembled from retrospective data from the Whole Genome Laboratory at Baylor
College of Medicine. We demonstrate the performance of our method for matching to
known diseases by comparing our computational OMIM disease matches for subjects
against the findings reported in exome reports. These tools can help improve the
quality of clinical care by more effectively utilizing available indication data, increasing
the value of the electronic health record (EHR), and better illuminating the
correspondence between the requisition content and the ultimate genetic diagnosis.
Contributors: James, Regis; Bainbridge Matthew; Eng, Christine; Shaw, Chad
Kelsey Elizabeth Jarrett
Integrative Program in Molecular and Biomedical Sciences
Advisor: William Lagor, Ph.D.-Department of Molecular Physiology & Biophysics
Obesity is a major problem in the United States; it is estimated that over 34%
of adults are obese and at increased risk for cardiovascular disease, stroke, and
diabetes. While several key genetic factors contributing to obesity are known, far less is
known of genes that contribute to leanness. ARE2 Required for Viability 1 (ARV1), a
transmembrane protein of the endoplasmic reticulum (ER), is a putative sterol
transporter. Loss of ARV1 in yeast results in buildup of early sterol intermediates and
ceramides in the ER, as well as reduced synthesis of complex sphingolipids. Yeast
lacking Arv1 have disorganized ER structure, increased ergosterol in the ER
membrane, and constitutive activation of the unfolded protein response. We found that
germline deletion of Arv1 in mice results in a 25% reduction in body weight, greatly
reduced white adipose tissue, increased energy expenditure, and improved glucose
tolerance. These data are mirrored in mice with conditional deletion of Arv1 by AP2-cre,
a cre recombinase primarily targeting adipose tissue; however, AP2-cre also deletes in
other tissues, particularly the brain. In contrast, deletion of Arv1 with Adiponectin-cre,
which is adipose-specific and does not delete in brain, fails to reproduce the phenotype
observed with AP2-cre mediated deletion. Given the importance of cholesterol and
sphingolipids in neuronal function, we hypothesize that loss of Arv1 in the brain may
alter sympathetic outflow to control whole body energy expenditure. To test this, we are
generating mice with targeted deletion of Arv1 in key neuronal populations.
Contributors: Gupta, Rajat; Fields, David;
Hsin-I Jen
Program in Developmental Biology
Advisor: Andrew Groves, Ph.D.-Department of Neuroscience
The majority of hearing loss is due to irreversible hair cell death in the cochlea of
the inner ear. Hair cells (HCs) are the sensory receptor cells for sound, and are
surrounded by supporting cells (SCs). During ear development, the differentiation of
HCs requires a transcription factor, Atoh1, which is evolutionarily conserved and is
necessary and sufficient for hair cell formation. With the ability to generate cochlear
HCs from SCs. Atoh1 based gene therapy has thought to be a promising treatment of
deafness. However, its ability to induce trans-differentiation is limited to early postnatal
stages. Ectopic expression of Atoh1 in mature SCs does not induce trans-differentiation.
To successfully regenerate HCs in the adult cochlea, it is important to understand the
mechanisms that prevent Atoh1 from inducing HCs in the mature animal. Since Atoh1 is
a transcription factor, we hypothesize that Atoh1 is unable to activate at least some HCspecific genes in adult SCs. This may be due to the alterations in the epigenetic state of
its target genes, or absence of transcriptional co-activators, or both.
We have identified 10 Atoh1 direct target genes in the mammalian ear using
RNA-seq, in situ screen and Atoh1 ChIP-PCR. We aim to study how these targets are
epigenetically regulated in neonatal and adult supporting cells. In addition, we are also
studying another important HC specific transcription factor, Gfi1, which has been shown
to synergize with Atoh1 and recruit repressive chromatin remodeling complexes. Our
preliminary data shows that Gfi1 may interact with Atoh1 and alter Atoh1 transcriptional
activity. Base on these finding, we hypothesize that Gfi1 might be an important factor in
HC formation and maturation, possibly through interaction with Atoh1. Contributors:
Xiqian Jiang
Department of Pharmacology
Advisor: Jin Wang, Ph.D.-Department of Pharmacology
Glutathione (GSH) plays an important role in maintaining redox homeostasis
inside cells. Currently, there are no methods available to quantitatively assess the GSH
concentration in live cells. In order to minimize the perturbance on the biological system
in live cell imaging, the probe concentration needs to be significantly lower than the
analyte concentration. Because of this, any irreversible reaction-based GSH probe will
exhibit the maximum response regardless of the GSH concentration. A reversible
reaction based probe with an appropriate equilibrium constant allows measurement of
an analyte at much higher concentration, and thus is a prerequisite for GSH
quantification inside cells. In this contribution, we report the first fluorescent probe
CouBro for quantitative imaging of intracellular GSH in live cells and demonstrate the
importance of using reversible reactions for ratiometric probe design in order to
quantitatively measure the analyte concentrations in biological systems.
Contributors: Jiang, Xiqian; Yu, Yong; Zhao, Mingkun; Matzuk, Alexander J.; Chen, Jianwei;
Tan, Xiao; Sizovs, Antons; Wang, Meng C.; Wang, Jin
Jennifer Leigh Johnson
Department of Neuroscience
Advisor: Mauro Costa-Mattioli, Ph.D.-Department of Neuroscience
As our population ages, cognitive decline and dementia are becoming more
prevalent. In the United States, there are currently 4.7 million people with dementia. By
2050, the number is expected to double. Memory declines with age indicating crosstalk
between these two processes. Yet, mechanisms are not fully understood nor have
shared components been identified. This information is critical as different mechanisms
of lifespan extension may benefit certain functions but be detrimental to others such as
cognition. One important example is the Target of Rapamycin Complex 2 (TORC2)
signaling pathway. Given the evolutionary conservation of TORC2 between flies and
humans and the relatively short lifespan of Drosophila melanogaster, we measured
lifespan in flies lacking rictor and sin1, essential components of TORC2 formation. We
found that TORC2 mutants are significantly long lived, suggesting that TORC2
regulates lifespan in the fly. In addition, given that TORC2 is required for long-term
memory (LTM) and mTORC2 activity declines with age in mice, we asked whether
TORC2 dysregulation contributes to age-related memory impairment. Intriguingly, we
found that a specific small molecule that activates TORC2, A-443654, restored the
impaired LTM in aged wild-type flies and mice. These results suggest that TORC2 plays
a role in age-related memory impairment via an evolutionarily conserved mechanism.
Ongoing work will determine if TORC2-regulated memory and aging processes can be
uncoupled by targeting distinct cell populations or neural circuitries. The knowledge
obtained in this study will lead to a better understanding of the molecular, cellular and
neuronal circuit mechanisms underlying aging and age-related cognitive impairment. In
addition, this study may also lead to the development of TORC2-based treatments to
boost memory in the old but also enhance lifespan.
Contributors: Johnson, Jennifer; Huang, Wei; Dierick, Herman; Roman, Gregg; Costa-Mattioli,
Alyssa N Johnston
Program in Translational Biology & Molecular Medicine
Advisor: Yi Li, Ph.D.-Department of Molecular & Cellular Biology
In 2014, approximately 297,000 new breast cancer cases will be diagnosed in the
United States alone with an estimated 39,620 deaths by year’s end. Breast cancer is
the most common cancer among women, as well as the second leading cause of
cancer death of women in the United States; reducing breast cancer incidence will have
a profound impact on saving lives and reducing the tremendous cost associated with
treatment. Epidemiological studies indicate that there is a link between increased risk of
breast cancer and pregnancy. Previous studies in the Li lab discovered that pregnancy
stimulates the progression of pre-existing early lesions to cancer via activation of
STAT5, which is normally expressed during pregnancy to maintain lactation. In normal
breast epithelium; pSTAT5 is down regulated after lactation to allow the breast to return
to a more normal, pre-pregnancy state. However, some pre-cancerous lesions can
activate STAT5 in response to pregnancy and lactation hormones and aberrantly
maintain high levels of pSTAT5 even after involution. The prolactin receptor (PRLR) is
an upstream regulator of pSTAT5 that is activated during pregnancy and later
inactivated following lactation. After ligand-mediated activation of PRLR, Janus kinase
(JAK) family tyrosine kinases can phosphorylate and activate STAT family proteins to
transduce cytokine-mediated signals. We believe that treatment of antipsychotic drugs
that suppress dopamine can increase breast cancer risk as these drugs remove the
negative feedback of prolactin. Based on previous studies we hypothesize the following:
Pregnancy promotes breast cancer via aberrant maintenance of PRLR-JAK/STAT
pathway and drugs that increase PRL levels, such as Pimozide, increase breast cancer
incidence while drugs that block PRLR/JAK/STAT signaling can prevent it. Aim one:
Determine the role of PRLR in pSTAT5 maintenance in parous early lesions. PRLR will
be selectively knocked down and then separately over expressed in our mouse model
to determine its effect on breast cancer development in the presence of an oncogenic
insult. Aim two: Elucidate the role of antipsychotic drug, Pimozide, on breast cancer risk.
Nulliparous mice were burdened with an oncogenic insult and then treated with either
Pimozide or DMSO control. Aim 3: Examine the dosage and efficacy of the clinically
used JAK inhibitor Ruxolitinib. We are currently testing whether Ruxolitinib treatment for
a short window of time can prevent STAT5 dependent parity-stimulation of
tumorigenesis in mice. This will also move to a phase two clinical trial in high risk
Contributors: Johnston, Alyssa; Li, Yi
Jason T Kaelber
Department of Molecular Virology & Microbiology
Advisor: Wah Chiu, Ph.D.-Department of Biochemistry & Molecular Biology
The family Reoviridae contains dsRNA viruses with 9-12 genome segments. In
some turreted reoviruses, 260 copies of the peripheral trimer protein cover the inner
shell with a T=13l second layer. In others, as few as 60 copies of the trimer are present.
Presently, the only exception has been the genus Cypovirus which lacks trimer
altogether; Yu and co-workers (2011) have proposed that an extra domain in the T=2
major capsid protein compensates for its absence by stabilizing the single-shelled
We describe the isolation and characterization of the first natural isolate of a
nine-segmented reovirus, tentatively designated Fako virus. Fako virus is a mosquitospecific reovirus that was isolated from several mosquito species and there is evidence
of vertical transmission of Fako virus in nature. Fako virus is a member of the genus
Dinovernavirus, sister clade to Cypovirus. We obtained a cryo-EM structure of Fako
virus capsids at subnanometer resolution. Like Cypovirus, Fako virus is a single-layered
turreted reovirus and has no peripheral trimers. However, it does not contain the
compensatory extra domain found in Cypovirus. Fako virus lacks the clamp protein at
the icosahedral three-fold axis, even though this feature is conserved among all other
turreted reoviruses.
Evolutionary analysis reveals that Dinovernavirus and Cypovirus evolved from a
more complex double-layered ancestor through stepwise loss of structural features. The
Fako virus capsid contains fewer polypeptides than any other reovirus capsid and does
not compensate with domain insertions. The structure and genome of Fako virus
demonstrate the dispensability of many conserved reoviral features previously
Contributors: Kaelber, Jason; Auguste, A. Jonathan.; Liu, Xiangan; Fokam, Eric; Guzman, Hilda;
Jakana, Joanita; Tesh, Robert; Weaver, Scott; Chiu, Wah
Deepti Karandur
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: B. Pettitt, Ph.D.-Biochemistry
Jianpeng Ma, Ph.D.-Department of Biochemistry & Molecular Biology
Intrinsically disordered proteins (IDPs) are proteins that do not fold into a stable,
three-dimensional, structure, and may only undergo ordering when interacting with other
molecules. IDPs tend to be rich in amino acids like glycine, which favor the proteins’
disorderliness. Hence, oligoglycines are a good model to study the behavior of IDPs in
aqueous solvent. Experimentally, the solubility of oligoglycine in water decreases as its
length increases until, when the peptide contains 5 glycines, it aggregates and falls out
of solution at mM concentration.
We present results of large scale simulations of over 3 million atoms of several
hundred short (five residue) oligoglycines at varying concentrations in explicit solvent.
We find that intermolecular interactions between oligoglycines are favored over
interactions between oligoglycine and water, leading to their aggregation, viz.
concentration effects play a significant role in driving oligoglycines to aggregate and/or
collapse. However, the interaction driving peptide associations, liquid-liquid phase
separation, are not predominantly hydrogen bonding. We hypothesize that the
thermodynamics of aggregation of short oligoglycines is equivalent to the
thermodynamics of collapse of longer oligoglycines and similar disordered domains in
water. We compare the aggregation of short oligoglycines with the collapse of longer,
single oligoglycines in water.
Contributors: Karandur, Deepti; Pettittt, B. Montgomery
Akash Kumar Kaushik
Department of Biochemistry & Molecular Biology
Advisor: Arun Sreekumar, Ph.D.-Department of Molecular & Cellular Biology
Androgen signaling is the central modulator of prostate cancer (PCa) development and
progression. UDP-glucuronosyltransferases (UGTs) are the major glucuronidation enzymes
known to participate in the inactivation of androgens in prostate cancer. Previously, we
nominated UGT2B28 to be associated with biochemical recurrence in PCa patients and verified
its higher expression in castration-resistant or metastatic PCa compared to androgen
dependent (AD) localized tumors. To investigate the role of UGT2B28 in AD PCa we knockdown (KD) the gene using two independent lentivirus shRNA clones in LNCaP PCa cells. The
KD resulted in an increased expression of androgen receptor (AR), elevated levels of
testosterone and its precursors as well as elevated levels of prostate specific antigen (PSA), a
classical AR regulated gene. Consistent with these, microarray analysis suggested increased
expression of genes involved in biosynthesis of androgens from cholesterol and concomitant
down regulations of genes regulating the conversion of cholesterol to bile acids. This apparent
increased flux towards steroid biosynthesis was consistent with low steady state levels of bile
acids in the KD cells. Interestingly, the KD cells exhibited significantly higher rate of proliferation
and migration in vitro while forming higher number of tumors in vivo. Mining the microarray data
further suggested that this tumor promoting function of UGT2B28 is potentially caused by
rewiring of metabolism towards a more bioenergetic state constituted by altered mitochondrial
activity. Targeted metabolic analysis showing elevated levels of citrate, isocitrate, glutamate and
glutamine further supported the increased mitochondrial activity. In addition to increase in
energy production, the microarray data also described the existence of a senescence signature
in the KD cells. Androgen action on prostate cancer cells has been long known to cause
proliferation and more recently suggested to be involved in inducing senescence. This is most
evident in the biphasic effect seen in LNCaP prostate cancer cells treated with higher
concentrations of androgen. This alludes to the importance of fine tuning androgen levels during
PCa development with higher levels potentially causing inhibition of cell growth or senescence.
Our data described above suggests higher levels of testosterone in UGT2B28 KD cells. In light
of the microarray-derived senescence signature, we reasoned that the KD cells may be more
sensitive to growth inhibition by low levels of exogenous androgens compared to controls, which
was indeed the case when tested. This novel finding is currently being validated in vivo. If
verified this will reveal a new therapeutic modality for early stage PCa involving down regulation
of UGT2B28 expression to perturb the androgen homeostasis coupled to testosterone therapy
to induce tumor growth arrest. Overall, our data for the first time suggests UGT2B28 to be a key
regulator of androgen levels in prostate cancer, modulating its proliferative and anti-proliferative
Contributors: Kaushik, Akash; Sonavane, Rajni; Putluri, Vasanta; Manikkam, Mohan; Gohlke,
Jie; Nagireddy Putluri; Nancy, Weigel; Michailidis, George; Palapattu, Ganesh; Sreekumar, Arun
Sara Elizabeth Kee
Department of Neuroscience
Advisor: Huda Zoghbi, M.D.-Department of Pediatrics
Learning and memory deficits are characteristic of Intellectual Disability
Disorders (IDDs), however, little is known about how this phenotype arises in so many
disorders with vastly different genetic causes. Our hypothesis is that different genetic
defects can cause functional deficits in the hippocampal (HP) circuit, during at least one
of the stages of memory processing: memory formation, consolidation, or retrieval. HP
neurons (place cells) encode spatial memories by firing at specific locations (place
fields) in an environment. Thus, to test our hypothesis we used tetrode recording to
examine the HP place cells in the CA1 region of naturally behaving animal models of
two clinically similar IDDs that are caused by different genes, Rett Syndrome (RTT) and
Angelman Syndrome (AS). Available rodent models of RTT (Mecp2+/-) and AS
(Ube3amat-/pat+) recapitulate the learning and memory deficits observed in human
patients. We have implanted tetrode hyperdrives on four pairs of RTT mice with wildtype (WT) controls and three pairs of AS mice with WT controls. Preliminary
examination of AS model place fields suggests reduced spatial information (SI) of place
fields as animals run on a novel linear track (p = 0.0001, rank-sum) as compared to WT
animals. There was also reduced correlation between place fields in a familiar open field
with a cue card and the same field with the cue card removed in AS mice, compared
with that in WT mice (Pearson correlation, p = 0.03, rank-sum). These results suggest
impaired memory formation and recall, respectively, in AS mice. Preliminary results
from RTT mice suggest that the amplitude of ripples, high frequency, high amplitude
events that occur in the local field potential of HP CA1, was decreased during slowwave sleep (p < 0.0001, rank-sum), as compared to WT. This suggests a deficit in
memory consolidation in RTT mice. Therefore, spatial memory code is disrupted in both
animal models, but in a different fashion. Our data suggest that genetic alterations in
both the Mecp2 and Ube3a genes can cause functional changes in HP, however, the
seemingly similar behavioral deficits caused by these two genetic manipulations are
mediated by different circuit mechanisms within the HP.
Aaron Josef Kelly
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Tsz-Kwong Man, Ph.D.-Department of Pediatrics
Osteosarcoma (OS) is the most common malignant bone tumor in children and
adolescents. Although survival rates have been relatively constant at 60% to 70% over the past
few decades, the prognosis of patients with metastasis at diagnosis is much worse.
Identification of biomarkers besides clinically observable metastatic lesions will help to facilitate
the risk-stratification of patients more effectively and identify the most appropriate treatment
option to improve their outcomes. However, due to complex and highly variable genomic
characteristics, coupled with a relatively low prevalence inherent in childhood cancers, it has
been exceedingly difficult to discover tumor biomarkers in OS. Circulating cytokines and
chemokines provide an excellent alternative to tumor biomarkers since they can be detected
non-invasively through patients’ peripheral blood samples. Levels of these known cancerrelated proteins may be tumor derived or they may correspond to immune responses that occur
during tumor pathogenesis. In this study, a luminex assay was employed to analyze 39
chemokines/ cytokines in two cohorts comprised of totally 290 OS patients. A regressive
partitioning method was used to binarize the variables in the training cohort (40 samples). 24 of
the candidate biomarkers were significant with respect to overall survival. The cutoffs derived
from the training cohort were applied to the validation cohort (250 samples) for each of the 24
variables. After Benjamini-Hochberg correction, CXCL10 significantly correlated with overall
survival (p=.047). After controlling for the known prognostic factor, i.e. initial metastasis,
CXCL10 remained significant (CXCL10: p=0.0037, Metastasis: p=0.00012), indicating that
CXCL10 is an independent prognostic factor. Furthermore, when the two prognostic factors
were combined, we found that the patients with no initial metastasis and low CXCL10 had a
71% survival rate, those with initial metastasis and high CXCL10 had 31% survival rate and
those with one or the other had 54% survival rate at 5 year follow-up. In the non-metastatic
patients alone, CXCL10 levels were able to further significantly risk stratify patients (p=0.019),
indicating that high CXCL10 can help to identify high-risk patients that did not have detectable
metastatic lesions at diagnosis. Lastly, compared to non-tumor controls, the “high CXCL10”
group of patients had significantly higher protein expression (p=3.4 e-05) whereas “low
CXCL10” patients were not significantly different (p=0.16). These analyses indicate that
circulating CXCL10 is a novel prognostic factor that is independent of metastasis at diagnosis.
Our results showed that they can be used in conjunction to provide a more accurate riskstratification of OS patients in a non-invasive manner. Finally, CXCL10 is a ligand of CXCR3,
which is known to be related to cancer progression and metastasis. A majority of primary OS
tumors express CXCR3, which is targetable and may lead to a personalized therapy for patients
with high levels of CXCL10.
Contributors: Kelly, Aaron; Flores, Ricardo; Nakka, Manjula; Li, Yiting; Lau, Ching; Hicks, John;
Man, Tsz-Kwong
Nicole M Kettner
Department of Molecular & Cellular Biology
Advisor: Loning Fu, Ph.D.-Department of Pediatrics
Hepatocellular carcinoma (HCC), a major type of liver tumor in humans, was
previously considered a rare cancer in Western countries, but shows an average
increase of 3.5% annually among men and woman since 1992 and is one of the top
cancer-related deaths in the U.S. This increase in HCC in the U.S. parallels the obesity
epidemic, and obesity-related metabolic disorders including nonalcoholic fatty liver
disease (NAFLD) and diabetes mellitus which have recently been identified as novel risk
factors for HCC worldwide. We have previously reported that chronic disruption of
circadian light cues following a schedule similar to human night shift-work schedule
promotes tumor development, and that HCC is the second most commonly observed
primary lesion induced by circadian disruption in mice. Further study of the mechanism
of chronic circadian dysfunction-induced cancer risk has led to the discoveries that
chronic circadian disruption increases the risk of obesity by disruption of homeostasis of
neuroendocrine function, which induces liver metabolic malfunction prior to HCC
development, including a dramatic increase in the risk of hepatomegaly, NAFLD, liver
inflammation, and fibrosis. Genome-wide microarray analysis identified a large number
of deregulated pathways prior to HCC detection in the livers of mice lacking circadian
homeostasis, including all core clock genes, well-established molecular markers for
human HCC, and those controlling the biosynthesis and metabolism of redox, steroids,
vitamins, nucleotides, carbohydrates, triglycerides, bile acids, and glycogen leading to
global deregulation of liver metabolism as detected by metabolomics studies. Thus, we
conclude that chronic circadian disruption is an independent risk factor for HCC.
Contributors: Kettner, Nicole M.; Katchy, Chinenye A; Finegold, Milton J.; Moore, David D.; Fu,
Jordan Kho
Program in Developmental Biology
Advisor: Brendan Lee, M.D./Ph.D.-Department of Molecular & Human Genetics
Osteocytes are the most abundant component of bone in the body, making up
nearly 95% of all bone cells. They are derived from osteoblasts and become embedded
under the mineralized bone matrix upon differentiation. Recent studies over the past
several years unraveled the diverse physiological roles of osteocytes in regulating bone
remodeling, mineral homeostasis, mechanosensing, hematopoietic stem cell
mobilization, and most recently, fat metabolism. Despite their important roles, little is
known about the molecular mechanisms that govern osteocyte differentiation. To
identify candidate transcription factors that are involved in regulating osteocyte
differentiation, we performed TRANSFAC analysis of the promoter regions of known
osteocyte-specific genes. We identified a 5-base pair CCACA motif that is highly
enriched in the promoter region of all of these genes. This motif is known to be the
consensus binding site for Kid3 (also called Zfp354c and AJ18), a Krüppel associated
box (KRAB) domain-containing C2H2 zinc finger transcription factor. Gene expression
analysis revealed that Kid3 is highly expressed in osteoblast and its expression is
significantly down-regulated upon osteocyte differentiation. To investigate how Kid3
regulates the expression of osteocyte genes, we generated a stable osteocyte cell line
(Ocy454) overexpressing Kid3 in an inducible manner. We found that expression of
sclerostin (SOST) and Dmp1, well-known markers of mature osteocytes, were
significantly decreased in Kid3-overexpressing Ocy454 cells at the late stage of
differentiation, suggesting a potential role of Kid3 in suppressing osteocyte
differentiation. To further understand in vivo roles of Kid3 during osteocyte
differentiation, we generated transgenic mice overexpressing Kid3 specifically in
osteoblast (driven by 2.3 kb mCol1a1 promoter) or osteocyte (driven by 10 kb Dmp1
promoter). Our current work focuses on the phenotypic characterization of these
transgenic mice.
Contributors: Bertin, Terry; Chen, Yuqing; Zeng, Huan-Chang; Bae, Yangjin; Spatz, Jordan;
Pajevic, Paola; Brunetti-Pierri, Nicola; Lee, Brendan
Cynthia J Kim
Department of Molecular & Human Genetics/M.D.-Ph.D. Program
Advisor: Benjamin Arenkiel, Ph.D.-Department of Molecular & Human Genetics
Our lab aims to uncover new regulatory pathways that control activity-dependent
survival and integration of neural precursors in the adult brain. These studies may be
useful towards development of cell repair or renewal approaches in neuro-regenerative
therapy. The rodent olfactory system features robust and lifelong neurogenesis and
thereby serves as a well-characterized model to examine the mechanisms of
synaptogenesis and circuit rewiring in the adult brain. We recently reported a novel
mechanism that influences adult-born neuron circuit integration. Namely, adult-born
neurons that receive pre-synaptic input from local interneurons, which secrete the
neuropeptide corticotropin-releasing hormone (CRH), show increased propensity for
survival and integration. These studies defined a new role for CRH, apart from its wellknown functions in the systemic stress response. Microarray gene expression analysis
of gain- and loss-of-function animal models of local CRH signaling revealed that local
CRH signaling dynamically regulates expression of the brain-specific Homeoboxcontaining transcription factor Brain-5 (BRN5). Overactive CRH signaling upregulates
transcription of BRN5, whereas decreased CRH signaling downregulates transcription
of BRN5. BRN5 is described as a pro-neural differentiation factor in embryonic neural
precursors, and its expression remains high in the neurogenic olfactory areas of the
adult brain. However, little is known about the function of BRN5 in adult-born neuron
development. To elucidate the role of BRN5 in neuronal development in the adult brain,
we use biochemical, genetic, and electrophysiological techniques to test the hypothesis
that BRN5 influences synaptic maturation in adult-born neurons.
Contributors: Kim, Cynthia; Garcia, Isabella; Arenkiel, Benjamin R
Ik Sun Kim
Integrative Program in Molecular and Biomedical Sciences
Advisor: Xiang Zhang, Ph.D.-Department of Molecular & Cellular Biology
Background: Tumors evolve immunosuppressive microenvironment to subvert
anti-tumor immunity. Among cancer promoting inflammatory effectors, tumor-associated
myeloid cells (TAMCs) represent a heterogeneous group that predominantly
orchestrates tumor-induced immunosuppression as well as many other hallmarks of
cancer. It has become increasingly clear that these immunosuppressive mechanisms
elicited by TAMCs are a major barrier to effective anti-tumor therapies. What remains
poorly understood, however, is why and how individual tumors evolve to employ
different mechanisms to suppress anti-tumor immunological response.
Experimental Design and Methods: To delineate the immune system disorder in
the context of heterogeneous breast cancer, we utilized p53-null syngeneic mammary
tumor models that highly resemble different human breast cancer subtypes. Various
lymphoid and myeloid cells have been profiled by multi-color flow cytometry in blood,
bone marrow, and primary tumor at different stages of tumor growth.
Results: We have demonstrated heterogeneous immunosuppression phenotype
across p53-null tumor models conferred by various expansion and differentiation of
CD11b+ Gr1+ myeloid cells. Interestingly, these myeloid cells exhibited inter- and intratumoral heterogeneity in terms of the initial expression of surface antigens, cellular
plasticity upon infiltrating tumors, and functional roles in tumor progression.
Conclusions: This work highlights the dramatic phenotypical and functional
heterogeneity of TAMCs among different subtypes of breast cancer. Identification and
intervention of such tumor-specific immune aberrations will likely provide a rationale to
classify patients for different cancer immunotherapies.
Contributors: Kim, Ik Sun; Welte, Thomas; Rosen, Jeffrey; Zhang, Xiang
Maria Hyoun Kim
Clinical Scientist Training Program
Advisor: Elizabeth Chiao, M.D./M.P.H.-Department of Medicine
Objective: In July 2011, Malawi implemented Option B+ (B+), lifelong
antiretroviral therapy (ART) for pregnant and breast-feeding women. We aimed to
describe changes in service uptake and outcomes along the antenatal PMTCT cascade
post B+ implementation.
Design: Pre/post study using routinely collected program data.
Methods: We compared testing of HIV-infected pregnant women at antenatal
care, enrollment into PMTCT services, receipt of ART and six month ART outcomes
pre- (Oct 2009-Mar 2011) and post- (Oct 2011-Mar 2013) B+.
Results: A total of 13,926 (pre) and 14,532 (post) women presented to antenatal
care. Post-B+ a smaller proportion were HIV tested (99.3% vs. 87.7% post-; p<0.0001).
There were 1654 (pre) and 1535 (post) HIV-infected women identified, with a larger
proportion already known to be HIV-infected (18.1% vs. 41.2% post-; p<0.001) and on
ART post-B+ (18.7% vs. 30.2% post-; p<0.001). A significantly greater proportion
enrolled into the PMTCT program (68.3% vs. 92.6% post-; p<0.001) and was retained
through delivery post-B+ (51.1% vs. 65% post-; p<0.0001). Amongst those not already
on ART at enrollment there was no change in the proportion newly initiating ART/ARVs
(79% vs. 81.9% post-; p=0.11); although median days to initiation of ART decreased
(48d [19,130] vs. 0d [0,15.5] post-; p<0.001). Amongst those newly initiating ART, a
smaller proportion was alive and on ART six months post-initiation (89.3% vs. 78.8%
post-; p=0.0004).
Conclusion: While several improvements in PMTCT program performance were
noted with implementation of B+, challenges remain at several critical steps along the
cascade requiring innovative solutions to ensure an AIDS-free generation.
Contributors: Kim, Maria H. *1,2,§; Ahmed, Saeed*1,2; Hosseinipour, Mina C. 3,4;
Giordano,Thomas P. 5; Chiao, MD, Elizabeth Y. 5; Yu, Xiaoying 6; Nguyen, Chi6;
Chimbwandira, Frank 7; Kazembe, Peter N. 2; Abrams, Elaine J. 8,9
1Baylor College of Medicine, Department of Pediatrics, Section of Retrovirology, Houston,
Texas, USA
2Baylor College of Medicine-Abbott Fund Children’s Clinical Center of Excellence, Lilongwe,
3University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
4UNC Project, Lilongwe, Malawi
5Department of Medicine, Baylor College of Medicine, Houston TX
6Department of Pediatrics, Epidemiology Center, Baylor College of Medicine, Houston TX
7Department of HIV and AIDS, Ministry of Health, Lilongwe, Malawi
8ICAP-Columbia University, Mailman School of Public Health, New York NY, USA
9College of Physicians & Surgeons, Columbia University, New York, NY, USA
Jana Knezevic
Department of Molecular & Cellular Biology
Advisor: Jeffrey Rosen, Ph.D.-Department of Molecular & Cellular Biology
Claudin-low tumors are a highly aggressive breast cancer subtype with no
targeted treatments and a clinically documented resistance to chemotherapy. The
significant enrichment in Cancer Stem Cells (CSCs) makes claudin-low tumors an
attractive model forstudying CSC behavior in order to develop different approaches to
minimize CSC therapy resistance. The epithelial-mesenchymal transition (EMT) is one
mechanism by which CSCs arise, and its reversal may provide a potential therapeutic
approach for increasing tumor chemosensitivity. We therefore investigated the miR-200
family of microRNAs in the regulation of the epithelial state within a primary p53null
claudin-low tumor model, normally deficient in miR-200 expression, and its subsequent
impact on stem-like properties and therapeutic response in vivo. We show that
restoration of miR-200c cluster within this claudin-low model changes the epithelial
state, and consequently, impedes CSC behavior within these mesenchymal tumors.
Moreover, these state changes are accompanied by a decrease in proliferation and
differentiation status. MiR-200c expression also forces a significant reorganization of
tumor architecture, affecting important cellular processes involved in cell-cell contact,
cell adhesion and motility. Accordingly, the chemosensitivity of the p53null claudin-low
tumors was significantly enhanced upon restoration of miR200c expression, along with
a decrease in the metastatic potential. Collectively, our data suggest that miR-200c reexpression in claudin-low tumors offers a potential therapeutic application to disrupt the
EMT program on multiple fronts within this mesenchymal tumor subtype, altering tumor
growth, chemosensitivity and metastatic potential in vivo.
Contributors: Knezevic, Jana; Pfefferle, Adam; Petrovic, Ivana; Perou, Charles; Rosen, Jeffrey
Kathleen Kong
Department of Molecular Virology & Microbiology
Advisor: Ronald Javier, Ph.D.-Department of Molecular Virology & Microbiology
Human adenoviruses cause acute illnesses associated with respiratory,
gastrointestinal, and ocular infections. In addition to their use as vectors for vaccination
and gene and cancer therapy, adenoviruses also serve as tools for revealing
mechanisms of cancer due to their tumorigenic potential in experimental animals. The
human adenovirus E4-ORF1 gene encodes an oncoprotein that enhances viral
replication by activating cellular phosphoinositide 3-kinase (PI3K). While the underlying
mechanism of activation is not known, this activity depends on a complex formed
between E4-ORF1 and the cellular PDZ protein Discs Large 1 (Dlg1).
Mass spectrometry analysis of cellular proteins associated in vitro with an
E4-ORF1 protein fused to glutathione S-transferase identified the PI3K regulatory
subunit p85 as a candidate binding partner of E4-ORF1. Confirming and extending this
observation, I demonstrated that in human epithelial cells, E4-ORF1 directly interacts
with both the p85 regulatory and p110 catalytic subunits of PI3K and elevates their
levels, the latter effect of which, like PI3K activation, requires Dlg1. I also showed that
E4-ORF1, PI3K, and Dlg1 assemble into a ternary complex located at the plasma
membrane. At this site, Dlg1 co-localized with the activated PI3K effector protein Akt,
supporting the idea that the ternary complex mediates PI3K signaling. Signifying the
functional significance of the ternary complex, the capacities of E4-ORF1 to induce soft
agar growth and focus formation in cells were ablated either by a mutation that prevents
E4-ORF1 binding to Dlg1 or by a PI3K inhibitor drug. These results reveal that
E4-ORF1 interacts with Dlg1 and PI3K to assemble a ternary complex where E4-ORF1
subverts the Dlg1 oncogenic function to relocate cytoplasmic PI3K to the membrane for
constitutive activation. This novel mechanism may serve as a paradigm to understand
PI3K activation mediated by other pathogenic human viruses, including influenza A
virus, human papillomavirus, and human T-lymphotropic virus type I, which likewise
target Dlg1 in infected cells. Thus, studies of human adenovirus E4-ORF1 may yield
mechanistic insights that aid development of new therapeutics to treat both viral
infections and human cancers.
Contributors: Kong, Kathleen; Kumar, Manish; Taruishi, Midori; Javier, Ronald T.
Robert Layne Kruse
Program in Translational Biology & Molecular Medicine/M.D.-Ph.D. Program
Advisor: Karl-Dimiter Bissig, M.D./Ph.D.-Department of Molecular & Cellular Biology
Fasiha Kanwal, M.D.-Department of Medicine
Hepatitis B Virus (HBV) is a major global health problem with 350 million
people chronically infected, resulting in increased risk of cirrhosis and hepatocellular
carcinoma. In order to evaluate new vaccines, drugs, and immunotherapies against
HBV, testing in an immunocompetent small animal model resembling human infection is
crucial – a model that currently does not exist since human HBV only productively
infects humans and chimpanzees.
The holy grail of HBV research would be the establishment of a transgenic
immunocompetent mouse model permissible to all stages of natural HBV infection. After
years of limited understanding of HBV entry, the HBV field was transformed over past
two years with the discovery of the first validated receptor for HBV, sodium taurocholate
cotransporting polypeptide (NTCP). The discovery of the receptor prompted speculation
that a small animal model infectious for HBV by expressing human NTCP (hNTCP)
could be generated. This goal is similar in spirit to the successful entry and infection of
human hepatitis C virus into mice expressing human CD81 and human occludin (Dorner
et al, Nature 2013).
Unfortunately, published results by several groups have indicated that
human NTCP alone does not confer permissiveness to HBV infection in murine liver cell
lines, in contrast to the results with hepatitis delta virus (HDV) harboring the same viral
envelope (Li et al, Cell Mol Immunol 2014). Our preliminary results in mice in vivo have
confirmed these results. Given that once the HBV genome is introduced inside the
mouse hepatocyte nucleus all subsequent steps of the viral life cycle are functional, this
suggests that one or more of the steps during HBV entry and covalently closed circular
DNA (cccDNA) genome formation are restricted in mice. Our research seeks to study
the basic biology of these potential restriction factors, in order to discover the point of
block during infection. We hypothesize that HBV is not restricted at the steps of nuclear
import and cccDNA formation and amplification, but rather an intracellular fusion step is
limiting. A novel intracellular receptor triggering PreS domain rearrangement and
exposure of the fusion loop within the viral envelope may be lacking in mouse
hepatocytes. This hypothesis does not exclude, however, that these other processes
are inefficient and might be limiting as well inside mouse hepatocytes.
Our first aim will be to characterize HBV entry mechanisms using our
humanized mouse model and virion labeling in vitro. To study intracellular restriction, I
will create novel mouse cell lines to study cccDNA in the murine context.
Contributors: Kruse, Robert; Minor, Marrisa; Slagle, Betty; Bissig, Karl-Dimiter
Amy Tsu Ku
Program in Translational Biology & Molecular Medicine
Advisor: Hoang Nguyen, Ph.D.-Department of Molecular & Cellular Biology
Abdul Diwan, Ph.D.-Department of Pathology & Immunology
Embryonic stem cell signature genes are activated in various types of cancers.
Sox11, a SRY box-containing transcription factor, plays a critical role in embryonic
development. Recent studies show that Sox11 has a tumor-promoting role in mantle cell
lymphoma, breast cancer and ovarian cancer. Since I found that Sox11 expression is
reactivated in the murine skin squamous cell carcinoma (SCC), I hypothesize that
aberrant expression of embryonic gene Sox11 contributes to skin tumorigenesis. To
determine the oncogenic potential of Sox11, I tested how gain and loss of function of
Sox11 affects skin tumorigenesis using the two-stage chemical carcinogenesis model of
skin SCC. I found that Sox11 overexpression increases tumor incidence and multiplicity;
whereas Sox11 deficiency reduces tumor formation and growth. These results
demonstrate that Sox11 has a tumor-promoting role in skin SCC. I then performed
transcriptional profiling to identify downstream target genes that are altered by Sox11
overexpression. I identified Lef/Tcf genes, which are known as β-catenin DNA-binding
partners in the canonical Wnt signaling pathway, as the downstream targets of Sox11. I
also found that Sox11 overexpression increases β-catenin transactivation activity. Our
data suggest that Sox11 reactivation promotes tumor growth by altering β-catenin
activity. In summary, our novel tet-inducible Sox11 transgenic mouse model, in
combination with the previously described Sox11 conditional knockout mouse, allow us
to establish for the first time the role of Sox11 as a tumor promoter in skin SCC. Our
findings could directly impact diagnosis, prognosis and treatment of this common
malignant form of skin cancer.
Contributors: Ku, Amy T; Miao, Qi; Nguyen, Hoang
Suhrab Kurbanov
Department of Pathology & Immunology
Advisor: Leonid Metelitsa, M.D./Ph.D.-Department of Pediatrics
Our group is developing a novel therapeutic cancer vaccine platform, which
utilizes molecular machinery of Salmonella for effective delivery of tumor-associated
antigens into the cytosol of the antigen presenting cells in situ. An attenuated strain of
S. typhimurium, MvP728 has been engineered to express human survivin and the
resulted vaccine (MvP728-survivin) induced potent CD8 T cell-mediated anti-tumor
responses in a highly aggressive murine A20 lymphoma model. The vaccine therapeutic
efficacy was further enhanced in combination with an exogenuous ligand for Natural
Killer T cells, leading to complete regression of 5-day established A20 tumor grafts in
mice and tumor-free survival for at least 60 days in 8 of 8 vaccinated animals. Although
this data indicates that exogenuous stimulation of NKT cells can augment the vaccineinduced anti-tumor immune response, the mechanistic role of the NKT/APC interaction
in response to Salmonella-based cancer vaccine remains elusive. To assess the direct
contribution of NKT cells in response to MvP728, the vaccine-specific CD8 T cellmediated immune response generation were compared in Ja18-/- (lacks type-I NKT
cells), Cd1d-/- (lacks all NKT cells), and WT mice. Surprisingly, we found that NKT
deficiency significantly augments the Salmonella-based cancer vaccine-induced
immune response, resulting in higher antigen-specific CD8 T cell-mediated immune
response in the blood and spleen of Black 6 mice. We also found that NKT deficiency
results in higher persistence of the vaccine in the spleen and mesenteric lymph nodes.
In summary, our results so far show that both exogenuous stimulation with potent NKT
ligands as well as the NKT deficiency enhance the vaccine-induced immune response.
We hypothesize that exogenuous stimulation of NKT cells with potent ligands renders
NKT cells unresponsive for any stimulation in the course of vaccination imitating the
NKT deficiency. Therefore, we will further investigate the mechanism of exogenuous
stimulation of NKT cells at different time points during the course of vaccination with the
Contributors: Suhrab Kurbanov, Xin Xu, Amy Courtney, Linjie Guo, Xiuhua Gao, and Leonid
Antonina V Kurtova
Program in Translational Biology & Molecular Medicine
Advisor: Keith Chan, Ph.D.-Department of Urology
Seth Lerner, M.D.-Department of Urology
Cytotoxic chemotherapy remains a major therapeutic option for a wide spectrum
of epithelial cancers including urothelial carcinoma. While chemotherapy is highly
effective in debulking the tumor mass and improving survival, certain patients show
initial response but their tumors eventually become unresponsive after multiple
chemotherapy cycles. Currently the identity of repopulating cancer cells following
chemotherapy is unknown, and the underlying molecular mechanisms that initiate tumor
repopulation remain poorly understood. Here we hypothesize that cancer stem cells
(CSCs) may be recruited to repopulate chemotherapy induced damage within residual
tumors, similar to how normal resident tissue stem cells mobilize to wound sites for
tissue repair.
To test this hypothesis we utilized our previous data showing that cytokeratin 14
(CK14) marks the most primitive urothelial carcinoma cells and abundance of CK14+
cancer cells in patients correlates with poor survival. We found that exposure to one
cycle of gemcitabine and cisplatin (GC) chemotherapy effectively reduced the size and
growth rate of tumors in vivo. We then followed the regular clinical regimen with a gap
period to allow recovery of normal tissues and found a generalized expansion of CK14+
cancer cells in residual tumors during the time between treatment cycles. Next we
investigated whether cancer cells are stimulated to proliferate in between chemotherapy
courses to repopulate residual tumors and demonstrated the increase of proliferating
CK14+ cells. We also found that prostaglandin E2 (PGE2) released by neighboring
apoptotic cells induced CSC expansion in a paracrine manner. This repopulation could
be abrogated by a PGE2 neutralizing antibody and Celecoxib drug-mediated blockade
of PGE2 signaling. In vivo administration of Celecoxib significantly attenuated
progressive manifestation of chemoresistance in xenograft tumors, including primary
xenografts derived from a patient who failed chemotherapy.
Here we demonstrated a new mechanism by which CSCs contribute to
therapeutic resistance, via repopulating residual tumors between chemotherapy cycles.
This repopulation occurs by the recruitment of quiescent CSCs to divide and “repair”
chemotherapy-induced damage. Celecoxib blocked CSC repopulation following the first
chemotherapy cycle and consequently improved chemotherapeutic response in a
second cycle of chemotherapy. These findings reveal a new mechanism in the
development of clinically relevant chemoresistant cancer, and provide a new paradigm
to enhance chemotherapeutic response by abrogating early “wound repair”-like CSC
repopulation. Contributors: Kurtova, Antonina; Xiao, Jing; Pazhanisamy, Senthil; Ho, Philip;
Krasnow, Ross; Lay Erica; Chan, Keith
Stephanie Marie Kyle
Department of Molecular & Human Genetics
Advisor: Monica Justice, Ph.D.-Department of Molecular & Human Genetics
David Nelson, Ph.D.-Department of Molecular & Human Genetics
Metabolic dysregulation can lead to downstream pathogenesis in nearly all
tissues and organ systems. In recent decades, a large body of data has implicated
metabolic perturbations in neurological development and degeneration. In particular,
dysregulation of cholesterol trafficking and biosynthesis are responsible for the onset of
Neimann-Pick type C and Smith-Lemli Opitz syndrome, respectively. Furthermore,
Fragile X syndrome, Alzheimer, Parkinson, and Huntington diseases have all been
linked to aberrant cholesterol homeostasis. Rett syndrome (RTT) is a progressive
neurodevelopmental disorder of females primarily caused by mutations in the X-linked
gene encoding methyl-CpG binding protein 2 (MECP2). To identify pathways in disease
pathology for therapeutic intervention, we carried out a dominant random mutagenesis
suppressor screen in Mecp2 null mice1. One suppressor identifies a stop codon
mutation in a rate-limiting enzyme in cholesterol biosynthesis, which ameliorates RTTlike symptoms and increases longevity in Mecp2 null mice by altering brain cholesterol
homeostasis. Although RTT has been classically labeled a neurological disorder, these
studies suggest that a metabolic component contributes to pathology. Here we show
that the Mecp2 mutation induces metabolic defects in mice including fatty liver,
increased lipolysis, and insulin resistance in muscle and adipose. These studies inform
highly targetable therapeutic pathways relevant to treating RTT; remarkably, statin drug
administration improves motor symptoms and confers increased longevity in Mecp2 null
mice. The suppressor mutation also suggests that symptoms may be modified in
patients by mutations in genes that affect metabolism. In support of this idea, a subset
of RTT patients has increased serum cholesterol and triglycerides, independent of body
mass index. Our ongoing studies point to additional metabolic pathways that are prime
targets in the pursuit of preventing morbidities associated with Rett syndrome.
Contributors: Kyle, Stephanie M.; Buchovecky, Christie, M.; Justice, Monica J.
Andrew Marc Laitman
Program in Structural and Computational Biology and Molecular Biophysics/M.D.-Ph.D.
Advisor: Mirjana Maletic-Savatic, M.D./Ph.D.-Department of Pediatrics
Multiple Sclerosis is an autoimmune neurodegenerative disorder that affects the
central nervous system and is characterized by demyelinating lesions separated by
space and time. Conventional Magnetic Resonance Imaging (MRI) can detect
demyelinating lesions in white matter areas, but only after significant pathology has
occurs. Similarly, Magnetic Resonance Spectroscopy (MRS) is able to distinguish
metabolic changes in between the white matter of the frontal lobe in healthy controls
(CTWM), the frontal lobe in patients with MS that appears to be normal by conventional
MRI (NAWM - normal appearing white matter), and periventricular non-enhancing
lesions (NELES). Furthermore, Diffusion Tensor Imaging (DTI) has been able to identify
abnormal diffusivity in NAWM in MS patients. Each modality seems to be able to detect
abnormalities that are not detectable by normal MRI. None of the current imaging
biomarkers for MS are sensitive nor specific enough to detect the pathological process
before symptoms occur. Since MS is characterized by recurrent attacks on the nervous
system, a way to predict the subsequent attack would be incredibly useful. We propose
to integrate these different modalities into a holistic model to classify patients. In MS
patients and healthy controls, we have collected MRI, MRS, and DTI data in the same
patient. While each modality has been used to compare MS patients and and healthy
controls, no model has been able to combine all 3 imaging modalities.
Contributors: Laitman, Andrew; Liu, Zhandong; Maletic-Savatic, Mirjana
Janakiraman Kausik Lakshminarasimhan
Department of Neuroscience
Advisor: Dora Angelaki, Ph.D.-Department of Neuroscience
Single neurons in the macaque medial superior temporal (MSTd) and ventral
intraparietal (VIP) cortices are tuned to the animal’s heading direction, but their relative
roles in heading perception are unknown. We inactivated area VIP in monkeys trained
to discriminate heading and found no deficits in performance. In contrast, earlier results
demonstrated that inactivating area MSTd is detrimental to behavior. This is surprising
because responses of VIP neurons are more correlated with behavioral choices than
those in MSTd. We attempted to resolve this paradox by analyzing neural data recorded
from these two areas to evaluate potential decoding strategies, in the context of a
mathematical theory of population codes. First, we demonstrate that the observed
pattern of choice probabilities (CPs) rules out strictly optimal decoding. Second, we
evaluate the degree of optimality by considering two encoding models with
fundamentally different noise correlations, one model having extensive information that
scales with population size, and the other having information that saturates to a smaller
value. We combine each model with the observed CPs to infer decoding weights and
decoding efficiency, and predict consequences of inactivating MSTd or VIP. The
decoding model with limited information is only mildly suboptimal, whereas the model
with extensive information is terribly suboptimal. Although both population models are
qualitatively consistent with inactivation results, the latter can account for our
inactivation results only under a fragile coincidence that offers a clear and surprising
prediction for future experiments: weak inactivation of VIP should improve performance,
while strong inactivation should not change performance.
Contributors: Lakshminarasimhan, Janakiraman Kaushik; Liu, Sheng; Gu, Yong; DeAngelis,
Gregory; Pitkow, Xaq; Angelaki, Dora
Sharon Lam
Department of Pathology & Immunology
Advisor: Catherine Bollard, M.B.,B.Ch.-Department of Pediatrics
Cliona Rooney, Ph.D.-Department of Pediatrics
Anti-retroviral therapy (ARVs)(??ART??) does not eliminate HIV from latently -infected
reservoirs, has long-term toxicities and fails to fully prevent immune attenuation. Therefore there
is a need for alternative therapies that will decrease dependency on ARTVs. Previous studies
have demonstrated the safety and feasibility of infusing single-epitope specific CD8 T cells or
artificial T cell receptor- transduced T cells to HIV+ patients. However, these T cells were
restricted to a single HLA restricted epitope and had limited persistence in vivo. Hence, we
hypothesized that broadly HIV-specific T cells could be expanded from patients on ARTV to
effectively target HIV infection using a non-HLA restricted, GMP-compliant approach. We
developed a method by which PBMCs from patients on ARTV were stimulated with antigen
presenting cells pulsed with gag, pol, and nef peptide libraries (pepmixes) in the presence of
growth, Th1, and memory promoting cytokines and co-cultured with co-stimulatory K562 cells.
Starting from 60 to 100 mL of blood, T cells expanded to clinically relevant numbers
(Mean=1.62e8 cells, Range (3.72e7, 2.87e8 cells), n=7) in the presence of ART to prevent
possible viral spread during expansion. The majority of the expanded T cells had an effector
memory phenotype (CD3+CD45RO+CD62L-) with approximately 10% suggestive of a central
memory phenotype (CD3+CD45RO+CD62L+) which is important for long-term persistence of T
cells in vivo. Post-expansion, 5 of 7 patient sample lines showed specific activity to all 3 HIV
antigens in IFNγ ELISPOT assays, with the remaining 2 showing specificity to 1 of 3 antigens.
The T cell lines were broadly -specific to gag (mean=99.33 SFC/10e5 cells), pol (mean=131.11
SFC/10e5 cells) and nef (mean=337.26 SFC/10e5 cells), and polyclonal as shown by flowbased Vβ usage analysis (mean usage= 14.67 of the 24 Vβ analyzed). Importantly, T cells
expanded from both ART patients were cytotoxic, as e. Expanded T cells lysed antigen loaded
autologous targets (mean=67.55% specific lysis compared to absence of antigen: mean=0.46%
at 10:1 effector target ratio) in 51Cr release assays. Expanded T cells from ART patients also
showed a greater ability to suppress HIV outgrowthreplication in vitro compared to unexpanded
CD8 T cells when co-cultured with reactivated resting CD4+ T cells from ART-suppressed HIV+
patients, the authentic latently infected cells that define viral latent reservoirs in treated patients
compared to unexpanded CD8 T cells, which presumably contains pre-existing HIV-specific T
cells. In 5 patients on ART a statistically lower recovery of virus from resting CD4+ cells was
seen in the presence of CTLs as compared to no effectors (p<0.006 by Mann Whitney), while
the unexpanded CD8 cells showed only a modest trend towards decreased recovery that was
not statistically significant (p>0.9). We have developed robust GMP-compliant methodologies
for expanding functional HIV-specific T cells from both HIV+ patients and HIVneg for autologous
and third-party use, respectively. We now plan to translate our approach to the clinical setting
where we will testuse HIV-polyspecific T cell products as a part of a strategy to fully eradicate
HIV infectionsterilizing therapeutic for patients with HIV.
Contributors: Lam, Sharon; Sung, Julia; Cruz, Conrad RY; Castillo, Paul; Ngo, Minh T; Rooney,
Cliona M; Margolis, David M; Bollard, Catherine M
Dylan James Laug
Program in Developmental Biology
Advisor: Benjamin Deneen, Ph.D.-Department of Neuroscience
Oligodendrocytes are responsible for providing the myelin sheath that is essential
for the rapid and efficient propagation of an action potential down the axon of a neuron.
Understanding the processes that regulate the differentiation of oligodendrocytes and
their myelination of neurons is important for determining possible treatments for demyelinating disorders, such as multiple sclerosis and cerebral palsy. Specifically, the
progression of chronic multiple sclerosis is partly due to the failure of intrinsic
mechanisms for the replacement of lost myelin and the leading cause of cerebral palsy
is the loss of myelin during early post-natal due to white matter injury.
Previously our lab demonstrated that NFIA is dynamically expressed in
differentiating oligodendrocytes in the embryonic spinal cord, where it is co-expressed
with Olig2+, oligodendrocyte precursors within the pMN domain of the embryonic spinal
cord and downregulated as these precursor populations differentiate into myelinating
oligodendrocytes. These observations suggest that NFIA functions to suppress
oligodendrocyte precursor differentiation. Our lab demonstrated that NFIA suppresses
oligodendrocyte differentiation during embryogenesis in mouse, chick, and in vitro
oligodendrocyte precursor models. Also, our lab found that overexpression of NFIA in
an adult mouse model of white matter injury and remyelination, suppressed
oligodendrocyte precursor differentiation and remyelination. This, coupled with our
observation that NFIA is expressed in oligodendrocyte precursors in human multiple
sclerosis lesions and neonatal white matter injury, suggests that it may also play an
important role in the suppression of differentiation and the failure of remyelination in
these disorders.
Late embryonic lethality of the NFIA knockout mice has limited our basic studies
to embryonic stages. However, the clinical application of these findings is that reduced
NFIA expression in oligodendrocyte precursors stimulates remyelination in cases of
white matter injury and multiple sclerosis in post-natal or adult humans. Therefore,
studies in the post-natal and adult mouse are crucial to assess whether the loss of NFIA
stimulates remyelination following injury. Recently, we have generated a conditional,
floxed NFIA allele that we have crossed with an oligodendrocyte precursor-specific cre
to delete NFIA in this population to study its role in adult myelination and remyelination
during white matter injury. In order to address the remyelinating capabilities of
oligodendrocyte precursors, we will use lysolecithin injection in the spinal cord of
oligodendrocyte precursor-specific NFIA knockout mice. Following lysolecithin-induced
demyelination, we will analyze for myelin repair through both staining for mature
oligodendrocyte markers, such as MAG and PLP, and observe the remyelination by
electron microscopy.
Contributors: Laug, Dylan; Glasgow, Stacey; Deneen, Benjamin
John Preston Leach
Department of Molecular Physiology & Biophysics
Advisor: James Martin, M.D./Ph.D.-Department of Molecular Physiology & Biophysics
The leading cause of death in the United States is heart failure brought on by
heart disease, and a loss of functional cardiac muscle. Because heart muscle
regenerates poorly, loss of cardiomyocytes leads to a weakening of the heart
culminating in heart failure. There is still paucity in understanding endogenous
mechanisms preventing heart regeneration; our objective is to clarify the molecular
pathways preventing meaningful cardiomyocyte renewal.
The Hippo signaling pathway and its down stream effector Yap are known
regulators of intrinsic organ size by modulating proliferation during development. Wnt/β
catenin signaling has been well characterized in stem cells, and modulation of Wnt
signaling like Hippo signaling presents an interesting potential for treating cardiac
disease. Multiple studies have established a link between Hippo and Wnt signaling
through activation and interaction of the down-stream transcription factors Yap and βcatenin. However the interaction of these two pathways during cardiac regeneration is
poorly understood. Thus, the specific effect of Hippo and Wnt signaling activity in
cardiomyocytes following myocardial infarction has yet to be determined. Thus, we
hypothesize Hippo/Wnt signaling effectors Yap/β-catenin regulate cardiomyocyte
We have used two methods of cardiac damage in the mouse: Apex resection
(AR) and LAD-ligation (MI). In a cardiomyocyte-specific inducible Salvador-knockout we
demonstrated Hippo signaling inhibits cardiac regeneration. In he same mice adult
cardiomyocytes re-enter the cell cycle as indicated by EDU assays and proliferation
markers: pHH3, AurkB, and Ki67. Furthermore, we see functional recovery and reduced
fibrosis in both the post-natal AR and MI models as well as the adult MI model. Others
have now shown similar results looking at over-expression and GOF for Yap. Our
current studies aim to clarify the role of beta-catenin during this Hippo knockout
mediated cardiac regeneration. Thus far, our findings have uncovered Hippo signaling
as an endogenous repressor of adult cardiomyocyte renewal and regeneration. Current
experiments aim to evaluate Wnt signaling using both a β-catenin LOF and GOF
Contributors: Leach, John; Heallen, Todd; Tao, Ge; Morikawa, Yuka; Zhang, Min; Martin, James
Yi-Chien Lee
Integrative Program in Molecular and Biomedical Sciences
Advisor: Brendan Lee, M.D./Ph.D.-Department of Molecular & Human Genetics
Osteogenesis Imperfecta (OI) is a brittle bone disease characterized by low bone
mass, bone deformities, and multiple bone fractures. Our laboratory and other groups
have found WNT1 mutations in OI patients. To establish a mouse model of OI caused
by WNT1 mutations, we studied swaying mice that carry a spontaneous single base
deletion in the Wnt1 gene. Interestingly, we found spontaneous bone fractures with
severe osteopenia in swaying mice. We believe that the swaying mouse model could
recapitulate the human phenotypes of OI caused by WNT1 mutations. Current
treatment options for OI mostly focus on bisphosphonate therapy. Because of its
questionable efficacy in mild OI patients and concerns about long-term administration, it
is necessary to develop a new treatment. Studies have shown that sclerostin could
directly inhibit WNT signaling through binding to LRP5/6, the co-receptor of WNT
signaling. Therefore, utilizing an antibody targeting sclerostin to enhance WNT signaling
and further increase bone formation becomes a potential therapeutic approach. We
hypothesize that anti-sclerostin antibody could be beneficial for treating WNT1 related
OI. In this study, we treat swaying mice and wild-type littermates with anti-sclerostin
antibody twice per week for six weeks. At six weeks, we assess the bone phenotypes of
mice. First, anti-sclerostin treated swaying mice exhibit reduced spontaneous fractures.
From MicroCT imaging and analysis, we find swaying mice treated with anti-sclerostin
antibody show significant increase of bone volume verses tissue volume in both femurs
and spines. Moreover, by three point bending analysis on femur, anti-sclerostin antibody
treated swaying mice show increased maximum load indicating the bone strength is
increased. Finally, we evaluate the biomechanical properties of bone and find antisclerostin antibody treated swaying mice have increased collagen in bone. The results
suggest that anti-sclerostin antibody treatment partially rescued the low bone mass
phenotype in swaying mice. Anti-sclerostin antibody may be a potential treatment for OI
caused by WNT1 mutations.
Contributors: Joeng, Kyu Sang; Bi, Xiaohong; Ambrose, Catherine; Lee, Brendan H
Yu-Ju Lee
Program in Translational Biology & Molecular Medicine
Advisor: Weei-Chin Lin, M.D./Ph.D.-Department of Medicine
Mothaffar Rimawi, M.D.-Department of Medicine
Breast cancer is the most common cancer and the second leading cause of
cancer death among American women. About 12% women in the US will develop
invasive breast cancer during their lifetime. Although the early diagnosis and target
therapy have significantly decreased the mortality rates of breast cancer, there is no
effective treatment of ER, PR and HER2 triple-negative breast cancer (TNBC) and
metastatic breast cancer. To develop a better treatment of breast cancer, we need to
understand the molecular mechanisms of the initiation and progression of breast
RNF197, also known as CGRRF1, is a p53-induced cell growth regulator with
RING-finger domain. p53 is a tumor suppressor and approximately 20~35% of breast
cancer patients have p53 mutations. Since RNF197 is implicated in the suppression of
cell growth and is regulated by p53, it might be related to cancer development. Indeed,
over-expression of RNF197 inhibits colony formation of colon carcinoma, ovarian
carcinoma and glioblastoma cell lines. However, the role of RNF197 on breast cancer is
still uncharacterized. We analyzed the correlation between the level of RNF197 and the
survival rates of breast cancer, and found that the low expression of RNF197 is strongly
correlated with a shorter survival. Compared with matched normal tissues, the level of
RNF197 is lower in several tumor types including breast cancer. The analysis of the
level of RNF197 in different subtypes of breast cancer showed a lower level of RNF197
in basal-like, mainly triple-negative, breast cancer. RNF197 contains a C3HC5 RINGfinger domain which is a binding motif of ubiquitin-conjugating enzymes. The activity of
most E3 ubiquitin ligases is specified by the RING-finger domain. However, studies
have not been done to examine the potential E3 ubiquitin ligase activity of RNF197.
Based on this information, the central hypothesis of my research is that RNF197
regulates the growth of breast cancer. In particular, I hypothesize that RNF197 inhibits
the growth of breast cancer through cell cycle regulation. To understand the mechanism
behind RNF197-inhibited cell growth, I hypothesize that RNF197 is an E3 ubiquitin
ligase and is able to degrade or regulate the biological function of its substrates
associated with breast cancer cell proliferation. Studying the potential E3 ubiquitin ligase
activity of RNF197, as well as its role in the development of breast cancer may help us
to identify potential therapeutic targets or pathways that would aid in breast cancer
Contributors: Lee, Yu-Ju; Lin, Weei-Chin
Laramie Denise Lemon
Integrative Program in Molecular and Biomedical Sciences
Advisor: Alison Bertuch, M.D./Ph.D.-Department of Pediatrics
Telomeres are nucleoprotein complexes that cap the ends of linear eukaryotic
chromosomes, protecting them from degradation, recombination and fusion, thus
contributing to genome stability. Telomeres are maintained by telomerase which, in
Saccharomyces cerevisiae, consists of an RNA component, TLC1, the reverse
transcriptase Est2, and Est1 and Est3, which are required for telomerase activity in vivo,
but not in vitro. Est1 recruits telomerase to the telomere during late S phase by
interacting with Cdc13, a single-stranded telomeric DNA binding protein. The
evolutionarily conserved Ku heterodimer also contributes to telomere length
maintenance by associating with TLC1 at a unique stem loop structure. In contrast to
strains lacking telomerase components, telomeres are short yet stable and cells do not
senesce in the absence of Ku or Ku:TLC1 interaction. Strains lacking Ku or Ku:TLC1
binding, such as yku80-135i, fail to localize TLC1 to the nucleus and experience
reduced levels of Est2 and Est1, possibly secondary to less Est2, at the telomere. Thus,
the primary role of Ku in telomere elongation has yet to be fully elucidated. Our data
support a model in which Ku’s major function in telomere length regulation is via effects
on Est1. Unlike Est2, when we tethered Est1 to the telomere via Cdc13, Ku’s role in
telomere elongation was bypassed, with telomeres progressively elongating in yku80∆
or yku80-135i strains comparable to wild type. Furthermore, we found Ku in a complex
with Est1; this interaction was dependent on Ku’s ability to bind TLC1, as the interaction
was abolished in a yku80-135i strain or upon treatment with RNase A. Although the
presence of both Est1 and Est2 at the telomere is mutually dependent, we found
tethering Est2 to the telomere via Cdc13 did not rescue Est1 levels at telomeres in a
yku80∆ or yku80-135i strain, despite partial restoration of TLC1 in the nucleus. These
data suggests that Ku’s primary role in telomere elongation lies in its interaction with
Est1, and not with Est2 recruitment or TLC1 nuclear localization. The mechanism
behind the role of Ku in Est1 recruitment to the telomere is currently under investigation.
Although the Ku:TLC1 interaction is important for Est1 localization to the telomere, it
remains unknown whether Ku binding to the DNA end is also required for Est1
association. The Est1:Cdc13 interaction at the telomere is promoted by Cdc13
phosphorylation at T308 and telomeres exhibit the same degree of shortness in yku80∆
cdc13-T308A and yku80∆ mutants. This suggests that Ku and the phosphorylation of
Cdc13 at T308 function in the same pathway at the telomere. We are currently testing
whether Ku DNA end binding activity modulates Cdc13 phosphorylation, thereby
promoting Est1 association with the telomere.
Contributors: Williams, Jaime; Ouenzar, Faissal; Chartrand, Pascal; Bertuch, Alison
Kelsey Elizabeth Lesteberg
Department of Pathology & Immunology
Advisor: George Makedonas, Ph.D.-Department of Pediatrics
Background: One mechanism by which a cytotoxic T lymphocyte (CTL) kills its
target relies on the exocytosis of specialized lysosomes called lytic granules, which
contain the pore-forming protein perforin. Traditional dogma states that a CTL may only
replenish its perforin content upon proliferation. However, it was demonstrated that CTL
may rapidly upregulate perforin within hours of stimulation, and that it is targeted to the
immunological synapse independently of lytic granules. In the current study, we aim to
define the means by which this newly synthesized perforin traffics to the immunological
Methodology: Antigen-specific CTL were generated by stimulating human
leukocytes with peptides from CMV, EBV, or influenza viruses. After 10 days of
expansion, the cells were re-stimulated with their cognate antigen for 2-6 hours. We
then utilized imaging flow cytometry to determine the intracellular trafficking mediators
involved in the transport of newly synthesized perforin. The cells were stained with
antibodies against CD8 and CD56, a panel of rab and SNARE proteins, and antiperforin clones D48 and δG9—D48 recognizes both new perforin and lytic granuleassociated perforin, whereas δG9 recognizes only granule-associated perforin. The
localization of both perforin types with rab and SNARE proteins was assessed through
the use of bright detail similarity (BDS) analysis, which assigns a score to each cell
based on the overlap of the probes of interest.
Results: We defined cells displaying significant colocalization between perforin
and trafficking molecules as those having BDS scores greater than 1.5. Newly
synthesized perforin (D48+ δG9-) localized to recycling endosome compartments, as
identified by rab8, vti1b, and the transferrin receptor (CD71). In addition, new perforin
also colocalized with rab37 and VAMP4, which are involved in transport to the synapse
and to endosomes, respectively.
Conclusions: Our data suggest that newly synthesized perforin is incorporated
into recycling endosomes, where it may be sorted to subsequent destinations: the
immunological synapse for immediate use, or to lysosomes to refill the cells’ store of
lytic granules. These results define an alternative intracellular trafficking pathway for
perforin that functions to sustain CTL cytotoxicity. Future work will investigate the
determinants of perforin sorting through this alternative pathway.
Contributors: Lesteberg, Kelsey; Makedonas, George
Kyle Joseph Edward Lewis
Department of Molecular & Cellular Biology
Advisor: Nikolaj Timchenko, Ph.D.-Department of Pathology & Immunology
Richard Sifers, Ph.D.-Department of Pathology & Immunology
Biological processes in the liver are regulated by a complex cooperation of
multiple signaling pathways. Disorganization of this cooperation is associated with
development of liver diseases such as liver cancer and non-alcoholic fatty liver disease.
We have previously found that alterations in the ubiquitin-proteasome system are
involved in development of liver cancer. A small subunit of the 26S proteasome,
Gankyrin (Gank), is activated during development of liver cancer and eliminates tumor
suppressor proteins Rb, p53, C/EBPα, and HNF4α through UPS-mediated degradation.
Quiescent livers express relatively low levels of Gank due to repression by Farnesoid X
Receptor (FXR) signaling. We found that long-lived, growth hormone-deficient mice
have expressed high levels of FXR and do not develop liver cancer with age and under
conditions of Diethylnitrosamine (DEN)-mediated carcinogenesis. Because these mice
do not increase levels of Gank, this led us to the hypothesis that Gank plays a key role
in liver proliferation and liver cancer. To further investigate the role of Gank in liver
biology, we have generated mice with liver-specific deletion of Gank, Gank LKO mice.
We first examined the role of Gank in liver regeneration after partial hepatectomy (PH).
These studies showed that the deletion of Gank leads to a dramatic reduction of liver
proliferation which is associated with much lower and delayed DNA replication and
almost complete inhibition of mitosis. We found that a powerful activator of liver
proliferation, cyclin D1, is identically elevated in both WT and Gank LKO mice; however,
this elevation is not sufficient to initiate DNA replication in Gank LKO livers. In addition,
we found that expression of PCNA and cdc2 is significantly reduced in livers of Gank
LKO mice after PH. Examination of liver injury and recovery after carbon tetrachloride
(CCl4) treatments also showed that deletion of Gank changes liver response to CCl4.
Taken together, our results showed that the FXR-gankyrin pathway plays an essential
role in liver cancer and in liver proliferation after surgical resections and injury.
Contributors: Lewis, Kyle; Jiang, Yanjun; Moore, David; Timchenko, Nikolai
Phoebe Elnora Lewis
Program in Translational Biology & Molecular Medicine
Advisor: Qizhi Yao, M.D./Ph.D.-Department of Surgery
Shital Patel, M.D.-Department of Medicine
Human Immunodeficiency Virus (HIV) continues to be a major public health
concern with almost 1.2 million people currently infected with the virus. Despite 3 phase
III clinical trials, an effective prophylactic HIV vaccine candidate has yet to be identified.
Results from previous clinical trials have revealed major safety concerns involving the
use of viral delivery vectors and their potential to increase risk of HIV infection. VirusLike-Particles (VLPs) offer a safe alternative for viral-vector based strategies as this
platform does not require the use of viral genetic material that may have the potential to
increase the risk for HIV infection.
The translatable potential of VLP based vaccines have been demonstrated
with the FDA approved Gardasil ™ vaccine against HPV. VLPs are structurally similar
to native virions and have the capability of eliciting both cellular and humoral immune
Our lab has successfully engineered chimeric VLPs containing
combinations of SIV or HIV gag, various strains of HIV env, and immuno-modulatory
molecules such as CD40 ligand (CD40L) or influenza hemagglutinin (HA). Adjuvants
provide an additional platform to further increase the immunogenicity of our chimeric
VLPs. In this study, we evaluated the potency and longevity of HIV-specific immune
responses induced by HIV GagIIIB/EnvBaL VLPs conjugated to Conjugatable Adjuvant
Lipid Vesicles (CALVs) containing a Toll-like-receptor 4 (TLR4) agonist in a C57B6
mouse model.
We demonstrate that the TLR4-agonist potently enhanced HIV-1IIIB Gag, HIV1BaL Env , and Gag/Env-VLP-specific total IgG responses up to 10-fold after intranasal
priming followed by three intracheek boosts with VLPs +/- TLR4 agonists.
Furthermore, our TLR4-agonist conjugated VLPs induced a predominant IgG1 subtype
response with levels 4-fold higher than specific IgG2a responses. Intracellular Cytokine
Staining demonstrated that Env and Gag peptide stimulation induced a potent IL-2
response, the primary cytokine involved in immunological memory formation. Mice
immunized with VLPs + TLR4-agonist had 3 fold higher IL-2 producing CD8+ cells than
the VLP only immunization group upon Env- peptide stimulation. Preliminary data also
suggests that TLR-4 agonist conjugated to VLPs can elicit persistent long-term antibody
responses as high levels of HIV-specific IgG1 were detected 90 days after the last
immunization. Our results indicate that TLR-4 agonists conjugated with our VLPs
induce potent and enduring IgG responses with a predominant IgG1 subtype.
Contributors: Lewis, Phoebe; Poteet, Ethan; Ho, Sam; Fujii, Gary; Chen, Changyi;Yao, Qizhi
Cheng-Lin Li
Department of Molecular & Human Genetics
Advisor: Gad Shaulsky, Ph.D.-Department of Molecular & Human Genetics
The cell-cell adhesion molecules, TgrB1 and TgrC1, mediate allorecognition and
cell differentiation during development in Dictyostelium. TgrB1 and TgrC1 are essential
for proper development as single-gene deletions (tgrB1-null or tgrC1-null) or singlegene replacement (e.g., replacing tgrC1AX4 with tgrC1QS45) lead to a developmental
arrest at the loose aggregation stage and lack of cell-type differentiation. Moreover,
TgrB1 and TgrC1 function as a receptor-ligand pair for kin recognition and are highly
polymorphic in natural populations. Cells carrying diverged allelic pair of tgrB1-tgrC1
segregate from one another during streaming.
The TgrB1-TgrC1 system presents a novel model to study the molecular basis of
rapid diversification of adhesion receptors. It is intriguing how adhesion receptors can
evolve rapidly despite the fact that they have essential functions in development. To
elucidate the evolvability of this system, we are dissecting the signal transduction
pathways that mediate TgrB1-TgrC1 signaling. We have devised screens for genetic
suppressors that rescue the developmental defects in tgrC1-null cells. We discovered
mutations in several candidate genes, including a glycosyltransferase (alg9) and stcA
(suppressor of tgrC1–), that suppress some of the tgrC1-null phenotypes. Inhibition of
N-linked glycosylation by insertional mutation in alg9 or by Tunicamycin treatment allow
tgrC1-null cells and the single gene replacement strain (tgrB1AX4 tgrC1QS45) to
sporulate (~10% sporulation efficiency). The mutation stcAins increases the sporulation
efficiency of tgrC1-null to 7% through an unknown mechanism. Co-development of the
suppressor mutants and the parental tgrC1-null cells rescued the development of the
latter, suggesting that suppression acts through a non-cell autonomous mechanism.
The essential function of TgrB1 and TgrC1 in development and the selection for
maintaining the compatibility between these two proteins restrict the molecular evolution
of the tgrB1-tgrC1 genes. However, genetic suppressors, such as alg9 and stcA, may
play a role in buffering the effects of novel tgrB1-tgrC1 mutations and allow the
organism to tolerate the associated fitness disadvantages. Therefore, these
mechanisms may promote allelic diversification and expand the allelic repertoires of
Contributors: Li, Cheng-Lin; Wang, Yue; Kuspa, Adam and Shaulsky, Gad
Lele Li
Program in Cardiovascular Sciences
Advisor: James Martin, M.D./Ph.D.-Department of Molecular Physiology & Biophysics
Pitx2 is localized on chromosome 4q25, variants of which are associated with
increased risk of atrial fibrillation. This locus locates close to the pituitary homeobox2
(Pitx2) gene. Pitx2 is a transcription factor, which plays a critical role in left-right
asymmetry establishment and maintenance of the heart. Our previous studies indicate
that Pitx2+/− mice show symptoms of atrial fibrillation when given programmed
electrical stimulation. However, role of Pitx2 in heart function is still not clear. Therefore,
we hypothesize that Pitx2 deficiency leads to cardiac arrhythmia and ARVC generation.
Conditional knockout mouse Pitx2flox/flox MCK-Cre mice, in which Pitx2
expression is specifically disrupted in mice cardiac/skeletal muscle after birth showed
sinus node dysfunction. ChIP-sequencing and microarray assay is done to find target
genes of Pitx2 and determine changes in genes expression levels. Finally, we find RNA
level of genes including genes related to ion channels, signaling pathways, transcription
factors is more than two fold in mutant than in wildtype mice. Majority of the genes
screened have an increase in RNA level in mutant mice, indicating Pitx2 may function
mainly as a repressor for the candidate genes. RNA level of a number of candidate
genes, which regulates cardiac function, are analyzed using qRT-PCR. Result indicated
upregulation of RNA level of majority of candidate genes and down regulation of a small
number of genes, indicating Pitx2 may function mainly as repressor in regulation of
genes transcription in cardiac function. Luciferase reporter assay was conducted and
confirmed Pitx2 targets. TEM showed that Pitx2 CKO mice have damaged mitochondria
and disrupted intercalated disc structure, suggesting Pitx2 regulates heart function by
stabilize integrity of intercalated disc structure.
Pitx2 F/+Nkx2.5-Cre adult mice showed phenotype of AV block in telemetry,
indicating the defect of Pitx2 is associated with cardiac conduction system. In situ
hybridization and immunostaining showed PItx2 F/+ Nkx2.5-Cre mice have abnormal
expression of cardiac conduction system markers, including HCN4 and Tbx3. ChIP-seq
and microarray overlay screened a set of genes related to mitochondria. Confirmation
by qPCR showed increased level of these genes in mutant mice.
Contributors: Tao, Ye; Tao, Ge; Zhang, Min; Martin, James
Tongchao Li
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M.-Department of Molecular & Human Genetics
Andrew Groves, Ph.D.-Department of Neuroscience
The auditory organs of Drosophila and vertebrates have a number of molecular
and functional similarities despite being widely separated in evolutionary time. To
identify novel genes that regulate the development and function of auditory organs, we
performed a mosaic screen of X-chromosome linked lethal mutations in Johnston’s
organ, the auditory organ of Drosophila. We identified mutations in ubr3, an E3 ubiquitin
ligase, that cause a detachment of the sensory scolopidia in Johnston’s organ. This
phenotype is similar to that seen in mutations in Drosophila myosin7a. Here, we show
that ubr3 genetically interacts with myo7a and that Ubr3 regulates Myo7a monoubiquitination via Cullin 1 (Cul1), another E3 ligase. Myosin7a mutants in humans are
associated with Usher Syndrome 1B, and we show that Ubr3, Cul1 and Myo7a
physically and genetically interact with Drosophila homologues of two other Usher
syndrome proteins, PCDH15 and Sans. Finally, we show loss of Ubr3 in zebrafish leads
to the mislocalization of three Usher syndrome proteins, Myo7a, Harmonin and
PCDH15, and causes hair-bundle and hearing defects. Our results demonstrate the
conservation of Usher syndrome protein interactions between vertebrates and
invertebrates in the assembly and function of mechanosensitive organs and identify a
novel pathway in which a cascade of E3 ligases regulates protein abundance through
Contributors: Tongchao Li, Bernardo Blanco, Nikolaos Giagtzoglou, Shinya Yamamoto, Manish
Jaiswal, Sonal N. Jaiswal, Daniel F. Eberl, Dorothea Godt, Monte Westerfield, Andrew K.
Groves, Hugo J. Bellen
Zao Li
Department of Biochemistry & Molecular Biology
Advisor: Zheng Zhou, Ph.D.-Department of Biochemistry & Molecular Biology
Cells injured pathologically undergo necrosis, a type of cell death distinct from
apoptosis in both morphology and mechanism. Like apoptotic cells, necrotic cells must
be swiftly removed from animal bodies to prevent harmful inflammatory and
autoimmune responses. In the nematode Caenorhabditis elegans, gain-of-function
mutations in certain ion channel subunits result in necrotic-like cell death of six touch
neurons. Necrotic touch neurons are engulfed and degraded inside engulfing cells. It is
unclear how necrotic cells are recognized by engulfing cells. Phosphatidylserine (PS) is
an important apoptotic cell surface signal that attracts engulfing cells. Using ectopically
expressed MFG-E8, a high-affinity PS-binding protein, we observed that PS was
present on the surface of necrotic touch neurons. In addition, phagocytic receptor CED1, whose function is needed for the efficient clearance of necrotic cells, clusters around
necrotic cells. The extracellular domain of CED-1 associates with PS as determined in
assays for an enzyme-linked immunosorbent assay-like solid-phase reaction and
surface plasmon resonance. We further identified a necrotic cell-specific function of
CED-7, the worm homolog of mouse ABC1 transporter, in promoting PS-exposure on
necrotic cell surfaces. In addition to CED-7, ANOH-1, the C. elegans homolog of
mammalian Ca2+-dependent phospholipid scramblase TMEM16F, plays an
independent and unique role in promoting PS-exposure on the surface of necrotic but
not apoptotic cells. The combined activities from CED-7 and ANOH-1 ensure sufficient
PS exposure for necrotic cells to attract their phagocytes. Our work demonstrates that
cells killed by different mechanisms (necrosis or apoptosis) expose a common “eat me”
signal that attracts their common phagocytic receptor CED-1. Furthermore, unlike
previously believed, we discovered that PS is actively exposed onto the outer surface of
necrotic cells through two distinct molecular mechanisms, rather than being leaked out
Contributors: Li, Zao; Venegas, Victor; Raghavan, Prashant; Nakanishi, Yoshinobu; Zhou,
Hong Lian
Department of Molecular & Human Genetics
Advisor: Hui Zheng, Ph.D.-Department of Molecular & Human Genetics
Abnormal NFκB activation has been implicated in Alzheimer’s disease (AD).
However, the signaling pathways governing the regulation and function of NFκB in the
brain are poorly understood. Here we demonstrate that NFκB activity is critically
controlled by its inhibitor protein IκBα in astrocytes, but not in neurons. We identify
complement protein C3 as an astroglial target of NFκB whose levels are increased in
response to heightened NFκB activity. Elevated astroglial C3, in a neuronal complement
receptor C3aR dependent manner, triggers aberrant intraneuronal calcium levels and
disrupts surface AMPA receptor-mediated excitatory synaptic function and dendritic
morphology. These effects can be rescued by C3aR blockade. Importantly, we show
that astroglial NFκB and C3 can be induced by A and are upregulated in AD brains.
Thus, deregulation of neuron-glia interaction through IκBα/NFκB/C3/C3aR/calcium
signaling may contribute to synaptic dysfunction occurring in AD and our study provides
support that C3aR antagonists may be therapeutically beneficial.
Contributors: Li Yang, David Shim, Allysa Cole, Jennifer Rodriguez-Rivera, Giulio Taglialatela,
Hui-Chen Lu and Hui Zheng
Dan Liang
Department of Pathology & Immunology
Advisor: William Decker, Ph.D.-Department of Pathology & Immunology
Of professional antigen presenting cells (APC), only the dendritic cells (DC) are
regarded as initiators of adaptive immune responses. Previously we identified a Th1promoting phenotype of DC simultaneously loaded with overlapping (homologous) MHC
class I and II determinants: tumor specific mRNA and cell lysate. This event, termed
“homologous antigenic loading”, augmented DC IL-12 production, generation of CD8+
cytolytic effectors, and IFN-γ T-cell secretion, all downstream hallmarks of Th1
immunity. We further confirmed that this Th1-promoting phenotype exists across a
variety of antigenic systems in which DC class I and II are loaded with determinants of
homologous origin, indicating DC intrinsic mechanisms to recognize and compare
antigenic epitopes. In an effort to characterize underlying mechanisms, we looked for
secreted factors in DC culture supernatants and detected high levels of AIMp1/p43
release from DC loaded with homologous lysate/mRNA, single protein antigen, or
overlapping viral peptides in comparison to singly-loaded or heterologous controls.
AIMp1 is a structural component of the multi-enzyme aminoacyl-tRNA synthetase
complex (mARS) which consists of at least 8 aminoacyl-tRNA synthetases and 3
structural proteins. Previous studies have shown that AIMp1 is released by both tumor
and macrophages under stress or TLR stimulus, upregulates proinflammatory gene
expression in monocytes and macrophages, and induces Th1 polarization of bone
marrow derived DC (BMDC). We subsequently established that the level of AIMp1
release is closely correlated to the degree of homology between loaded class I and II
epitopes. In vitro studies confirmed that siRNA knockdown and genetic knockout
reduced the expression of costimulatory markers and Th1 cytokines from BMDC.
Further, AIMp1 deficiency abolished the ability of DC vaccines to mediate Th1 immune
response against B16 tumors in vivo. These data suggest secreted AIMp1 is critical for
Th1 immune responses induced by homologous loading of DC.
AIMp1 also colocalizes with MHC class I and II molecules as determined by coIP
and mass spectrometry, suggesting association with antigen recognition machinery. In
future studies, we will determine molecular and cellular mechanisms of AIMp1-mediated
crosstalk between DC and T-cells. We are looking for candidate cell surface receptors
and downstream signal pathways both in DCs and T cells. Additionally, we will
determine how AIMp1 release by DC is regulated and the role of other mARS
components in this process. Finally, we seek to apply AIMp1 as a novel vaccination
adjuvant and will test its potential in anti-tumor immunity and infectious diseases
models. Most critically, these studies will underscore the importance of homologous
antigenic signals in DC polarization.Contributors: Liang, Dan; Halpert, Matthew M; Konduri,
Vanaja; Levitt, Jonathan M; Decker, William K
Steven Donald Lien
Department of Neuroscience
Advisor: J. Dickman, Ph.D.-Department of Neuroscience
The dramatic escalation of conflict has led to higher incidences of exposure to
blast-related traumatic brain injury. Active service members who have been deployed to
conflict zones have reported high rates of vestibular dysfunction, such as vertigo,
imbalance, and dizziness, accumulating evidence suggests that blast wave trauma
causes damage to both the peripheral and central vestibular system. However, the
mechanism by which blast-wave exposure induces vestibular dysfunction remains
unclear. Previous work has established that blast-wave exposure is capable of causing
damage to the auditory hair cells of the inner ear. Our goal for this study is to identify
whether blast-wave exposure will damage the vestibular hair cells of the inner ear. To
determine whether blast-wave exposure causes damage to the peripheral vestibular
system, we plan on analyzing the horizontal vestibulo-ocular reflex (hVOR) and vertical
vestibulo-ocular reflex (vVOR) after exposure to a mild blast-wave. Analyzing the hVOR
and vVOR may give us further insight into the mechanism of vestibular dysfunction after
blast-related traumatic brain injury.
Contributors: Lien, Steven; Dickman, David
Angelique Lin
Integrative Program in Molecular and Biomedical Sciences
Advisor: Daisuke Nakada, B.A.Sc.-Department of Molecular & Human Genetics
Hematopoietic stem cells (HSCs) are the stem cells found in bone marrow that
generate all the functional hematopoietic cells. The ability of HSC to self-renew and
maintain multipotency is critical to sustain life and thus the identification and study of
genes that regulate HSC function are important for HSC research. The recently
developed CRISPR/Cas9 system is a powerful gene-editing tool and has been widely
used to study gene regulation. Delivering Cas9 mRNA and sgRNA targeting gene-ofinterest into HSCs would provide a fast and precise system to screen for genes that are
critical for HSC function. However, the transfection of HSCs is challenged with low
efficiency. For this purpose, our lab has optimized the transfection condition for HSCs
using the Neon transfection system. As an initial optimization step, we used c-kit
positive cells isolated from the bone marrow of ROSA26-LSL-eYFP mouse and
transfected Cre mRNA to induce eYFP expression as reporter for transfection
C-kit positive cells were separated using an autoMACS. Prior to electroporation,
c-kit positive cells were cultured for different time periods. The cultured cells were then
mixed with different amount of Cre mRNA and transfected by electroporation with Neon
transfection system. Transfected cells were cultured overnight and the transfection
efficiency was analyzed by flow cytometry for YFP expression. We found that different
culture condition time and the amount of mRNA affected transfection efficiency. The
transfection efficiency of the bulk population was about 20% for cells cultured for 1 hr
prior to electroporation with 1 ug of mRNA. Notably, the efficiency was about 50% for
lineage-negative population and about 60% for the LSK (lineage-negative, Sca-1+, ckit+) population. Electroporation of HSCs with Neon transfection system is an effective
way to transfect these cells with mRNA directly to study gene function in HSCs. Based
on these results, CRISPR/Cas9 system might be used for screening genes important for
HSC function by delivering Cas9-mRNA and sgRNA into HSCs.
Contributors: Nakada, Daisuke; Kitano, Ayumi; Takeichi, Makiko; Saitoh, Yusuke; Chapple,
Richard Harrison
Chih-Chun Lin
Department of Molecular & Human Genetics
Advisor: Meng Wang, Ph.D.-Department of Molecular & Human Genetics
The abilities of environmental sensation and adaption determine the fitness of
organisms in the constantly changing world. Whether and how animals anticipate
environmental changes remain largely unknown. Among many environmental variables,
food availability is the most critical one. In our studies, we utilize Caenorhabditis elegans
as a model organism to understand how organisms anticipate nutritional restriction in
the environment through perceiving dietary metabolite signals from their preys, and
adjust their metabolic strategies for a better survival under food scarcity.
As soil-dwelling nematodes, C. elegans experience their diet of bacteria as both
a source of nutrition and a source of sensory input. We found that C. elegans fed with
bacteria that are grown in a nutrient deprived condition dramatically increases fat
storage, and these pre-starved C. elegans survives better upon actual starvation. This
presents the first evidence that organisms are able to anticipate environmental changes
from interacting with their preys. We further demonstrated that this nutrient anticipation
response is mediated by an orphan nuclear hormone receptor, NHR-25, homolog of
mammalian LRH-1. We showed that NHR-25 regulates fatty acid de novo synthesis
pathway (fat-5, fat-7) and triacylglyceride synthesis pathway (dgat-2), which together
lead to increased fat storage. Our data also suggest that oleic acids and phospholipids
are the upstream signaling molecules that regulate NHR-25 activity.
This integrative gene-environment interaction study unveils a novel
mechanism by which organisms sense future environmental starvation from current
dietary cues and subsequently remodel their energy metabolic strategy to enable better
fitness in the time ahead. Through further dissecting this new molecular mechanism, we
expect to identify beneficial probiotic supplements that boost metabolic health in human.
Contributors: Wang, Meng
Krithika Lingappan
Clinical Scientist Training Program
Advisor: Bhagavatula Moorthy, Ph.D.-Department of Pediatrics
Background: Sex-specific differences in pulmonary morbidity in adults and
preterm infants are well documented. Hyperoxia contributes to lung injury in
experimental animals and humans. Cytochrome P450(CYP)1A enzymes have been
shown to play a mechanistic role in hyperoxic lung injury (HLI) in animal models.
Whether CYP1A enzymes contribute to sex-specific differences in relation to HLI is
unknown. In this investigation, we tested the hypothesis that mice will display sexspecific differences in HLI, and that this phenomenon will be altered in mice lacking the
genes for Cyp1a1 or 1a2.
Methods: Eight week-old male and female wild type (WT) (C57BL/6J) mice,
Cyp1a1-/-, and Cyp1a2-/- mice were exposed to 72 hours of hyperoxia (FiO2>0.95).
Lung injury and inflammation was assessed and pulmonary and hepatic CYP1A1 and
CYP1A2 levels were quantified at the enzyme activity, protein and mRNA level.
Results: Upon exposure to hyperoxia, WT males showed greater lung injury and
neutrophil infiltration. Analysis of liver and lung microsomal proteins showed higher
pulmonary CYP1A1 (apoprotein level and activity) in WT females compared to WT
males and a greater induction in hepatic CYP1A2 mRNA levels and activity in WT
females after hyperoxia exposure. The sex based female advantage was lost or
reversed in Cyp1a1-/- and Cyp1a2-/- mice.
Conclusion: This suggests an important role for CYP1A enzymes in the sexspecific modulation of hyperoxic lung injury
Contributors: Weiwu, Jiang; Wang, Lihua; Couroucli, Xanthi; Moorthy, Bhagavatula
Alexandra Litvinchuk
Integrative Program in Molecular and Biomedical Sciences
Advisor: Hui Zheng, Ph.D.-Department of Molecular & Human Genetics
Alzheimer`s disease (AD) is the most common form of dementia that affects millions of
people worldwide. The pathological changes of AD include the formation of amyloid plaques of
Aβ and neurofibrillary tangles (NFT) of hyperphosphorylated Tau. However, the functional link
between Aβ and NFTs is poorly understood.
A common feature of AD is the activation of the immune response in the brain. The
activation of complement pathway has been implicated in this process. In particular,
complement component C3 inhibitor sCrry has been reported to modulate both Aβ and NFT
pathologies in mouse models. Moreover, reduced levels of phospho-Tau were shown in a Taurelated model of AD, P301L/sCrry double-transgenic, suggesting the beneficial role of
complement inhibition in AD.
Ongoing studies in our lab have shown that NF-kB can activate complement C3
component in astrocytes but not neurons. Moreover, elevated astroglial C3 through C3aR in
neurons can affect intraneuronal calcium levels, and dysregulation of calcium homeostasis has
been implicated in the pathogenesis of AD. Importantly, astroglial NF-kB and C3 can be induced
by Aβ and are upregulated in AD brains. These data raise the intriguing possibility that astroglial
C3-mediated neuroinflammatory pathway may serve as a functional link between Aβ and NFT
pathologies in AD.
Therefore, we hypothesize that Aβ-mediated astroglial activation of complement induces
Tau phosphorylation in neurons via C3aR and this in turn leads to the formation of neurofibrillary
tangles and subsequent neurodegeneration. We further hypothesize that inhibition of the
C3/C3aR pathway will be therapeutically beneficial.
Aim1. To investigate the role of C3 and C3aR receptor on formation of NFTs and Tau
phosphorylation in vitro and vivo.
Soluble C3 will be administered to primary neurons from wild-type or PS19 mice, that
overexpresses human mutant Tau. Complement inhibition will be achieved by treatment of cells
with C3aR antagonist. For in vivo experiments PS19 and C3aR KO/PS19 mouse lines will be
generated. Levels of phospho-Tau will be quantified by western blot and immunohistochemistry
with total Tau, C3, AT8, PHF1 and MC1 antibodies. NFT formation will be confirmed with ThT
staining. Behavioral and electrophysiological studies will be performed to characterize the
phenotype of C3aR KO/P301S mice.
Aim 2. To identify downstream effectors of C3 and C3aR that promote Tau
The activation of potential kinases that are responsible for increased Tau
phosphorylation in complement C3-dependent model will be tested by western blot,
immunohistochemistry and ELISA. Validation of potential targets will be achieved by treatment
of primary neurons with specific kinase inhibitors or siRNA.
Aim 3. To establish the functional link between Aβ and Tau in development of Taurelated pathology in AD through C3 and C3aR.
Primary astroglia from wild type mice will be co-cultured with neurons from wild-type,
C3aR KO, PS19 and C3aR KO/PS19 mice. Activation of complement will be achieved by
administering Aβ oligomers and fibrils to astrocytes prior to starting the co-cultures. In in vivo
experiments Aβ microinjections in the hippocampal area of 5-months old PS19 and C3aR
KO/PS19 mice will be performed. Levels of total Tau, phospho-Tau, and C3 will be quantified by
western blot, immunohistochemistry and ELISA.
Kuanqing Liu
Department of Molecular Virology & Microbiology
Advisor: Jue Wang, Ph.D.-Department of Molecular & Human Genetics
Anthony Maresso, Ph.D.-Department of Molecular Virology & Microbiology
The nucleotide (p)ppGpp is a ubiquitous stress resistance mediator in bacteria,
but its targets and underlying mechanisms of action vary among bacterial species and
remain incompletely understood. Here we characterize the molecular interaction
between (p)ppGpp and guanylate kinase (GMK) and reveal its remarkable conservation
across many species and its importance in adaptation to starvation. Combining
structural and kinetic analyses, we show that (p)ppGpp binds the GMK active site and
inhibits the enzyme by a competitive mechanism. Abolishing the (p)ppGpp-GMK
interaction results in defective adaptation to amino acid starvation, highlighting its
physiological importance. A survey of GMKs from phylogenetically diverse bacteria
shows that the (p)ppGpp-GMK interaction is conserved in members of Firmicutes,
Actinobacteria, Deinococcus-Thermus, but not in Proteobacteria where (p)ppGpp
regulates RNA polymerase (RNAP). We propose that GMK is an ancestral (p)ppGpp
target and RNAP evolved more recently as a direct target in Proteobacteria.
Contributors: Liu, Kuanqing; Myers, Angela; Claas, Kathy; Satyshur, Kenneth; Keck, James;
Wang, Jue
Lucy Liu
Department of Neuroscience
Advisor: Hugo Bellen, Ph.D./D.V.M.-Department of Molecular & Human Genetics
An increase in lipid droplets (LD) has been implicated in some metabolic
disorders but their role in neurodegeneration is ill defined. We show that several genes
that affect mitochondrial function lead to an accumulation of LD in glia prior to or at the
onset of neurodegeneration in Drosophila. This LD accumulation is caused by increased
reactive oxygen species (ROS), which promotes c-Jun-N-terminal Kinase (JNK) and
Sterol Regulatory Element Binding Protein (SREBP) activity, and neuronal activation of
this pathway is sufficient to cause glial LD accumulation. LD accumulation can be
reduced by lowering ROS, JNK, or SREBP levels, or by overexpressing lipases. These
manipulations significantly delay the onset of neurodegeneration. Furthermore, a similar
pathway leads to glial LD accumulation in Ndufs4 mutant mice, suggesting that LD
accumulation following mitochondrial dysfunction is an evolutionarily conserved
phenomenon. In summary, our studies show that increased ROS leads to LD
accumulation in glia, and that preventing LD accumulation delays neurodegeneration.
Contributors: Lucy Liu, Ke Zhang, Hector Sandoval, Shinya Yamamoto, Manish Jaiswal,
Elisenda Sanz, Zhihong Li, Brett H. Graham, Albert Quintana and Hugo J. Bellen
Flora Lo
Integrative Program in Molecular and Biomedical Sciences
Advisor: Xiang Zhang, Ph.D.-Department of Molecular & Cellular Biology
20-40% of breast cancer patients eventually develop metastasis to the bone.
When presented clinically, breast cancer bone metastases are predominantly osteolytic.
Cancer cells secrete factors that activate osteoclasts (bone-breaking cells), and the
resulting degradation of bone matrix in turn releases factors that promote cancer cell
growth. This forms a vicious cycle that drives aggressive metastatic outgrowth.
Interestingly, in early stage metastasis, disseminated cancer cells likely first remain as
dormant micrometastases in the bone, and later re-activate and progress to overt
lesions. However, very little is known about the regulation of metastasis dormancy and
Recent work in our lab suggested that bone metastasis initially reside in a
microenvironment with features of osteogenesis, implying it later switches to an
osteolytic microenvironment by an unknown mechanism. We are investigating the
hypothesis that hypoxia serves as a mechanistic switch for micrometastases to
progress to overt metastases via the vicious osteolytic cycle.
Why hypoxia? To fuel the vicious cycle, cancer cells that have metastasized to
the bone secrete osteolytic factors, such as parathyroid hormone-related protein
(PTHrP) and connective tissue growth factor (CTGF). Evidence suggests that the
expression of these factors is modulated by hypoxia response proteins in various cell
types, but this has not been studied extensively in breast cancer cells. Moreover, our
lab discovered that early metastases are found in osteogenic microenvironment niches.
These are associated with hypoxic regions in the bone, supporting the role of hypoxia in
metastasis progression. It is possible that as the micrometastasis slowly expands, the
microenvironment becomes even more hypoxic and promotes the induction of the
osteolytic cycle.
Using a range of in vitro and in vivo techniques, I aim to identify osteolytic factors
that are up-regulated in expression under hypoxic conditions. I will also investigate the
effects of hypoxia-mediated changes in osteolytic factor secretion on the bone
microenvironment, and cancer cell dormancy and growth.
Contributors: Yu, Cuijuan; Wang, Hai; Muscarella, Aaron; Zhang, Xiang
Yuan-Hung Lo
Integrative Program in Molecular and Biomedical Sciences
Advisor: Noah Shroyer, Ph.D.-Department of Pediatrics
Colorectal cancer (CRC) is one of the most lethal cancers worldwide. Under
physiological conditions, the Notch and Wingless/Int (Wnt) signaling pathways modulate
homeostasis and differentiation of the intestinal epithelium. In intestinal cancers, these
pathways are frequently dysregulated. Constitutive activation of canonical Wnt signaling
resulting in high β-catenin transcriptional activity is believed to drive CRC
tumorigenesis, however current molecular targeted therapies have limited efficacy.
Therefore, identifying transcription factor networks between Notch and Wnt pathways
associated with β-catenin in CRC is critical for further developing new therapeutic
targets and strategies. We have identified that SAM Pointed Domain Ets transcription
Factor (SPDEF), a downstream target of ATOH1, plays an important role in Notchdriven cell cycle exit and terminal differentiation in the adult intestinal epithelium. In
CRC, we have recently published that SPDEF functions as a tumor suppressor. Our
data indicated SPDEF re-expression in colon tumors is sufficient to prevent colon tumor
cell proliferation by negatively regulating Wnt/β-catenin signaling, resulting in
downregulation of β-catenin target genes in vivo. Moreover, consistent with this
observation, transfection of human CRC cell lines with SPDEF inhibits β-catenin
transcriptional activity in vitro. Here, we proposed a model that SPDEF inhibits β-catenin
transcriptional activity through protein-protein interaction at the chromatin level. To
investigate the mechanism of SPDEF-mediated inhibition of β-catenin transcriptional
activity, tagged wildtype SPDEF or truncated SPDEF mutants were overexpressed in
human CRC cell lines followed by β-catenin transcriptional activity assay, coimmunoprecipitation (co-IP), and immunofluorescent staining. Our data suggested that
SPDEF inhibits β-catenin transcriptional activity and the expression of β-catenin targets
in human CRC cell lines, which is consistent with our previous studies. Moreover,
SPDEF interacts with nuclear β-catenin in both human CRC cell lines in vitro and
mouse intestinal crypts under physiological conditions in vivo. The binding regions for
SPDEF and β-catenin interaction were mapped by using truncated SPDEF and βcatenin mutants. Interestingly, our results indicated that the DNA-binding domain of
SPDEF is unnecessary for it to bind to β-catenin or to inhibit β-catenin transcriptional
activity. Finally, our chromatin immunoprecipitation (ChIP) data suggested that SPDEF
binds to β-catenin target sites on chromatin, resulting in the disruption of β-catenin—
chromatin interaction. Taken together, these results support a model wherein SPDEF
may be a pivotal link between Notch and Wnt/β-catenin pathways through interacting
with β-catenin in chromatin to repress proliferation and regulate differentiation in the
Contributors: Lo, Yuan-Hung; Noah, Taeko; Shroyer, Noah
James Anthony Loehr
Department of Molecular Physiology & Biophysics
Advisor: George Rodney, Ph.D.-Department of Molecular Physiology & Biophysics
Duchenne Muscular Dystrophy (DMD) is an X-linked progressive degenerative
disease caused by a mutation in the gene encoding dystrophin. Dystrophic muscle is
characterized by increased reactive oxygen species (ROS) production and Ca2+ influx,
decreased contractile function and morphological alterations. In mdx mice, a model of
DMD, nicotinamide adenine dinucleotide phosphatase (NADPH oxidase or Nox2) ROS
production is elevated early in the disease progression, resulting in increased Ca2+
influx and decreased muscle function. We hypothesized that inhibiting Nox2 ROS would
protect against the pathophysiological changes associated with dystrophy, which would
be maintained as the mice aged. Genetic inhibition of Nox2 activity resulted in
decreased ROS production and partial recovery of force in young (5-7 wks) mice. The
increased force was maintained with age (15-18 wks) while mdx force declined. We also
demonstrate that decreasing Nox2 ROS reduced Ca2+ influx and central nuclei while
maintaining CSA and fiber type distribution in young and mature mice. Our data support
our hypothesis that Nox2 inhibition improves the pathophysiology of mdx muscle and
identify Nox2 as a potential therapeutic target to preserve muscle function in DMD.
Contributors: Pal, Rituraj; Abo-Zahrah, Reem; Rodney, George G.
Hengyu Lu
Integrative Program in Molecular and Biomedical Sciences
Advisor: Kenneth Scott, Ph.D.-Department of Molecular & Human Genetics
Fusion genes arising from chromosomal translocations, deletions and inversions
play a central role in the development of cancer. Next generation sequencing efforts by
the TCGA and others are identifying numerous fusion gene transcripts in tumors, the
majority of which remain to be functionally characterized due to significant technical
challenges related to their cloning. To circumvent this bottleneck, we have developed a
completely novel strategy to build fusion genes that leverages our platform of >32,000
ORF clones and multi-fragment DNA recombination. We have used this approach to
successfully validate bona fide fusion drivers (e.g., BCR-ABL, EML4-ALK and ETV6NTRK3) in proof-of-concept studies demonstrating our ability to rapidly deliver fusion
genes with functional activity. In a pilot study of a panel of novel fusion genes identified
in melanoma, our work revealed potent transforming activity of BRAF fusions,
TAX1BP1-BRAF, CDC27-BRAF, and ATG7-BRAF, which strongly activate the MAPK
signaling and confers sensitivity to BRAF and MEK inhibitors. This may indicate use of
MAPK inhibitors therapy for patients with tumors harboring oncogenic BARF fusions.
We anticipate scaling this cloning strategy to functionalize numerous fusion genes
discovered by TCGA.
Contributors: Scott, Kenneth
Hsiang-Chih Lu
Program in Developmental Biology
Advisor: Huda Zoghbi, M.D.-Department of Pediatrics
Expansion of glutamine-encoding CAG repeats in ATAXIN-1 (ATXN1) causes
spinocerebellar ataxia type 1 (SCA1). Research over the past 20 years has provided
insight into SCA1 pathogenesis, whereby genetic studies have demonstrated that SCA1
is mostly caused by a gain-of-function mechanism. Little is known, however, about the
specific function of ATXN1. Thus, our lab sought out protein interactors of ATXN1 to
understand its function better. We discovered that the most salient of the interactors
(that also modify the SCA1 phenotype) are ATXN1’s functionally redundant paralog
Ataxin-1-like (ATXN1L) and the transcriptional repressor Capicua (CIC). These three
proteins, all highly expressed in brain, form a co-repressor complex in vivo, and depend
on one another for stability. Moreover, loss-of-function mutations in ATXN1 or CIC have
been implicated in developmental disorders such as autism spectrum disorder and
intellectual disability, suggesting that this protein complex is important for the
development of the nervous system. Atxn1-/-; Atxn1l-/- (double knockout, DKO) mice
and Cic-/- mice die perinatally with defects in multiple organs, demonstrating that these
proteins have critical roles during development. We therefore propose that ATXN1-CIC
complex is important for the proper development and function of the nervous system.
To test this hypothesis and to understand the function of this complex in the brain
we must bypass the perinatal lethality. Accordingly, we used the Cre-lox technology to
delete the genes encoding this complex conditionally in the nervous system. We
generated conditional knockout of either Atxn1-Atxn1l or Cic in the developing forebrain
using an Emx1-Cre. Loss of either Atxn1-Atxn1l or Cic causes multiple behavioral
abnormalities including increased motor activity, reduced anxiety, and impaired
memory. Histological studies showed that while the layering architecture of the cortical
neurons is largely preserved in these animals, the thickness of superficial cortical layers
(layer II-IV) is reduced. Moreover, there is an increased number of glia, suggesting that
there is a disturbance in the balance of neuron/glia differentiation. These results show
that ATXN1-CIC complex is indispensable for normal brain development and its
absence results in multiple behavioral deficits. We are now analyzing the defects in the
timing of neuro/gliogenesis transition in the embryonic cortex. In addition, we are
performing transcriptomic analysis to further dissect the molecular mechanism that
leads to these defects.
Contributors: Lu, Hsiang-Chih; Liu, Xiuyun; Zoghbi, Huda
Wen Lu
Department of Pathology & Immunology
Advisor: David Corry, M.D.-Department of Medicine
Emphysema is one of the most common causes of death worldwide without any
effective treatments. Emphysema is mainly caused by cigarette smoke and particle
inhalation and has a strong correlation with lung cancer. Cigarette/particle induced lung
antigen presenting cell (APC) activation and downstream T helper 17 cell (TH17)
development have been identified as key causative steps of emphysema development.
We report that microRNA-22 (Mir22), an oncogenic microRNA identified in lung cancers,
is also required for emphysema pathogenesis. Loss of Mir22 protects mice from
emphysema. Neutralizing Mir22 in lung using sponge Lock Nucleic Acid (LNA) is able
to reverse fully developed emphysema. Mir22 is highly expressed in APCs and its
expression in lung APCs correlates with emphysema disease severity in both human
and mouse. Mir22 deficient lung APCs induce less TH17 cells correlated with their less
pro-inflammatory cytokine secretion and lower expression level of surface costimulatory molecules. Mice with selective Mir22 deficiency in CD11c+ APCs are also
protected from emphysema. Adoptively transferring Mir22 sufficient lung APCs from
emphysematous donors is able to induce emphysema in Mir22 deficient recipients,
suggesting Mir22 in APC is both sufficient and necessary for emphysema development.
Utilizing gene expression microarray we identify histone deacetylase 4 (HDAC4) as the
target of Mir22 in lung APCs. We further demonstrate HDAC4 negatively regulate c-Jun
expression, a critical transcriptional subunit regulating pro-inflammatory gene
expression. In this study, Mir22 is the first microRNA functionally linked to emphysema
pathogenesis. We have identified a novel Mir22/HDAC4/c-Jun axis regulating APC
activation and innate immunity, with great therapeutic potentials treating emphysema
and other autoimmune diseases.
Contributors: Lu, Wen; You, Ran; Yuan, Xiaoyi; Yang, Tianshu; Tour, James; Kheradmand,
Farrah; Corry, David;
Yang Lu
Integrative Program in Molecular and Biomedical Sciences
Advisor: Jeffrey Rosen, Ph.D.-Department of Molecular & Cellular Biology
While miRNAs play an important role in normal development and cancer, little is
known about the functional role of miRNAs in mammary gland development. The
analysis of miRNA expression in the mouse mammary epithelial cells has revealed high
expression of miR-205 in basal stem cell-enriched population. Consistent with this
observation, miR205 over-expression in the mouse mammary epithelial cell (MEC) line
COMMA-DβGeo leads to an expansion of the Sca-1+ progenitor cell population,
decreased cell size, increased cellular proliferation and colony-forming potential. Based
upon these results, we hypothesize that miR205 is required in mammary gland
development and stem cell maintenance. By using mice with a conditional floxed
miR205 allele containing a Neo-LacZ cassette, we are able to show by X-gal staining
that miR205 is preferentially expressed in the cap cell layer of terminal end buds.
Furthermore, functional cell transplantation assays of miR205 null cells display severely
impaired outgrowths in the first generation. Rudimentary miR205 null transplants lack a
K5+ basal cell layer and laminin staining, indicating disruption of basement membrane
deposition as well as the loss of a basal stem/progenitor cell population. This might be
the underlying reason for the impaired repopulating capacity of miR205 null cells. The
observed growth disadvantage was further verified using in vitro mammosphere assays.
miR205 null cells show a slight self-renewal defect in primary assays, with
mammosphere formation efficiency (MFE) of 0.4%. However, the MFE of miR205 null
cells dramatically decreases to 0.005% in the secondary assays, while WT cells still
exhibit a 0.1% MFE.
In addition, microarray data from Dr. Elsa Flores lab shows that knockout of p63
results in down-regulation of miR205 in skin. Therefore our next step is to study if the
p63-miR205 regulatory axis is associated with the ability of miR205 to influence
mammary gland stem cell fate.
To conclude, our preliminary finding suggests a pivotal role of miR205 in
maintaining the mammary gland stem cell population and outgrowth potential.
Unraveling miR205’s function in normal development will further help us to study its role
in promoting tumorigenesis possibly through regulating the generation and function of
cancer stem cells. Supported by NIH grant CA16303.
Contributors: Lu, Yang, Rosen, Jeffrey
Jeannette Galande Lumaban
Department of Molecular & Human Genetics
Advisor: David Nelson, Ph.D.-Department of Molecular & Human Genetics
Fragile X syndrome results from loss of FMR1 expression. Individuals with the
disorder exhibit not only intellectual disability, but also an array of physical and
behavioral abnormalities, including sleep difficulties. Studies in mice demonstrated that
Fmr1, along with its paralog Fxr2, regulate circadian behavior, and that their absence
disrupts expression and cycling of essential clock mRNAs in the liver. Recent reports
have identified circadian genes to be essential for normal metabolism. Here we describe
the metabolic defects that arise in mice mutated for both Fmr1 and Fxr2. These mice
have reduced fat deposits compared with age- and weight-matched controls. Several
metabolic markers show either low levels in plasma or abnormal circadian cycling (or
both). Insulin levels are consistently low regardless of light exposure and feeding
conditions and the animals are extremely sensitive to injected insulin. Glucose
production from introduced pyruvate and glucagon is impaired and the mice quickly
clear injected glucose. These mice also have higher food intake and higher VO2 and
VCO2 levels. We analyzed liver expression of genes involved in glucose homeostasis
and found several that are expressed differentially in the mutant mice. These results
point to the involvement of Fmr1 and Fxr2 in maintaining the normal metabolic state in
Contributors: Lumaban, Jeannette; Nelson, David
Jessica Leigh Lunsford
Department of Pathology & Immunology
Advisor: Jonathan Levitt, Ph.D.-Department of Pathology & Immunology
Src homology region 2 domain-containing phosphatase-1 (SHP-1) is a tyrosine
phosphatase which inhibits NF-κB signaling through interference in MyD88/IRAK4dependent toll-like receptor (TLR) signaling. However, the specific substrates of SHP-1
in this pathway are unknown. Our lab has previously identified IRAK4 as a binding
partner of SHP-1, despite zero reports of tyrosine phosphorylated IRAK4 in the
literature. Further, expression of a dominant negative form of SHP-1 leads to
accumulation of tyrosine phosphorylated IRAK4 in dendritic cells. We hypothesized that
SHP-1 interacts with IRAK4 to inhibit TLR-dependent NF-κB signaling. We have
identified three tyrosine residues in the IRAK4 structure which are consistent with SHP1 binding motifs: Y48, Y371 and Y413. Since phenylalanine resembles a tyrosine which
cannot be phosphorylated we mutated these three tyrosine sites to phenylalanines,
singly and in double and triple mutant permutations. RAW264.7 cells, a murine
macrophage like cell line, were transfected with various IRAK4 mutations, and activated
overnight with LPS. Activation was assayed by IL-6 ELISA and NFkB activity.
Compared to transfected WT IRAK4, all mutations reduce activation of RAW264.7 cells.
Continuing experiments will explore causes of downregulation in the endogenous TLR
pathway, the kinase responsible for tyrosine phosphorylation after TLR activation, and
the involvment of SHP-1 in this pathway.
Contributors: Konduri, Vanaja; Decker, William; Levitt, Jonathan
Boxue Ma
Department of Biochemistry & Molecular Biology
Advisor: Wah Chiu, Ph.D.-Department of Biochemistry & Molecular Biology
Chaperonin TRiC is the essential protein-folding machine in eukaryotic cells. It is
a hetero-oligomeric complex containing two rings sitting back to back, which serve as
folding chambers that sequester substrate from the cellular environment. Each ring
consists of eight homologous subunits, known as CCT1-CCT8. In vivo and in vitro
studies have shown that the eight subunits have varied affinity to different substrates.
Therefore, it is important to identify the subunits for understanding the mechanism of
how TRiC interacts with substrates. Several studies have been carried out, however,
due to the high structural similarity among the eight subunits, it is difficult to distinguish
them directly even at near atomic resolution. Thus, we propose to perform cryo electron
microscopy single particle analysis to reconstruct the structure of an engineered yeast
TRiC which has a calmodulin binding peptide (CBP) fused into one of the eight subunits.
The CBP tag will bind to calmodulin, which serves as a label on the specific subunit in
cryo-EM reconstruction. Combining with the two-fold symmetry between the two CCT
rings and the asymmetric feature within each ring, labeling and identifying subunit CCT2
or CCT6 will confirm the arrangement of all the eight subunits. Such labeling allows the
recognition of the subunits of apo-state TRiC at medium resolution. Furthermore, the
labeled TRiC will be a powerful tool for future structural study to reveal the substratebinding sites on TRiC.
Contributors: Ma, Boxue; Gestaut, Dan; Chiu, Wah
Simran Madan
Program in Translational Biology & Molecular Medicine
Advisor: Brendan Lee, M.D./Ph.D.-Department of Molecular & Human Genetics
Brett Graham, M.D./Ph.D.-Department of Molecular & Human Genetics
Deficiency of urea cycle enzyme argininosuccinate lyase (ASL) is is
characterized by hyperammonemia, vomiting, seizures and if left untreated, can lead to
coma and death. Despite early intervention and management, some patients still
develop long term complications such as hypertension, liver dysfunction, and
neurocognitive deficits. In this study, we are characterizing the liver pathology in a
hypomorphic mouse model of ASL. Plasma from ASL deficient mice show significantly
elevated liver enzymes AST and ALT, which is an indication of liver damage. Electron
microscopy on livers from ASL hypomorphic mice showed dysmorphic mitochondria and
an unusually large number of lysosomes. Histological analysis show increased glycogen
mobilization. Previously, we have shown that ASL is an important regulator for nitric
oxide production. Proteomic analysis of livers from ASL hypomorphic mice compared to
WT mice showed that loss of ASL results in loss of nitrosylation of several key
metabolic enzymes in the citric acid cycle and the electron transport chain. Currently,
we are investigating if this loss of nitrosylation causes altered mitochondrial energetics
and mitochondrial dysfunction which may explain the liver dysfunction seen in ASL
deficient mice and patients.
Contributors: Madan, Simran; Burrage, Lindsay; Chen, Yuqing; Bertin, Terry; Graham, Brett;
Lee, Brendan
David Russell Magnan
Integrative Program in Molecular and Biomedical Sciences
Advisor: David Bates, Ph.D.-Department of Molecular & Human Genetics
All cells must initiate new rounds of DNA replication once and only once per cell
division in order to maintain genomic stability. E. coli strictly regulates initiation timing
and periodicity through multiple mechanism including staged assembly of replication
initiation factors on the replication origin oriC and cell-cycle dependent changes to oriC
structure. Additional data suggests that oriC position in the cell influences capacity of
origins to fire. To date however no mechanistic link between cell division and replication
initiation has been discovered in E. coli. In order to study these models we developed a
system to disrupt position and structure of specific chromosomal loci. A TetR (DNA
binding) fusion protein normally used to visualize loci in living cells was fused to a
membrane-inserted chemotaxis protein, creating a physical linkage between the
chromosome and the cell membrane at specific loci of our choosing. Tethering
chromosomal loci to the cell membrane using this system inhibits cell division and
slowly reduces cell viability. Tethering does not cause DNA damage or disrupt
membrane integrity. Tethering does not inhibit replication elongation or nucleoid
segregation but does strongly inhibit replication initiation. Surprisingly, tethering oriCdistal loci inhibit initiation to the same extent as tethering oriC-proximal loci, albeit in a
delayed manner. Global chromosome structure is disrupted in tethered strains,
suggesting we may have disrupted a long-sought mechanistic link between cell division
and replication initiation.
Contributors: Magnan, David; Joshi, Mohan; Bates, David
Sangeetha Kaveri Mahadevan
Program in Translational Biology & Molecular Medicine
Advisor: Ignatia Van Den Veyver, M.D.-Department of Obstetrics & Gynecology
Carlos Bacino, M.D.-Department of Molecular & Human Genetics
Maternal effect mutations in NLRP7 and less commonly, KHDC3L, cause
Biparental Hydatidiform Moles (BiHM), a rare form of Gestational Trophoblastic Disease
characterized by degenerate cystic villi, hyperproliferative trophoblast, abnormally high
β-HCG levels and absent fetal development. Unlike their more common
androgenetically derived counterparts, BiHM pregnancies are recurrent, biparentally
inherited and tissues from these pregnancies exhibit loss of methylation at maternally
imprinted loci. Because NLRP7 is not present in rodents or other commonly used
animal models, we generated NLRP7 knockdown and overexpressing hESC lines which
have enabled us to gain insight into NLRP7's function in an in vivo system that most
closely resembles germ cells. Whole genome methylation and RNA-Seq studies on the
NLRP7 knockdown hESC have revealed altered methylation and expression at several
hundred genes suggesting loss of NLRP7 has a more genome wide influence on DNA
methylation and expression that is not restricted to imprinted loci. Expression studies on
these cells further revealed a cell cycle dependent increase and intracellular
redistribution of both BiHM proteins NLRP7 and KHDC3L. Nuclear staining was
observed during the mitotic phase of the cell cycle which is a novel observation given
that NLRP proteins are known to be cytoplasmic in localization. This observation of
nuclear staining also supports the genome wide alterations in DNA methylation and
expression observed in NLRP7 knockdown cells. A yeast two hybrid screen and
hypothesis driven candidate approach was employed to detect interacting partners of
NLRP7 and this revealed positive interactions with NPM1 and CFP1. Given that the
expression of both these CFP1 and NPM1 oscillates during the cell cycle, further
investigations into the interaction of NLRP7 with these proteins could uncover the
mechanisms, by which (i) NLRP7’s expression is regulated during the course of the cell
cycle, (ii) NLRP7 impacts DNA methylation and expression at imprinted and nonimprinted loci. These studies are currently ongoing.
Contributors: Mahadevan, Sangeetha; Wan, Ying-Wooi; Liu, Zhandong; Van den Veyver,
Christina Susan Mahanic
Integrative Program in Molecular and Biomedical Sciences
Advisor: Weei-Chin Lin, M.D./Ph.D.-Department of Medicine
E2F1 is a transcription factor that plays a critical role in diverse cellular pathways
such as cell cycle progression, apoptosis induction, and the DNA damage response.
E2F1 has been shown to be necessary for inducing apoptosis in a chemotherapeutic
response by transcriptionally activating target genes such as p73. The mechanism by
which E2F1 differentially regulates transcription of proliferative genes and apoptotic
genes remains unknown. Our co-IP/MS experiment identifying potential interacting
proteins of E2F1 has identified a deubiquitinating enzyme (DUB), UCHL5, as a novel
E2F1 interacting protein. Through further investigation, I have shown that E2F1
interacts with ectopically overexpressed UCHL5 in 293T cells with a slight induction
after treatment with a chemotherapeutic drug, adriamcyin. In 293T cells, the
accumulation of ubiquitinated E2F1 (K63-chain specific) is decreased in the presence of
ectopic UCHL5. This suggests that UCHL5 is a potential DUB for E2F1. Through using
a semi-endogenous ubiquitination assay, E2F1 is shown to be ubiquitinated through
K63-specific linkages and when E2F1 and UbK63 are co-expressed, E2F1
transcriptional activity decreases. Conversely, a reporter assay has shown that E2F1
transcriptional activity is increased when UCHL5 is present, and Q-RT-PCR analysis
has shown that p73, an E2F1 target gene, is upregulated in the presence of UCHL5.
This functional role of UCHL5 has been verified using a stable knockdown cell line with
the reporter assay and Q-RT-PCR. As a functional assay, a Caspase 3/7 reporter assay
was used to show that UCHL5 knockdown cell lines have less Caspase 3/7 activity
when compared to the scrambled control, suggesting UCHL5 has a role in apoptosis.
Finally, I have shown through chromatin immunoprecipitation (ChIP) that UCHL5 is
localized at the site of E2F1-specific pro-apoptotic promoters such as APAF1, caspase
3 and 7, and PARP1. Prior studies have shown UCHL5 is able to interact with the
INO80 chromatin-remodeling complex, also placing UCHL5 at the site of transcription. I
hypothesize that UCHL5 is able to interact with E2F1 on the chromatin, and
deubiquitinate E2F1, thus enabling an upregulation of transcriptional activity of genes
required for apoptosis. I propose that ubiquitination is an additional regulation of
transcriptional activity of E2F1. Discovering a mechanism in which E2F1 transcriptional
activity is increased could lead to a possible target therapy that induces apoptosis in
cancer cells. In conclusion, I have found that a DUB, UCHL5, interacts with E2F1 and
deubiquitinates E2F1 in vivo and knockdown of UCHL5 decreases an apoptotic
response when DNA damage is present.
Contributors: Mahanic, Christina; Budhavarapu, Varija; Lin, Weei-Chin
Tabassum Majid
Program in Translational Biology & Molecular Medicine
Advisors:Robia Pautler, Ph.D.-Department of Molecular Physiology & Biophysics
Rachelle Doody, M.D./Ph.D.-Department of Neurology
Current therapy for Alzheimer’s disease (AD) focuses on delaying progression,
illustrating the need for more effective therapeutic targets. A target of interest in
Alzheimer’s disease is HDAC6. Of all the HDAC classes, this enzyme has been shown
to have roles in the clearance of protein aggregates, interact with microtubule-stabilizing
proteins, and recruit chaperones for axonal motor proteins. In addition, HDAC6 is
elevated in Alzheimer’s patient brains. HDAC6 genetic depletion studies in pre-clinical
AD models have demonstrated ex vivo axonal transport is recovered. However, HDAC6
inhibitors have been met with limited success in pre-clinical models due to a lack of
blood-brain-barrier penetration by selective HDAC6 inhibitors. Our previous work has
demonstrated that axonal transport deficits are detectable in vivo using manganeseenhanced MRI (MEMRI) in Alzheimer’s models prior to the onset of amyloid deposits.
Due to the crucial role of microtubule-based axonal transport in Alzheimer’s
disease and our ability to measure it using MEMRI, we investigated the potential of a
specific, potent HDAC6 inhibitor (ACY-738) in a mouse model of Alzheimer’s disease.
ACY-738 has been shown to have specificity to HDAC-6 inhibition, permeation of the
blood brain barrier, and its rapid clearance from brain and plasma in mouse models.
Using a chow-based treatment of ACY-738, we sought to determine the effects of
HDAC6 inhibition on axonal transport, behavior, and pathology in the APP/PS1 mouse
model of AD.
Group 1 received a treatment of ACY-738 for 21 days and were evaluated at 3
months of age. After treatment, we measure therapeutic levels in both brain and plasma
of 3 month-old mice. We also demonstrate a recovery in axonal transport deficits after
21 days of chow treatment. Group 2 was treated with ACY-738 and evaluation was
completed at 6 months of age. Therapeutic levels of ACY-738 are present in both the
plasma and brain of APP/PS1 mice. We demonstrate a recovery in short-term fearassociated learning and memory deficits and axonal transport deficits after 3 months of
treatment with ACY-738. Our findings implicate the development of specific, targeted
HDAC6 inhibitors for AD patients. The completion of this pre-clinical proof of concept
study in the APP/PS1 mouse model of AD indicate a recovery in short-term learning and
memory and axonal transport deficits. Further investigation into the effects of HDAC-6
inhibitors in AD will allow for the development of more effectively targeted
therapeutic interventions to combat the deleterious effects of this devastating disease.
Contributors: Majid, Tabassum, Griffin, Deric, Criss II, Zachary, Pautler, Robia G.
Keng Hou Mak
Integrative Program in Molecular and Biomedical Sciences
Advisor: Jue Wang, Ph.D.-Department of Molecular & Human Genetics
Meng Wang, Ph.D.-Department of Molecular & Human Genetics
The ability to sense and signal stress is essential for an organism to survive.
Intracellular nucleotides are an important group of signaling molecules that relay diverse
environmental or metabolic information. Among them, the highly phosphorylated
nucleotides guanosine tetra(penta)phosphate ((p)ppGpp) play a major role in stress
signaling in prokaryotes and are required for adapting bacterial physiology to changing
environments, such as nutrient starvation and pathogenesis. It has been a longstanding mystery whether (p)ppGpp has physiological functions in metazoans.
Recently, a protein family of (p)ppGpp regulatory enzymes was identified in higher
animals, with one member each in the genomes of human, Drosophila and C. elegans.
In Drosophila, MESH1 mutants have altered starvation tolerance, and the protein
product hydrolyzes (p)ppGpp in vitro. However, no (p)ppGpp was detected in vivo, and
the functions of MESH1 on the tissue and cellular levels are still unknown. This
knowledge will provide key insight into physiological functions beyond starvation
response and guide future efforts to identify (p)ppGpp or other MESH1 substrates in
C. elegans serves as a good model because of its power to uncover gene
function on both cell and tissue levels. To first test the physiological role of mesh-1
(encoding the uncharacterized C. elegans MESH1), we examined the starvation
tolerance of mesh-1 mutants. Surprisingly, mesh-1 mutants show no change in
starvation tolerance and display no apparent growth and morphology defects. However,
they have enhanced tolerance to pre-freezing coldness, which can be complemented by
overexpressing the wildtype gene, demonstrating the role of mesh-1 in cold sensitivity.
Tissue-specific RNAi knockdown indicates mesh-1 activity in either the epidermis or
intestine is functionally required for the phenotype. Because (p)ppGpp regulatory
enzymes function in chloroplasts in plants, I am currently testing the hypothesis that
MESH-1 regulates mitochondrial functions associated with cold tolerance. Future
studies include identifying MESH-1 substrate(s) in vivo. Taken together, our results
suggest a tissue-specific role for MESH-1 in thermal adaptation and extend the function
of MESH1 beyond starvation response.
Contributors: Mak, Keng Hou; Wang, Meng; Wang, Jue
Kathleen Seger Manning
Integrative Program in Molecular and Biomedical Sciences
Advisor: Thomas Cooper, M.D.-Department of Pathology & Immunology
Myotonic dystrophy (DM) is the most common form of muscular dystrophy in
adults. DM is characterized by myotonia, muscle wasting, cardiac conduction
abnormalities, and central nervous system dysfunction, among other symptoms.
Myotonic dystrophy type I (DM1) is caused by an unstable CTG repeat expansion in the
3’ UTR of the muscle kinase gene DMPK. The expanded CTG repeat is transcribed into
CUG expanded repeat (CUGexp) RNA transcripts, which are retained in the nucleus
and aggregate into distinct foci. CUGexp RNA sequesters Muscleblind-like (MBNL)
family splicing factors and alters the activity of CUGBP Elav-like family (CELF) RNA
binding proteins, leading to defects in post-transcriptional gene regulation for multiple
genes. However, while misregulation of MBNL or CELF proteins is each sufficient to
induce a subset of DM1 symptoms in mouse models of disease, some effects of
CUGexp RNA are independent of MBNL and CELF dysfunction. To identify novel
mechanisms of pathogenesis in DM1, we propose a high-throughput
immunofluorescence-based assay to objectively screen for proteins associated with
CUGexp RNA foci. While these CUGexp RNA foci are a characteristic feature of DM1,
their full protein composition is unknown. A library of monoclonal antibodies will be
robotically screened in immortalized cell lines from DM1 patients to identify antibodies
that co-localize with CUGexp RNA foci by immunofluorescence. Positive hits will be
identified by mass spectrometry. A comprehensive understanding of the protein-protein
and protein-RNA interactions associated with CUGexp RNA foci will improve therapeutic
targeting and may reveal novel mechanisms of pathogenesis in DM1. This project will
also provide a framework for future studies of foci and aggregate composition in other
disease models.
Dongxue Mao
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M.-Department of Molecular & Human Genetics
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease
that affects both upper and lower motor neurons, leading to muscle atrophy and
paralysis. There is no cure for ALS and the pathogenesis is still poorly understood.
VapB is a highly conserved type II integral membrane protein associated with the
Endoplasmic Reticulum (ER). It is implicated in lipid transport, muscle mitochondrial
dynamics and ER stress. Mutations in VapB are identified through linkage analysis in
families with ALS and spinal muscular atrophy. VapB mRNA or protein level is
decreased in the spinal cord of sporadic ALS patients as well as in ALS mouse models,
indicating there is correlation between VapB and ALS.
Previous data suggest that the N-terminal MSP domain of VapB is cleaved and
secreted and ectopically expressed VapBP56S aggregates in the ER, leading to the
elevation of ER stress and the interference of MSP secretion in Drosophila
melanogaster. In C. elegans, secreted MSP binds to the Ephrin receptor (Eph), Lar-like
protein-tyrosine-phosphatase (CLR) and Roundabout (Robo) in the muscle. This
interaction is required for proper cytoskeleton organization, which affects mitochondrial
morphology and localization in the muscle. In SODG93A transgenic mice, a wellestablished animal model for ALS, we find that full length VapB protein level is elevated
in muscle but the cleaved MSP is reduced. This indicates that insufficient MSP cleavage
or secretion may lead to muscle atrophy and a feed-back mechanism is required to upregulate VapB protein synthesis to maintain the MSP pool. The MSP cleavage is
present in most mouse tissue such as nervous system, heart and liver. Secreted MSP is
also detected in human blood serum and Cerebrospinal Fluid (CSF). Addition of
recombinant MSP induces tyrosine phosphorylation of RTKs, such as EGFR and FGFR
in HEK293 cells. In summary, the neuronal or muscular degeneration in ALS patients or
disease model correlates with loss of function of VapB or defect in MSP secretion.
Therefore, we propose that MSP function as a hormone. It is a beneficial factor that
prevents muscle atrophy by modulating RTKs.
Contributors: Mao, Dongxue; Lin, Guang; Tepe, Burak; Bellen, Hugo
Miguel Marin
Department of Neuroscience
Advisor: Matthew Rasband, Ph.D.-Department of Neuroscience
Myelinated axons are divided into several distinct domains, which includes the
axon initial segment (AIS), nodes of Ranvier, paranodes, and juxtaparanodes. The AIS
serves as both a physical barrier between the axonal and somato-dendritic
compartments of the neuron and as the site of action potential (AP) initiation. Nodes of
Ranvier are responsible for the rapid and efficient propagation of APs along the axon.
Disruption of the AIS or nodes of Ranvier by genetic and/or pharmacological
manipulation has a dramatic impact on the central nervous system. With this in mind,
we have designed a series of experiments, which will allow us to assess the efficacy of
neuroprotective paradigms upon axons of the central nervous system after insult. Using
the optic nerve crush injury model, we have established a timeline for degenerative
events of the nodes of Ranvier of the optic nerve and AIS of retinal ganglion cells. We
have established that loss of nodes of Ranvier begins 6 hours after injury and
progresses both distal and proximal to the injury site. A total loss of nodes of Ranvier
occurs 1 week after injury and persists 1-month post crush. Loss of AIS in retinal
ganglion cells begins 24 hours after injury and persists 1-month post crush. We have
assessed the neuroprotective efficacy of MDL-28170 - a calpain inhibitor that has been
shown to protect from AIS degeneration after ischemic injury. MDL-28170 spares nodes
of Ranvier and AIS from degeneration 24 hours post injury.
Contributors: Miguel Marin1 & Matthew N. Rasband1 & 2
Jarrod Don Martinez
Department of Molecular & Cellular Biology
Advisor: Jianming Xu, Ph.D.-Department of Molecular & Cellular Biology
PTEN loss occurs approximately 33-48% across different molecular subtypes of
breast cancer (BC) and has correlations with poor prognosis and disease related death.
In conjunction, P53 alterations or deletions in BCs has been reported to be as high as
80%. To date few studies have examined the effects of P53 and PTEN dual deletions
and have yielded important insights. Most importantly these studies show that there is
an important selective pressure to deactivate or delete P53 when PTEN is lost. Our
model system will use the RCAS-CRE/TVA system from which we will be able in infect
mammary epithelial cells and subsequently cause the deletion of both P53 and PTEN
alleles and the cleavage of the RFP to GFP reporter. Sequential genomic profiling of the
cancers pathological progression from initiation to distant metastases will allow us to
obtain a comprehensive view of the genomic changes that occur throughout the course
of the disease. Despite the heterogeneity of mutations an genomic alterations that
accompany cancer progression, we hypothesize that there are certain genomic changes
that must occur sequentially during the progression of the disease that allows for BC
cells to initiate progress, and metastasize to distant organs. To test this hypothesis we
will use the following aims: Aim 1: To characterize and profile the malignant progression
of the resulting PTEN and P53 null tumors from our novel TVA+ R/G+ PTENf/f P53f/f
mouse model. Aim 2: To determine the genomic changes at DNA/RNA levels that occur
after the deletion of PTEN and P53 in various stages of tumorigenesis and metastasis
of the transformed mammary epithelial cells. Approximately 90 percent of BC related
deaths are attributed to metastasis, making it the greatest threat to overall patient
survival. The understanding of the molecular mechanisms regulating the various stages
of tumorigenesis and metastasis is therefore essential in the development of high risk
management and new therapies for patients.
Contributors: Qin, Li Ph.D; Xu, Yixiang; Li, Yi Ph.D.; Xu, Jianming Ph.D.
Heidi Martini-Stoica
Program in Translational Biology & Molecular Medicine/M.D.-Ph.D. Program
Advisor: Hui Zheng, Ph.D.-Department of Molecular & Human Genetics
Joshua Shulman, M.D./Ph.D.-Department of Neurology
Presently, tau-targeting therapies to treat Alzheimer’s disease (AD) are limited by
efficacy and safety. To date, the majority of therapeutic trials have focused on Aβ
because of the strong genetic evidence linking mutations in APP and presenilins to
familial AD. However, tau-based neurofibrillary tangle (NFT) pathology correlates more
closely with cognitive decline than Aβ plaques. Thus, the failure of Aβ directed AD
therapies may in part be due to their inability to target AD’s NFT pathology. Thus, the
long-term goal of this work is to gain a better understanding of the cellular mechanisms
of NFT clearance in order to develop more effective therapies. The overall objective of
this proposal is to dissect the multiple roles of transcription factor EB (TFEB), a critical
regulator of lysosomal biogenesis, in clearing these pathological forms of tau. The
central hypothesis is that neuronal exocytosis of tau and subsequent astroglial uptake
are crucial components of TFEB-mediated clearance of aberrant tau in diseases of
tauopathy, in addition to TFEB’s cell-autonomous effect in enhancing the autophagylysosomal pathway (ALP). This hypothesis has been formulated based on recent work
demonstrating the therapeutic potential of TFEB in ameliorating tau pathology in a
mouse model of tauopathy. In addition, TFEB transcriptionally activates lysosomal
exocytosis genes to promote cellular clearance, as well as endocytosis genes to
promote cellular uptake. These known targets of TFEB suggest an extracellular
mechanism of aberrant tau clearance that may involve neurons discarding tau
extracellularly through lysosomal exocytosis and astrocytes subsequently taking up tau
through endocytosis. In the astrocytes, tau could then undergo clearance through the
ALP, also enhanced by TFEB. In order to accomplish the overall objective for the
current proposal, the central hypothesis will be tested by pursuing the following specific
aims: 1) Determine how TFEB affects tau handling in neurons using in vitro and in vivo
overexpression of TFEB in neurons while measuring extracellular release of tau. 2)
Determine how TFEB affects the role of astrocytes in tauopathies by measuring TFEBmediated astrocyte uptake of tau in vitro and determining the therapeutic efficacy of
astrocyte specific TFEB overexpression in a mouse model of tauopathy. The successful
completion of these aims will define the extracellular mechanisms of TFEB’s action and
provide valuable insight into the poorly defined role of astrocytes in tauopathies.
Clarification of the extracellular mechanisms of TFEB-mediated pTau clearance is
expected to identify novel therapeutic targets to guide the development of new
therapeutic strategies in the treatment of AD.
Contributors: Martini-Stoica, Heidi; Zheng, Hui
Ruchi Masand
Department of Molecular & Human Genetics
Advisor: Brett Graham, M.D./Ph.D.-Department of Molecular & Human Genetics
The voltage-dependent anion channel (VDAC or porin) is an integral membrane
protein present in the mitochondrial outer membrane (MOM). VDACs are not only the
predominant determinant of MOM permeability but also integrate mitochondrial function
and other cellular pathways by interacting with various mitochondrial and cytoplasmic
proteins; however, details of these functions as well as the interacting pathways remain
poorly understood. VDACs have also been implicated to play a pathogenic role in
several human diseases including cancer, diabetes, Alzheimer’s and cardiac ischemiareperfusion injury. Flies mutant for porin (the predominant VDAC in Drosophila)
demonstrate energy metabolism defects, neurologic dysfunction with abnormal
mitochondrial distribution in motor neurons and male infertility.
A pilot modifier screen identified multiple deletions that suppress male infertility
in porin mutants, including one deletion that also suppressed neuronal dysfunction in
porin mutants. Deletion mapping identified Lsp2, a hexamerin expressed in the fat body,
as a suppressor of porin mutant CNS phenotype. Expression microarray analysis and
qRT-PCR on adult porin deficient flies revealed extremely high levels of Larval Serum
Proteins (LSPs) as well as Fat Body Proteins (FBPs), all of which are typically highly
expressed in the larval Drosophila fat body, which is analogous to the adipose tissue
and liver in mammals as the major site of energy metabolism and fat storage in the fly.
In addition, we also observed significantly lower levels of triglycerides (TAG) in flies
deficient for porin. Elevated levels of LSPs and FBPs have also been reported in
Drosophila mutant for technical knockout (tko), previously reported as showing
respiratory chain deficiency, developmental delay, and neurological abnormalities.
A p-element insertion allele of Lsp2 was able to rescue the increased bang
sensitivity as well as the secondary complex I deficiency phenotypes seen in porin
mutants. Also, both CNS and fat body specific expression of porin in the mutant flies
was able to rescue the increased bang sensitivity phenotype.
Since tissue specific expression of porin in the fat body, the key metabolic tissue
in the fly, is sufficient to rescue the CNS phenotype in porin mutants and some of the
key genes expressed almost exclusively in the fat body are elevated in both porin and
tko mutants, our data suggests a possible link between mitochondria, fat body (i.e,
adipose tissue) metabolism and neurological function.
Contributors: Masand, Ruchi; Graham, Brett H.
Melinda Mata
Department of Pathology & Immunology
Advisor: Stephen Gottschalk, M.D.-Department of Pediatrics
Adoptive transfer of T-cells expressing chimeric antigen receptors (CARs) has
shown promising anti-tumor activity in early phase clinical studies, especially for
hematological malignancies. However, several patients developed adverse events after
CAR T-cell infusions, which were not predicted by preclinical studies since most murine
models do not reliably mimic human disease. Dogs are excellent models of human
disease since canine tumors exhibit similar genetic abnormalities, tumor location, and
comparable metastatic spread in a natural tumor environment. We reasoned that
developing an adoptive T-cell therapy approach for spontaneous tumors occurring in
dogs, such as osteosarcoma (OS), would more closely reproduce the condition in
human cancer and allow us to further evaluate CAR T-cell therapy prior to human
clinical trials. To generate CAR-expressing canine T-cells we developed expansion and
transduction protocols that allow for the generation of sufficient numbers of CARexpressing canine T-cells for future clinical studies in dogs within 2 weeks of ex vivo
culture. Seven days post-activation of canine T-cells with irradiated K562-APC, PHA,
and IL21, cell lines consisted predominately of CD3-positive T-cells (83 ± 10.7%) with a
mixture of CD4- and CD8-positive subsets. Restimulation of T-cells in Grex tissue
culture devices with the addition of IL2 resulted in 100-fold expansion of cells. Since
canine OS tumor biopsies and cell lines express the tumor antigen HER2, we retrovirally
transduced canine T-cells 3 days post-activation with GALV-pseudotyped retroviral
particles encoding a HER2-specific CAR. Post-transduction up to 38% of canine T-cells
expressed HER2-CARs and were able to recognize canine HER2, secrete IFNγ, and
lyse HER2+ canine OS tumor cell lines in an antigen dependent manner. To reduce the
potential immunogenicity of the CAR we evaluated a CAR with canine-derived
transmembrane and signaling domains, and found no functional difference between
human and canine CARs. Hence, we have successfully developed a strategy to
generate CAR-expressing canine T-cells for future preclinical studies in dogs. Testing Tcell therapies in an immunocompetent, outbred animal model may improve our ability to
predict their safety and efficacy prior to conducting studies in humans.
Contributors: Mata, Melinda; Vera, Juan; Gerken, Claudia; Rooney, Cliona M.; Miller, Tasha;
Pfent Catherine; Wang, Lisa L.; Wilson-Robles, Heather M.; Gottschalk, Stephen
Allison Mayle
Department of Molecular & Human Genetics
Advisor: Margaret Goodell, Ph.D.-Department of Pediatrics
DNA METHYLTRANSFERASE 3A (DNMT3A) is mutated in hematological
malignancies affecting myeloid, mixed, and lymphoid lineages and is associated with
poor prognosis. Past studies in mice revealed Dnmt3a-KO HSCs had increased selfrenewal, but no leukemia was observed. Here, all lethally irradiated mice transplanted
with Dnmt3a-deleted HSCs died within one year, most developing hematopoietic
malignancies across a spectrum as observed in patients. In some cases, Dnmt3a-KO
HSCs acquired secondary mutations similar to those identified in patients, further
validating this model. In this study, loss of Dnmt3a led to disturbed methylation patterns
unique to different diseases, rather than to a uniform methylation profile, pointing to
lineage-specific methylation aberrations promoted by Dnmt3a loss. Global
hypomethylation was observed in all of the malignancies but lymphoid malignancies
also exhibited hypermethylation specifically at promoter regions. This mouse model
underscores the important role of Dnmt3a in normal hematopoietic development, and
demonstrates that Dnmt3a loss-of-function confers a preleukemic phenotype on murine
HSCs. In patients, preleukemic stem cells with DNMT3A mutations are clinically silent,
and outcompeted by malignant clones during disease presentation. These cells are
reported to survive chemotherapy, and may contribute to increased rates of relapse.
Our mouse model may prove valuable for developing targeted strategies for eliminating
preleukemic cells for prevention and treatment of hematological malignancies in the
Contributors: Mayle, Allison; Yang, Liubin; Rodriguez, Ben; Zhou, Ting; Chang, Edmund; Curry,
Choladda V; Challen, Grant A; Li, Wei; Wheeler, David; Rebel, Vivienne; Goodell, Margaret
Ryan Mayle
Department of Molecular & Human Genetics
Advisor: Grzegorz Ira, Ph.D.-Department of Molecular & Human Genetics
A major source of a spontaneous double stand break (DSB) in replicating cells is
a single strand nick which is encountered by a replication fork, creating a single ended
DSB break, commonly called a broken fork. Break-induced replication (BIR) is a
pathway of homologous recombination known to repair single ended DSBs, and has
been hypothesized to be responsible for repairing broken replication forks. However,
BIR has been studied at DSBs outside the context of a replication fork, thus is remains
unclear whether BIR has similar mechanistic and enzymatic requirements as the repair
of broken replication forks. The hallmarks of BIR in yeast are its long duration, taking
hours to complete, high mutagenicity, and a requirement of proteins which stimulate a
D-loop migration mode of repair synthesis, including Pol32, the non-essential subunit of
Polδ, and the helicase Pif1. We used a site-specific nick-induced DSB repair assay to
study the process of broken replication fork repair, and compare this to BIR. By
studying the genetic requirements and mutagenicity of broken fork repair at two distinct
genomic loci, we can determine how closely this pathway resembles traditional BIR.
We have placed nick sites between two origins as well as between the first origin and
the telomere. The latter situation presents the cell with only one option for repair;
synthesis primed from the single end break. We find that the role of BIR in repair at both
break locations is minimal, as Pif1 and Pol32 are not required. However, two HR
endonucleases, Mus81 and Yen1, are required for repair. This is consistent with repair
mediated by cleavage of the D-loop formed at the break, leading to re-establishment of
a normal, high fidelity, fork. In contrast to BIR, where mutagenesis remains high
throughout repair, broken fork repair results in no increase in mutations away from the
break. When Mus81 in absent, mutagenesis is elevated away from the break, and has
similar characteristics to BIR. Therefore, we propose that Mus81 regulates repair
pathway choice at broken forks by cleaving the D-loop, preventing BIR, and promoting
repair utilizing a high fidelity fork.
Contributors: Mayle, Ryan; Ira, Grzegorz
Janielle P Maynard
Program in Translational Biology & Molecular Medicine
Advisor: Sundararajah Thevananther, Ph.D.-Department of Pediatrics
John Goss, M.D.-Department of Surgery
Hepatocellular carcinoma (HCC) is the third most lethal cancer worldwide, but
molecular mechanisms of its pathogenesis are not well understood. Recent studies
suggest that extracellular ATP-mediated activation of P2Y2 purinergic receptor induces
hepatocyte proliferation in response to partial hepatectomy and ATP treatment alone
was sufficient to induce hepatocyte proliferation in vitro. The purpose of this study was
to characterize extracellular nucleotide effects on HCC cell proliferation and to examine
the role of P2 purinergic signaling in the pathogenesis of HCC in patients and Mst1/2-/-,
a mouse model of HCC. Hypothesis: Dysregulation of purinergic signaling facilitates
aberrant cell proliferation underlying hepatocellular carcinogenesis. Methods. HCC
human-derived Huh7 cells, maintained in serum free media for 24h, were treated with
ATP S, or ADP (100µM) for different time intervals. SP600125 pretreatment was used
to inhibit c-Jun N-terminal Kinase (JNK) signaling. Western blotting, qRT-PCR and 5Bromo-2'-deoxy-uridine (BrdU) incorporation analysis were done. Mst1/2-/- and WT
mouse livers (1, 3, & 6 months) and HCC patient livers (n=27) were analyzed by qRTPCR for all 15 P2 purinergic receptor isoforms. Results. Extracellular nucleotide
treatment alone was sufficient to induce cell cycle progression in Huh7 cells, evidenced
by increased BrdU incorporation and increased cyclin D3, E, and A mRNA and protein
expression. We observed downregulation of cyclin D1 mRNA, however, as previously
reported in a subset of HCC with high tumor grade. JNK inhibition attenuated
nucleotide-induced cyclin D3, E and A protein expression, but enhanced downregulation
of cyclin D1. Mst1/2-/- mouse tumors (at 3-6 months) exhibit dysregulated expression
of multiple P2 purinergic receptor isoforms as compared to WT. In HCC patients,
multiple P2 purinergic receptor isoforms were elevated ≥2-fold in liver tumors as
compared to uninvolved areas in up to 52% of patients. P2 purinergic receptor
upregulation was more prevalent among HCC patients infected with hepatitis C virus
(HCV) (75%) as compared to non-viral groups (20%) identifying a unique subset of viralinduced HCC overexpressing P2 receptors. Conclusions. Our results suggest that
extracellular nucleotides are potent mitogens in Huh7 cells, inducing downregulation of
cyclin D1 and upregulation of cyclin E, which are associated with poor prognosis in HCC
patients. Our analysis of HCC patient and Mst1/2-/- mice livers has uncovered a likely
role for purinergic signaling in the pathogenesis of HCC, highlighting P2 purinergic
receptors as potential biomarkers and novel therapeutic targets for HCC.
Contributors: Johnson, Randy L.; Lee, Ju-Seog; Lopez-Terrada, Dolores; Goss, John A;
Thevananther, Sundararajah
Tyler Jordan McCue
Integrative Program in Molecular and Biomedical Sciences
Advisor: Joseph Petrosino, Ph.D.-Department of Molecular Virology & Microbiology
Catheter-associated urinary tract infections (CAUTI) are most common infections
in nursing homes and rank in the top three of hospital-acquired infections. There is
currently no effective strategy in place to prevent CAUTI in long-term catheterized
patients and with a continuing increase in the elderly population this problem will only
grow in the coming years. We hypothesized that colonizing the bladder with a benign
strain of E. coli (HU2117) isolated from a subject with asymptomatic bacteriuria would
be a safe and effective means of using bacterial interference to prevent CAUTI in older,
catheterized adults. Urine was collected longitudinally from 4 patients that were
inoculated with HU2117 via receiving an indwelling catheter that had been pre-coated
with E. coli HU2117. Aliquots of samples were monitored for cultivatable organisms for
28 days or until the loss of E. coli HU2117 using standard microbiological methods.
Each of the 4 subjects had a different outcome: infection developed without HU2117
colonization, infection developed during HU2117 colonization, no infection or HU2117
colonization, and no infection during HU2117 colonization. In order to better understand
the dynamics of the microbial communities in these patients’ bladders we extracted
DNA and performed 16S gene targeted sequencing to identify the bacteria present.
Analysis of the microbial communities present in the subject samples revealed a
significant decrease in the diversity of bladder flora in subjects that developed an
infection. It was found that colonization with E. coli HU2117 did not prevent infection
and did not lead to any significant changes in bladder flora diversity. These results
suggest that microbial diversity may play a protective role against invasive infection of
the catheterized bladder.
Contributors: McCue, Tyler; Horwitz, Deborah; Mapes, Abigail C.; Ajami, Nadim J.; Petrosino,
Joseph F.; Trautner, Barbara W.
Pooja Chetan Mehta
Department of Pathology & Immunology
Advisor: C Smith, M.D.-Department of Pediatrics
γδ T cells are resident in adipose tissue and increase during diet-induced obesity.
Their possible contribution to the inflammatory response that accompanies diet-induced
obesity was investigated in mice after a 5-10 week high milk fat diet (HFD). The HFD
resulted in significant increases in CD44hi, CD62Llo and TNF-α+ γδ T cells in
epididymal adipose tissue (eAT) of wildtype (WT) mice. Mice deficient in all γδ T cells
(TCRδ-/-) or only Vγ4 and Vγ6 subsets (Vγ4/6-/-) were compared with wildtype mice
with regard to pro-inflammatory cytokine production and macrophage accumulation in
eAT. Obesity among these mouse strains did not differ, but obese TCRδ-/- and Vγ4/6-/mice had significantly reduced eAT expression of F4/80, a macrophage marker, and
inflammatory mediators CCL2 and IL-6 compared to WT mice. Obese TCRδ-/- mice had
significantly reduced CD11c+ and TNF-α+ macrophage accumulation in eAT after 5 and
10 weeks on the HFD, and obese Vγ4/6-/- mice had significantly increased CD206+
macrophages in eAT after 5 weeks on the diet and significantly reduced macrophages
after 10 weeks. Obese TCRδ-/- mice had significant reductions in systemic insulin
resistance and inflammation in liver and skeletal muscle after longer-term HFD feeding
(10 and 24 weeks). In vitro studies revealed that isolated γδ T cells directly stimulated
RAW264.7 macrophage TNF-α expression, but did not stimulate inflammatory mediator
expression in 3T3-L1 adipocytes. These findings are consistent with a role for γδ T cells
in the pro-inflammatory response that accompanies diet-induced obesity.
Contributors: Mehta, Pooja ; Smith, C. Wayne
Shrenik Chetan Mehta
Department of Pharmacology
Advisor: Timothy Palzkill, Ph.D.-Department of Pharmacology
β-lactam antibiotics are the most commonly prescribed class of drugs for treating
bacterial infections. β-lactamase enzymes are the most important source of resistance
to these drugs as they hydrolyze the antibiotic and render it inactive. The rapid
dissemination and evolution of these enzymes has resulted in many classes of βlactamases capable of hydrolyzing a broad range of antibiotic substrates. The KPC
group of β-lactamases is one of the most threatening groups of enzymes as they are
capable of hydrolyzing the carbapenem class of β-lactam antibiotics. The carbapenems
currently serve as our last line of defense against bacterial infections. Variants of KPC
β-lactamases differing by one to two amino acids have been isolated from patients all
over the world. Despite the identification of these enzyme variants from the patients,
there is a lack of information about their substrate profiles and catalytic mechanism,
which could help in deciding treatment regimens for such infections. In this study, we
have characterized nine KPC variants that differ from KPC-2 (pseudo wild-type) by one
to two amino acids. The results indicate that both single and double mutant enzymes
hydrolyze the carbapenem substrates as proficiently as KPC-2. When tested for
ceftazidime, a 3rd generation cephalosporin antibiotic, the bacterial cells expressing the
single mutants showed a minimum inhibitory concentration (MIC) up to 6-fold higher
than KPC-2 while the bacterial cells expressing the double mutants showed a 3 to 100
fold increase in MIC. The purified double mutant enzymes also exhibited a 7 to 75-fold
increase in catalytic efficiency for hydrolyzing ceftazidime while the single mutants had
a catalytic efficiency 7 to 11-fold higher than KPC-2. 3rd generation cephalosporins are
the drug of choice for treating unknown bacterial infections in patients; thus, the
evolution of KPC to better hydrolyze ceftazidime is likely the result of selective pressure
due to antibiotic therapy. In addition, this improvement in ability to hydrolyze ceftazidime
does not significantly alter their ability to hydrolyze carbapenems. Thus, mutations
acquired by KPC-2 in natural isolates of bacteria significantly broaden its substrate
profile leading to multi-drug resistance.
Contributors: Mehta, Shrenik; Kacie Rice; Palzkill Timothy
Xiangling Meng
Department of Neuroscience
Advisor: Huda Zoghbi, M.D.-Department of Pediatrics
Background: Rett Syndrome (RTT) is a postnatal neurological disorder caused
by loss of function mutations in the gene encoding methyl-CpG-binding protein 2
(MeCP2). Deleting Mecp2 only from brain tissue at embryonic day 12 leads to
phenotypes identical to those of the null mutation, indicating that loss of MeCP2 from
the CNS is responsible for the RTT phenotypes. Deletion of Mecp2 only from inhibitory
GABAergic neurons recapitulates many RTT phenotypes including the stereotypies and
altered social interaction, but does not replicate anxiety-like behaviors and tremor. The
role that excitatory glutamatergic neurons play in the pathogenesis of RTT has not been
explored in detail.
Method: We conditionally deleted Mecp2 from glutamatergic neurons in the
mouse brain using a vGlut2-Cre line, and characterized the mice by a comprehensive
battery of behavioral tests as well as neurophysiological methods.
Results: The glutamatergic conditional knockout mice (CKO) became obese, and
developed impaired acoustic startle and motor deficits. Interestingly, unlike the
GABAergic CKO, the glutamatergic CKO showed anxiety-like behaviors as early as 5
weeks of age, and developed severe tremor. Furthermore, they died early with half of
them dead by 10 weeks. These phenotypes are identical to the disease progression
pattern of the Mecp2 null mutation. The glutamatergic CKO mice also showed deficits in
conditional fear memory and impairment of excitatory synaptic plasticity measured by
hippocampal CA1 long-term potentiation.
Conclusion: These data demonstrate that dysfunction of MeCP2 in excitatory
glutamatergic neurons contributes to numerous neuropsychiatric phenotypes.
Especially, it drives the onset of anxiety-like behaviors, tremor, and obesity in RTT,
indicating an excitatory neuron-dependent mechanism underlying these phenotypes of
Rett syndrome.
Contributors: Meng, Xiangling; Lu, Hui; Wang, Wei; Chen, Hongmei; Zoghbi, Huda.
Phillip Jay Minnick
Department of Biochemistry & Molecular Biology
Advisor: Susan Rosenberg, Ph.D.-Department of Molecular & Human Genetics
Mutagenesis mechanisms controlled by stress responses increase mutation
rates during stress, potentially accelerating adaptation to stressors such as nutrient
limitation, antibiotics, antifungal drugs and hypoxia, in bacteria, yeast and human cancer
cells. Inhibiting these mechanisms could provide new ways to combat cancer and
infectious disease. One stress-induced mutagenesis mechanism in Escherichia coli is
mutagenic repair of DNA double-strand breaks (DSBs). Under stress, or if the general
(RpoS) stress response is activated artificially, repair of DSBs switches from use of the
high fidelity DNA Pol III to use of error-prone Pol IV causing base-substitution and indel
(“point”) mutations, or use of Pol I causing copy-number variations. Mutagenic repair of
DSBs localizes new mutations both in time (under stress), and in genomic space (near
DSBs). Three stress responses promote mutagenic break repair in E. coli: the
envelope-protein stress response promotes break formation; the DNA-damage
response upregulates Pol IV (promoting point mutagenesis); and the general stress
response licenses use of Pols IV, II and I in repair. Here we demonstrate that a fourth
stress response must be activated for mutagenic break repair to occur: the stringent
(starvation) response. We show that stringent-response-defective ∆relA or ∆dksA cells
are defective for both point mutations and gene amplification. Moreover, the stringent
response promotes mutagenesis independently of the other three stress responses.
First, DksA is still required if DSBs are given artificially with I-SceI enzyme. Thus, the
stringent response role is not in formation of DSBs (role of the envelope-protein-stress
response). Second, the RelA/DksA role is not in DNA damage-response induction
because it is not suppressed by expression of Pol IV(the sole role of the damage
response). Third, mutations that allow general-stress-response-independent
mutagenesis do not compensate for RelA/DksA, demonstrating a role independent of
the general stress response. We conclude that the stringent response constitutes a
fourth, independent, stress response input into mutagenic break repair. The stringent
response presumably regulates the expression of a currently unknown protein(s), critical
to mutagenic break repair. Our data imply that cells regulate mutagenesis very
carefully, requiring four independent stress response inputs to unleash their
mutagenesis “program”.
Contributors: Frisch, Ryan; Gibson, Janet; Terwiliger, Austen; McCue, Tyler; Darrow, Michele;
Herman, Christophe; Rosenberg, Susan
Marissa Mie Kehaulani Minor
Department of Molecular Virology & Microbiology
Advisor: Betty Slagle, Ph.D.-Department of Molecular Virology & Microbiology
Chronic Hepatitis B virus (HBV) affects over 400 million people worldwide and is
a major risk factor for the development of hepatocellular carcinoma (HCC). Our
laboratory studies the HBV regulatory protein HBx, which is required for virus replication
in infection models. We and others have demonstrated that an interaction between HBx
and cellular damaged DNA binding protein 1 (DDB1) is critical for virus replication.
DDB1 functions as an adapter to the Cullin 4A E3 ubiquitin ligase complex. DDB1
recruits DDB1 Cullin Associated Binding Factors (DCAFs) to the Cullin 4A E3 ubiquitin
ligase complex, and the DCAFs in turn recruit substrates to be ubiquitinated and
degraded. Each of the ~90 human DCAFs share a 16 amino acid DDB1-binding WD40
motif. The HBx contains a sequence similar to the DDB1-binding WD40 motif and has
been shown through crystal structure to interact directly with DDB1, suggesting that it is
a viral DCAF. The DDB1 function affected by HBx binding is unknown and there are
few biologically relevant systems to study this interaction. We will be using human liver
chimeric mice (immunodeficient mice that have been engrafted with human
hepatocytes) as an infection model in which to study the HBx-DDB1 interaction.
We hypothesize that HBx binding to DDB1 may alter the DCAF profile, and that
this will lead to changes in the array of cellular factors that are ubiquitinated and
degraded, thereby creating a cellular environment that benefits virus replication.
Aim 1: The first aim of this project will determine the DCAF profiles of normal,
uninfected livers as well as HBV-infected livers and comparing the two to determine the
impact of HBV replication. This will be done using human liver chimeric mice of the
same hepatocyte donor. Immunoprecipitation/mass spectrometry will be used to
identify DCAFs that are bound to DDB1 in both infected and uninfected hepatocytes.
Aim 2: The second aim of this project will determine the impact of the HBx-DDB1
interaction on DDB1 functions such as the DNA damage response and innate immunity.
We will analyze uninfected and HBV-infected liver tissue for the DNA damage marker
γH2AX to determine if the DNA damage response is being activated during HBV
infection in vivo. We will also be using a variety of methods to analyze how HBV may
interact with DDB1 in order to dampen the immune response.
Contributors: Minor, Marissa
Patrick Gerald Mitchell
Integrative Program in Molecular and Biomedical Sciences
Advisor: Wah Chiu, Ph.D.-Department of Biochemistry & Molecular Biology
Microtubule associated protein tau (MAPT, or tau) is implicated as an associated
factor in the pathology of Alzheimer’s Disease (AD), as well as several other
neurodegenerative disorders, collectively referred to as tauopathies. The disease
associated form of tau aggregates in paired-helical filaments (PHF), which interfere with
tau’s ability to bind and stabilize microtubules, and cause higher-order intracellular tau
aggregate formation. These aggregates, or neurofibrillary tangles (NFT’s), are a
common feature of neurological diseases characterized by neuronal dysfunction and
eventual cell death. While it is well supported that pathologically modified tau and tau
aggregates can play a role in neurodegeneration, the specific neurotoxic species, and
the mechanisms underlying their pathology, remain unclear.
Although tau has been the subject of increasing interest in the Alzheimer’s
Disease research community, a comprehensive structural study of tau aggregates has
yet to be performed. Recent technological advances in cryo-electron microscopy (cryoEM) allow for the elucidation of structural features of tau fibrils at a resolution that has
not previously been attainable using this technique. A detailed structural study can
provide insight into the important differences between wild-type tau and aggregation
prone mutant tau.
In addition to high-resolution features of the tau fibril, the role of tau aggregation
within the cell is also an important consideration in our understanding of tau pathology.
Eukaryotic cells have been conventionally considered to exceed the maximum
specimen thickness for investigation by cryo-EM. However, as freezing techniques and
imaging technologies have improved, it has been demonstrated that cryo-EM is a useful
technique for imaging of some eukaryotic cells, especially in thin cellular projections,
such as neurites. As studies of larger specimens have become more common in the EM
community, a hybrid technique known as correlative light and electron microscopy
(CLEM) has emerged as a useful bridge between light microscopy, and the molecular
resolution of EM. This technique will be useful to visualize the structural characteristics
of tauopathies, as well as the mechanistic action of potential treatments.
Contributors: Mitchell, Patrick; Li, Hongmei; Martini-Stoica, Heidi; Dai, Wei; Zheng, Hui; Chiu,
Sayantan Mitra
Department of Biochemistry & Molecular Biology
Advisor: B Prasad, Ph.D.-Department of Biochemistry & Molecular Biology
Influenza viruses cause acute highly contagious respiratory disease and affect
500 million people annually worldwide. Currently the highly lethal H5N1 strain posing a
serious threat for worldwide pandemic, along with recent emergence of influenza virus
strains resistant to the available antiviral drugs make it necessary to identify potential
new drug targets. One of the promising targets is the non-structural protein 1 (NS1) of
influenza virus, which is important for virus replication, spread, and pathogenesis. It is a
potent antagonist of interferon-mediated host antiviral response. It consists of an RNA
binding domain (RBD) and an effector domain (ED) separated by a flexible linker. It
interacts with the F2F3 domain of cleavage and polyadenylation specificity factor
(CPSF30) to inhibit 3’ end processing of cellular pre-mRNA including IFN-β pre-mRNA,
thus inhibiting host antiviral immune response. Although the structure of NS1 ED with
F2F3 has been determined, the structural characteristics of the full-length (FL) NS1
suggest these structures might not be relevant. To determine the structure of H5N1 FLNS1 and F2F3 by X-ray crystallography, both the proteins have been purified
individually and after forming complex, it has been used for initial crystallographic
screening. Currently, optimization of the favorable conditions are been carried out. The
binding studies of H5N1 FL-NS1 and H5N1 NS1-ED with F2F3 by Isothermal titration
calorimetry (ITC) have also suggested FL-NS1 interacts with F2F3 more strongly than
ED alone. This might be due to a different and/or additional set of interactions that
allows for increased F2F3 binding affinity with theFL-NS1 than with ED alone. . In
addition to providing a better mechanistic understanding of how FL-NS1 interacts with
F2F3 and what are the characteristics of these interacting regions, they will also provide
a rational basis for the design and development of new small molecule drugs that
disrupt NS1 interaction with F2F3 and thus the ability of NS1 to antagonize the host
antiviral response.
Contributors: Mitra, Sayantan; Prasad, BVV
Teresa Monkkonen
Department of Molecular & Cellular Biology
Advisor: Michael Lewis, Ph.D.-Department of Molecular & Cellular Biology
Hedgehog signaling mediates organogenesis, and stem cell function in
mammals, and often is misregulated in cancers, including breast. Breast lesions show
decreased expression of Patched 1 (Ptch1), an inhibitor of signaling, and increased
expression of Smoothened (Smo), an activator of signaling. Previous studies using mice
homozygous for a hypomorphic Ptch1 allele showed stunted, dysplastic ducts, while
mice heterozygous for a null allele showed hyperproliferative, hyperplastic mammary
glands with filled-in ducts. Transplantation experiments with these mutants showed a
requirement for epithelial, stromal, and systemic Ptch1; but the true Ptch1 loss-offunction phenotype in each compartment is unknown. We hypothesize that Ptch1
mediated inhibition of canonical hedgehog signaling has discrete epithelial, stromal, and
systemic functions to regulate mammary gland morphogenesis, histology, and
To test this hypothesis, we performed tissue compartment-specific deletion of
Ptch1. Outgrowths from transplanted Adenovirus-Cre treated, conditional null Ptch1fl/fl
mammary epithelial cells yielded hyperbranched, hyperproliferative mammary ducts.
Stromal loss of Ptch1 via Fsp-Cre deletion in fibroblasts and immune cells produced
different phenotypes. Heterozygotes for the null allele were hyperbranched and
hyperproliferative. Homozygous null Fsp-Cre, Ptch1fl/fl animals showed stunted ductal
outgrowths and loss of proliferation in mature ducts and terminal end buds. These
animals recapitulated the filled-in duct phenotype of Ptch1 +/- animals, indicating this
phenotype was due to a stromal function of Ptch1. Both stromal mutants had aberrant
microlumen formation and multiple layers of luminal cells. Interestingly, Fsp-Cre,
Ptch1fl/fl animals had increased ER expression, but complete loss of PR at 8 weeks.
Whole gland transplantation rescued the stunted duct and loss of side branching
phenotypes, but filled-in ducts and microlumens were still present.
In summary, we have shown that epithelial Ptch1 is required to suppress
branching and proliferation. Ptch1 in a systemic Fsp positive cell promotes branching
and proliferation; and Ptch1 in the mammary stroma regulates ductal histomorphology.
These data underscore the importance of tissue-tissue crosstalk in the mammary gland.
Contributors: Monkkonen, Teresa; Landua, John; Visbal, Adriana; and Lewis, Michael T.
Tanner Oliver Monroe
Department of Molecular Physiology & Biophysics
Advisor: James Martin, M.D./Ph.D.-Department of Molecular Physiology & Biophysics
George Rodney, Ph.D.-Department of Molecular Physiology & Biophysics
Traditionally, cardiac hypertrophy was thought to be solely attributed to growth of
the resident cardiomyocytes (CM). However, recent reports indicate an increased level
of cell cycle re-entry and proliferation of resident cardiomyocytes during physiological
hypertrophy. The Hippo pathway is an evolutionarily-conserved negative regulator of
organ growth. In the heart, it activates during late development, facilitating cell cycle
exit, which restricts CM proliferation, thereby controlling the final CM number. Further
growth of the organ is attributed to hypertrophy of the individual myocytes.
There are three main causes of post-natal hypertrophy: exercise, pregnancy
(physiological), and chronic pressure overload (pathological). We believe that during
physiological hypertrophy, where an increase in CM division has been reported, a set of
CMs within the heart re-enter the cell cycle due to the down-regulation of Hippo
pathway kinases, in a crosstalk with the Wnt-Akt signaling axis. Our data, and that of
publicly available data sets indicate that cell-cycle related genes controlled by the Hippo
pathway nuclear effector, Yap, are up-regulated during physiological hypertrophy. In
contrast, during pathological hypertrophy, up-regulation of cell cycle-related transcripts
is absent.
Steady-state CM renewal is thought to be critically important to maintenance of
the health of the organ, with baseline CM replenishment approximately 1% of the cells
per year, diminishing progressively during aging. If our findings are genuine, we believe
we have found a natural way to increase CM renewal, as well as a previously
unreported mechanism for post-developmental heart growth. Further, we will gain
valuable knowledge about the differences that separate pathologic and physiological
hypertrophy, with the aim of targeting these pathways therapeutically.
Contributors: Monroe, Tanner; Zhang, Min; Heallen, Todd; Leach, John; Rodney, George;
Martin, James
Jessica M Moore
Department of Biochemistry & Molecular Biology
Advisor: Susan Rosenberg, Ph.D.-Department of Molecular & Human Genetics
Philip Hastings, Ph.D.-Department of Molecular & Human Genetics
E. coli under stress such as starvation increase mutation rate transiently under
the control of stress responses, until a mutation occurs that allows adaptation. The
mechanism of stress response-controlled switch to mutagenic repair of DNA breaks is
well characterized. lac frameshift-bearing cells starved on lactose medium acquire
compensating frameshift (“point”) mutations, or amplifications of the leaky lac allele to
20-50 copies, which confers sufficient enzyme activity for growth. The mutagenesis
requires (spontaneous) double-stranded DNA breaks and their repair. We show that
reactive oxygen species, specifically persistent oxidative lesions in DNA that result from
them, are required for mutation formation under stress in E. coli. Addition of exogenous
reducing agents, use of constitutively activating alleles for the oxidative damage
responses, and over-expression of katG to scavenge hydroxyl radicals dramatically
reduce mutation formation. We show that reactive oxygen species are not required in
stress-induced mutation to oxidize proteins, but to form oxidative lesions in DNA. These
lesions are not required for the formation of DNA double-stranded breaks or to titrate
out mismatch repair components, and the effect on mutation is lost if lesions are
excised from DNA. Together, these data show a novel requirement for unrepaired
oxidative base changes in DNA for the formation of environmental stress-induced
mutants separately from the requirement for DNA double-stranded breaks and stress
response regulation. We suggest that lesions in DNA pause the replicative DNA
polymerase, allowing error-prone translesion polymerases to become active, leading to
mutation formation.
Contributors: Moore, JM; Mojica AK; Rosenberg SM; Hastings PJ
Christina Narie Morra
Integrative Program in Molecular and Biomedical Sciences
Advisor: James Versalovic, M.D./Ph.D.-Department of Pathology & Immunology
Amino acid metabolism by the gut microbiome is critical for human health.
Commensal bacteria, such as Lactobacillus reuteri, produce biologically active
compounds that are capable of modulating host immunity. The decarboxylation of Lhistidine to histamine by HdcA in L. reuteri reduces the production of the proinflammatory molecule, TNF. Currently is it unknown whether metabolism of histidine by
L. reuteri affects its growth or whether L. reuteri produces other host-modulating
products. Using the stable isotope, 13C615N3 L-histidine in bacterial media we have
begun to elucidate histidine metabolism by L. reuteri ATCC 6475 and identify novel
histidine metabolism pathways. Analysis of 72 hr growth curves of wild type and two
HdcA L. reuteri mutants in defined (LDMIV), semi-defined (LDMIII) and rich (MRS)
media revealed no significant changes in growth or viability. This indicates that histidine
metabolism is not essential for L. reuteri growth and viability. To assess the mechanism
of histidine metabolism by L. reuteri, the semi-defined, LDMIII medium was
supplemented with the heavy isotope, 13C615N3 L-histidine and analyzed by mass
spectroscopy. A preliminary mass spectrometry screen for metabolites determined that
the addition of carnosine (β-alanyl-L-histidine) in the medium significantly increases
histamine production. Together this data has demonstrated that histidine metabolism,
although providing benefit to the host, is not required for L. reuteri growth and that the
addition of carnosine improves histamine production in vitro. Future work will involve coculturing of L. reuteri with human ileal enteroids to determine the effects of L. reuteri and
histamine metabolism on the human intestinal epithelium.
Contributors: Morra, Christina; Versalovic, James
Stephen Christopher Murray
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Steven Ludtke, Ph.D.-Department of Biochemistry & Molecular Biology
Over the last three years, the Cryo-EM community has begun shifting focus from
improving resolution, to validation of results. EMAN2.1 represents the results of major
new developments in EMAN2’s single particle reconstruction and single particle
tomography workflows. The new tools such as e2refine-easy, integrate “gold standard”
resolution assessment into the refinement process along with a number of new
optimizations, both speeding the refinement process, and eliminating the need for
empirical filtration of reconstructions by users. EMAN2.1 also integrates support for tiltpair validation and “true resolution” testing to insure self-consistency among data and
final 3-D maps. This is one of the few methods which can identify incorrect maps at low
Another approach often applied to increase confidence in a 3-D structure is to
reprocess the same data using multiple algorithms, preferably based on different
mathematical methods. EMAN2.1 includes an interface for converting data and
metadata into the appropriate format for reprocessing in Relion[2] or FreAlign[3], two
alternative single particle reconstruction packages. Once these packages complete their
refinements, the results can be imported back into EMAN2.1 for comparison and
analysis. As an alternative, EMAN2.1 can perform the opposite process as well. A
refinement completed originally in one of these other packages can be converted into
an EMAN2.1 project, which can then be used to re-refine the data from scratch.
Another important area of improvement is single particle tomography. Rather
than the traditional approach of reconstructing large numbers of 2-D images of identical
particles in random orientations, in single particle tomography, tomographic data is
collected for fields of particles, producing a low resolution and incomplete, but 3-D
reconstruction for each individual molecule. EMAN2.1 now incorporates tools for
subtomogram extraction, and a variety of different approaches for alignement and
averaging of particles. This approach is a powerful alternative to single particle analysis
particularly in cases where the particles are flexible or heterogeneous in solution
Contributors: Murray, Stephen; Galaz-Montoya, Jesus; Tang, Grant; Flanagan IV, John; Ludtke,
Jaclyn Nicole Murry
Department of Molecular & Human Genetics
Advisor: Ignatia Van Den Veyver, M.D.-Department of Obstetrics & Gynecology
Autism spectrum disorder (ASD) comprises a set of neurodevelopmental conditions
manifesting as repetitive behaviors and social and communication impairments. Genes
affecting synaptic function have been found to be causative in a subset of ASD patients. Early
exposure to environmental factors either by themselves or in conjunction with genetic factors
has also been implicated in ASD, and in combination may play a role in ASD penetrance. To
minimize confounding factors, our lab uses mouse models to answer this question. The overall
goal of my project is to investigate how gene and prenatal environment interact (GXE) to
influence ASD-like behavior in a Shank3 mutant mouse model. I chose the Shank3 null mice
based on their ASD-like behaviors. Furthermore, deletions in SHANK3 cause PhelanMcDermid Syndrome, whereas mutations cause isolated ASD. Our overarching hypothesis is
that ASD-associated risk alleles combined with certain common prenatal exposures can
modulate neurodevelopment, altering maternal care and resulting in offspring behavior
disturbances. This combination might cause an individual to reach a “phenotypic threshold”
resulting in increased ASD penetrance. I will address this hypothesis through two specific aims.
SPECIFIC AIM 1: To assess the effects of prenatal CUMS exposure on maternal care in
Shank3 mutant dams. Preliminary data from our lab indicates that mutant male offspring of
HET Shank3 dams exposed to gestational chronic unpredictable mild stressors (CUMS) exhibit
an atypical response when compared to non-stressed gender and genotype-matched littermates
in the three-chamber social behavior test. Because studies indicate that gestational stress can
alter postpartum maternal care and offspring development, I am examining the effects of
gestational stress on maternal care in WT and mutant Shank3 dams. Proposed experiments
will likely answer how stress combined with genetic susceptibility contributes to observed
SPECIFIC AIM 2: To assess the effects of prenatal FLX exposure of Shank3 HET dams
on offspring neurodevelopment. For the second aim, I am investigating if other prenatal
stressor-genotype combinations will result in altered social behavior in offspring. I am focusing
on Fluoxetine (FLX), a Selective Serotonin Reuptake Inhibitor, regularly prescribed during
pregnancy, and because maternal intake of (FLX) in the first trimester is associated with
increased incidence of ASD. Therefore, I will examine whether FLX exposure in Shank3-mutant
dams affects penetrance of ASD features in offspring through behavioral assessments. Postbattery, offspring brains are being harvested and processed for brain morphology, histology,
gene and protein expression. In doing so, we will be assessing whether these factors exhibit
additive defects. Proposed experiments will likely answer whether prenatal drug exposure
combined with genetic susceptibility can play a role in ASD penetrance.
Contributors: Murry, Jaclyn; He, Fang; Engler, David; Balasa, Alfred; Van den Veyver, Ignatia
Aaron M Muscarella
Integrative Program in Molecular and Biomedical Sciences
Advisor: Xiang Zhang, Ph.D.-Department of Molecular & Cellular Biology
Background: Most deaths from breast cancer are not due to the primary tumor
but from metastasis to secondary sites, the most common of which is the bone. The
Zhang lab recently founds that disseminated tumor cells (DTCs) that arrive in the bone
and progress into multi-cell micrometastases are almost always in proximity to, and in
direct contact with, Osteoblasts, pre-osteoblasts, and their precursor mesenchymal
stem cells (MSCs). It was also shown through in vitro co-culturing and in vivo xenograft
methods that the breast cancer cells receive proliferative advantages and activation of
mTOR signaling from these osteogenic cells, and that this relationship is dependent on
direct contact, calcium, and E-cadherin. This was especially curious as MSCs are not
known to express E-cadherin, only N- and OB-cadherins, and heterotypic cadherin
adherens junctions (AJs) are rarely described in the literature. Little has been
established about the initial, pre-clinical colonization of the bone by single DTCs. If we
are able to learn more about the molecular mechanisms of this niche, there is potential
for developing therapeutic strategies against their progression into fully pathogenic bone
metastases. Experimental design and methods: I performed coated plate assays,
where tissue culture plates were coated with IgG control or E-, N-, or OB- cadherin
fusion proteins. I identified candidate Receptor Tyrosine Kinases (RTKs) that could
activate mTOR signaling in the micrometastatic lesions using a phospho-tyrosine array,
and explored these candidates using qPCR, western blot, and inhibitor treatments. In
addition, I prepared CRISPR knockout constructs and shRNA constructs for AJ
components and these RTKs to use in functional assays. Results: Cancer cell lines
with strong E-cadherin expression proliferated selectively more on N-cadherin coated
surfaces, and cell lines without strong E-cadherin expression grew selectively better on
OB-cadherin coated surfaces. Furthermore, it has been demonstrated that E-cadherinexpressing cells form heterotypic E:N-cadherin AJs both in vitro using a pull-down
assay, as well as in vivo using immunohistochemistry and the blocking E- and Ncadherin. Western blot indicated that both ALK and TrkA are not expressed by our
luminal models, eliminating them as candidate RTK’s, while Ros1 inhibitor Foretinib was
effective at inhibiting growth in cells in contact with MSCs in vitro. Conclusion: Luminal,
E-cadherin expressing breast cancer cells form heterotypic, E:N-cadherin adhesion
junctions in the bone, and relationship activated mTOR signaling through Ros1 or some
other receptor on the cancer cell surface.
Contributors: Muscarella, Aaron; Wang, Hai; Yu, Cuijua; Zhang, Xiang H.F.
Christopher Gary Myers
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: B. Pettitt, Ph.D.-Biochemistry
We have shown that excluded volume, electrostatic forces and surface-induced
correlations are sufficient to predict most of the major features of the current structural
data of DNA packaged within viral capsids without assuming any elastic conformational
ordering. Current models assume highly-ordered, even spooled conformations, based
on interpretation of cryo-EM density maps. We have shown that surface-induced
ordering of unconnected DNA polymer segments is the only necessary ingredient in
creating ringed densities consistent with experimental density maps. This implies that
the ensemble of possible conformations of polymeric DNA within the capsid consistent
with cryo-EM data may be much larger than implied by the traditional interpretation that
such rings indicate a highly-ordered spool conformation. This suggests a more
disordered, entropically-driven view of phage packaging may be possible.
We have also shown the electrostatics of the DNA contributes a large portion of
the internal hydrostatic and osmotic pressures of a phage virion, suggesting that nonlinear elastic anomalies might be responsible reduction of overall elastic bending
energies, allowing more disordered conformations to be free-energetically favorable.
Currently MC path sampling techniques, phage genomic data, and sequencedependent DNA elasticity predictions are being employed to accurately model the
elastic bending contributions. Biophysical prediction of the pressure and structures of
DNA confined within phage capsids will aid prediction of DNA expression in vivo, as well
as the design of phage sequence-delivery methods.
Contributors: Myers CG1,2, Pettitt BM1,2
Amritha Nair
Department of Molecular & Human Genetics
Advisor: Thomas Westbrook, Ph.D.-Department of Biochemistry & Molecular Biology
Background:Tyrosine kinases are known oncogenic drivers of cancer. The role of
the antagonizing enzymes namely the Tyrosine Phosphatases has been less
characterized. Our lab identified PTPN12, a tyrosine phosphatase, to be a bonafide
tumor suppressor in breast cancer. It is frequently inactivated in Triple negative breast
cancer [TNBC] and negatively regulates several oncogenic TKs. Furthermore the
tumorigenic potential of PTPN12- deficient breast cancer cells is severely impaired upon
restoration of PTPN12 function. This suggests that loss of the tumor suppressor
PTPN12 drives cooperative TK activation in many breast cancers and that identifying
these PTPN12-regulated kinases and further perturbing them in combination might
ameliorate these cancers.
Experimental design and methods: Using multiple orthogonal genetic and
biochemical approaches we chose to identify the PTPN12-regulated kinome. We tested
the role of PTPN12-regulated kinases in TNBC proliferation and identified combinatorial
TKi treatments that impair survival of these cancers in vitro. We further utilized
comprehensive testing of xenografts and patient-derived tumorgrafts [PDXs] models to
validate our pharmacological findings in vivo.
Results: Using a confluence of techniques including BiFC, RTK arrays and IP we
have identified a narrow number of oncogenic RTKs that are regulated by PTPN12,
including PDGFRB, MET, NTRK1, NTRK2, ALK and others. We report that PTPN12
sensitive breast cancers respond in vitro to TK inhibition using a combination of the
drugs Sunitinib & Crizotinib that together inhibit a broad spectrum of the PTPN12regulated kinome. Due to the frequency of PTPN12 loss in TNBC we have expanded
our pre-clinical testing to rigorously test the efficacy of this combination on a panel of 16
PDXs. Our studies show that PDXs with low levels of PTPN12 [determined by IHC]
seem to correlate with better response to the drug combination.
This study clarifies a paradigm wherein cancers that lack
conventional biomarkers for TK dependency like amplifications/mutations, could still be
responsive to combination Tki therapy due to loss of a key regulator of tyrosine kinase
signaling and activation like PTPN12.
Contributors: Nair, Amritha1; Sun, Tingting2; Tyagi, Siddhartha2; Dominguez-Vidana, Rocio3;
Dobrolecki, Lacey.E. 4 ; Petrovic, Ivana 4 ; Schmitt, Earlene 3; Osborne, C.Kent 4 ; Schiff, Rachel 4 ;
Shaw, Chad.A. 1; Lewis, Michael.T. 4; Westbrook,Thomas.F. 1,2,3,4.
1Department of Molecular and Human Genetics, Baylor College of Medicine, TX. 2The Verna and Marrs
McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX.
3Interdepartmental Graduate Program in Cell and Molecular Biology, Baylor College of Medicine,
Houston, TX, 4Lester & Sue Smith Breast Center, Baylor College of Medicine, Houston, TX.
Maria Namwanje
Department of Molecular & Human Genetics
Advisor: Chester Brown, M.D./Ph.D.-Department of Molecular & Human Genetics
Obesity is a growing epidemic worldwide; including the United States, where 35%
of adults are classified as obese. Several members of the TGF-beta superfamily play
important roles in body composition, adiposity and energy metabolism, including
protection from diet-induced obesity. Activins (A and (B are TGF-beta family members
with demonstrated roles in adipocyte differentiation and function. Activin (A inhibits
adipocyte differentiation of 3T3-L1 cells and human preadipocytes by decreasing the
expression of C/EBP(, which results in increased preadipocyte proliferation. Increased
expression of activin (B in mice and humans is associated with obesity. Additionally,
activin (B decreases lipolysis and down-regulates expression of lipolytic genes. In this
study, we seek to explore direct roles of activins in white adipose tissue. We utilize
mouse models with an adipose-specific conditional knockout of Inhba (activin (A) and a
global Inhbb (activin (B)-null mutation to understand the combinatory effects of activin
deficiency in adipose tissue and metabolism. Loss of either Inhbba or Inhbb has a
modest suppressive effect on diet-induced obesity. A combination of adipose-selective
deletion of Inhbba on the Inhbb-null background (activin double mutant mice) leads to
severe weight loss and reduced adiposity. Combined activin (A and activin (B deficiency
up-regulates the gene expression of Cidea, Cpt1b, FoxO1 and Pgc1-( in brown adipose
tissue, liver and skeletal muscle in activin double mutant mice when compared to wild
type controls. Moreover, we found that activin (A and activin (B deficiency induces
‘britening/beiging’ predominantly in visceral white adipose tissue, where the white
adipose tissue adopts a gene expression profile and morphological characteristics of
brown adipose tissue. Together our results suggest that disruption of activin signaling
reduces body growth and adipostity by up-regulating expression of genes involved in
metabolism and mitochondrial function. The ‘britening’ of white adipose tissue suggests
that combined loss of activin (A and activin (B probably alters the function of white
adipose tissue from storage to energy expenditure in the activin double mutant mice.
Together, these data highlight activins as suitable therapeutic targets, especially in
visceral white adipose tissue where they might be useful in promoting energy
Contributors: Namwanje, Maria; Huang, Lihua; Bournat Juan, Brown, Chester
Andrea Kathleen Nash
Department of Molecular Virology & Microbiology
Advisor: Joseph Petrosino, Ph.D.-Department of Molecular Virology & Microbiology
As the incidence of Clostridium difficile infection (CDI) increases, the use of
highly successful fecal microbiota transplants continues to grow. Concerns with this
procedure, though, including the need for a healthy stool donor, the potential for
pathogen transmission, and the inability to standardize a treatment regimen, have lead
to exploration of alternative approaches. One approach involves the transplant of a
defined bacterial community, which avoids the concerns that come with using feces
mentioned above. A man with chronic co-morbidities was admitted to the VA Hospital in
Houston, TX because of C. difficile-associated diarrhea that began while receiving
augmentin. Stools were C. difficile toxin positive by PCR and enteric pathogen negative.
Vancomycin was given with minimal effect. After 7 days he received a Bacteroides
transplant, which contained 109 cells of three different Bacteroides species
resuspended in 200 mL of preservative-free saline (with 1% human albumin), infused
into the proximal intestine via an ultraslim endoscope. He improved rapidly and within
24 hours was clinically well. He was discharged after 12 days with one formed bowel
movement every other day and has remained without diarrhea for more than one year.
Assessment of changes in the stool microbiota was done by 16S rRNA gene
sequencing using DNA isolated from samples collected on the day of treatment (before
treatment), one to eleven days post-treatment, and from a final sample collected at a
day 40 follow-up visit. Treatment with the triple species Bacteroides infusion resulted in
marked changes in the microbial communities found in the stool of the subject during
the first 10 days of treatment. By day 40, a dramatic rise in Bacteroides was observed.
Whole genome shotgun sequencing studies revealed that all of the three strains were
present up to 40 days after treatment. Therefore, relapsing chronic CDI was
successfully treated with Bacteroides replacement therapy, which avoided the additional
risks of using human feces. In contrast to fecal microbiota transplants, only three
bacterial strains were required for successful amelioration of disease. Additional clinical
studies await receipt of a pending IND.
Contributors: Nash, Andrea; Wong, Matthew C; Smith, Daniel; Ajami, Nadim J; Attumi, Tariq;
Opekun, Antone; Metclaf, Ginger A; Muzny, Donna; Highlander, Sarah; Graham, David Y;
Petrosino, Joseph F
Nicholas Jay Neill
Department of Molecular & Human Genetics
Advisor: Thomas Westbrook, Ph.D.-Department of Biochemistry & Molecular Biology
c-MYC (MYC) overexpression or hyperactivation is one of the most common
drivers of human cancer; however, despite intensive study, the MYC oncogene remains
recalcitrant to therapeutic inhibition. Therefore, the development of targeted therapies
for MYC-driven cancers remains an important challenge facing the field of cancer
therapeutics. Non-oncogene addiction, the dependence of cancers on support pathways
that allow cell survival in the presence of oncogene-induced stress, is a concept that
can be exploited to develop targeted therapies for undruggable cancer drivers such as
MYC. Our laboratory has identified several such non-oncogene addictions in MYCdriven breast cancer, including the cellular sumoylation machinery and the spliceosome.
Importantly, we have also discovered that genetic background strongly influences the
dependencies of MYC-driven tumors. Given the diverse spectrum of tumor-derived
mutations that exist within any particular patient population, it is crucial to consider the
effect of genetic background on dependencies that are intended to be translated into
clinical therapies. The goal of this work is to identify genetic backgrounds that enhance
the dependency of MYC-driven cancers on known non-oncogene addictions of MYC
and to discover novel dependencies that are maintained across multiple genetic
backgrounds. To do this, we will perform pooled shRNA screens in human mammary
epithelial cells that have been engineered to allow inducible MYC hyperactivation in four
separate genetic backgrounds. The results of these screens will allow us to identify
cellular pathways that MYC-driven cancers are dependent on for survival under many
different conditions, so-called “conserved” dependencies. The identification of these
types of dependencies is important because they represent therapeutic targets that are
potentially relevant to a larger patient population than dependencies that are restricted
to a specific genetic background. In addition, it will be critical to determine whether
known dependencies of MYC-driven cancers, such as the sumoylation and splicing
machinery, are conserved across multiple genetic backgrounds or restricted to a
particular set of circumstances.
Contributors: Neill, Nicholas; Hsu, Tiffany; Dominguez-Vidana, Rocio; Karlin, Kristen;
Westbrook, Thomas
Chinh Thi Quynh Nguyen
Department of Molecular Virology & Microbiology
Advisor: Anthony Maresso, Ph.D.-Department of Molecular Virology & Microbiology
Whereas DNA provides the information to design life, and proteins the materials
to construct it, the metabolome can be viewed as the physiology that powers it. As such,
metabolomics, the field charged with the study of dynamic small molecule fluctuations in
response to changing biology, is now being used to study the basis of disease. The use
of metabolomics has been increasingly gaining interest in the field of infectious
diseases. The metabolome of an organism reflects all metabolic fluctuations and can
give powerful insight into characterizing infection as well as fuel the discovery of new
biomarkers. Here, we describe the first metabolomic analysis of a systemic bacterial
infection using Bacillus anthracis, the etiological agent of anthrax disease, as the model
pathogen. Whole organ and blood analysis identified more than 200 different
metabolites that changed in response to infection, with most drastic alterations in lipid
metabolites and their associated biosynthesis pathways. Bioinformatic analysis revealed
two prominent lipid pathways affected during infection. Products of the polyunsaturated
fatty acid and lysolipid biosynthesis pathways, potent mediators of the innate immune
response that rely on the activity of host phospholipase A2 (PLA2) enzymes, were
reduced in infected mice compared to uninfected control. Metabolite changes were
detected as early as 1 day post-infection, well before onset of disease or the spread of
bacteria to organs. These findings suggests a novel mechanism in which B. anthracis
actively suppresses innate immune response by downregulating the levels of proinflammatory lipid mediators produced by PLA2 enzymes, thus preventing the initiation
of robust host defense mechanisms. Functional studies confirmed the importance of
PLA2 in host survival as drug inhibition of PLA2 activity potentiated disease severity and
increased mortality in mice infected with B. anthracis compared to animals given
bacteria or drug alone. Collectively, this study provides a blueprint for using
metabolomics as a diagnostic platform that grades the stage and type of infection, as
well as an identifier of novel pathogen- and host-induced processes that contribute to
bacterial pathogenesis.
Contributors: Nguyen, Chinh Thi Quynh; Maresso, Anthony
Lena H Nguyen
Department of Neuroscience
Advisor: Anne Anderson, M.D.-Department of Pediatrics
Rationale: Cortical dysplasia (CD) is a malformation of cortical development that
is a prevalent cause of severe and intractable pediatric epilepsy. Hyperactivation of the
mechanistic target of rapamycin (mTOR) pathway has been demonstrated in human CD
as well as animal models of epilepsy. Although inhibition of mTOR signaling early in
epileptogenesis suppresses epileptiform activity in the neuron subset-specific Pten
knockout (NS-Pten KO) mouse model of CD, the effects of mTOR inhibition after
epilepsy is fully established have not been investigated in this model. Here, we
evaluated whether mTOR inhibition suppresses epileptiform activity and other
neuropathological correlates in older NS-Pten KO mice with severe and well-established
Methods: The progression of mTOR pathway dysregulation and hippocampal
gliosis were evaluated using western blotting in 2, 4, 6, and 8-9 week-old NS-Pten KO
mice. Antibodies against p-S6 (S240/244) and p-AKT (S473) were used as markers for
mTORC1 and mTORC2 activation, respectively. GFAP and IBA1 were used as markers
for astrocytes and microglia, respectively. NS-Pten KO mice were treated with the
mTOR inhibitor rapamycin (10 mg/kg i.p., 5 days/week) starting at postnatal weeks 9
and monitored with video-electroencephalography (EEG) recordings for epileptiform
activity. Western blotting was also performed to evaluate the effects of rapamycin on the
expression of the glial markers.
Results: In parallel to the previously reported progressive epilepsy phenotype, we
found elevated protein levels of p-S6, p-AKT, GFAP, and IBA1 in NS-Pten KO mice that
became increasingly different from age-matched WT mice with age (p<0.05; n=6-15).
Treatment with rapamycin significantly suppressed epileptiform activity (p<0.001; n=5-6)
and increased survival (p<0.05; n=7-20) in severely epileptic NS-Pten KO mice
compared to naïve and vehicle-treated controls, suggesting that aberrant mTOR
signaling may play a crucial role in the maintenance of epilepsy in this model. At the
molecular level, rapamycin treatment was associated with a reduction in the levels of pS6 and p-AKT as well as GFAP and IBA1 (p<0.05; n=7-14).
Conclusions: Late inhibition of mTOR suppresses established, severe epilepsy
and associated hippocampal gliosis in NS-Pten KO mice. These findings reveal a wide
window for successful therapeutic interventions with mTOR inhibition in the NS-Pten KO
mouse model and further support mTOR inhibition as an effective treatment for latestage epilepsy associated with CD.
Contributors: Brewster, Amy; Clark, Madeline; Regnier-Golanov, Angelique; Sunnen, C. Nicole;
Patil, Vinit; Anderson, Anne
Bryan C Nikolai
Department of Molecular & Cellular Biology
Advisor: Bert O'Malley, M.D.-Department of Molecular & Cellular Biology
Approximately 20% of early-stage breast cancers display amplification or
overexpression of the ErbB2/HER2 oncogene, conferring poor prognosis and resistance
to endocrine therapy. Specific targeting of HER2+ tumors with trastuzumab or the
receptor tyrosine kinase (RTK) inhibitor lapatinib drastically improves survival, yet tumor
resistance and progression of metastatic disease can develop over time. While the
mechanisms of cytosolic HER2 signaling are well studied, nuclear signaling components
and gene regulatory networks that bestow mechanisms of therapeutic resistance and
proliferation are less well understood. Here, we use biochemical and bioinformatics
approaches to identify effectors and targets of HER2 transcriptional signaling in BT-474
cells. Phosphorylation and activity of the Steroid Receptor Coactivator-3 (SRC-3) is
reduced upon HER2 knockdown, and recruitment of SRC-3 to regulatory elements of
endogenous genes is diminished. Transcripts regulated by HER2 signaling are highly
enriched with E2F binding sites and define a gene signature associated with
proliferative breast tumor subtypes, cell cycle progression, and G1 to S phase transition.
Interestingly, pathway and network analysis identifies a cyclin dependent kinase (CDK)
signaling node that, when targeted using the CDK4/6 inhibitor PD 0332991, is
cooperative with other signaling pathways and potential therapeutic inhibitors thereof.
This work shows that HER2 signaling drives proliferation in breast cancer cells through
regulation of E2F target genes, at least in part through phosphorylation of SRC-3.
Moreover, these results have implications for logic-based discovery of pharmacological
combinations in pre-clinical models of adjuvant treatment and therapeutic resistance.
Contributors: Lanz, Rainer B; Creighton, Chad J; Hilsenbeck, Sue G; Lonard, David M; Smith,
Carolyn L; and O’Malley, Bert W.
Ilya Novikov
Department of Biochemistry & Molecular Biology
Advisor: Olivier Lichtarge, M.D./Ph.D.-Department of Molecular & Human Genetics
Functional non-coding RNAs play a vital role in a variety of subcellular processes
including RNA and DNA modification, maintenance of genome stability, and gene
regulation. In this work we aim to gain better insight into the evolutionary relationship
between sequence, structure, and function of these molecules. In particular, we
hypothesize that functionally-important nucleotides in these molecules can be identified
by analyzing the patterns of sequence and phylogenetic variation displayed by the
molecule though its evolutionary history. We then combine this evolutionary information
with structural analysis and discover that the more evolutionarily-important nucleotides
tend to form well-defined, non-random clusters on the structure of the molecule. We
also show that these clusters often constitute known functionally-relevant regions, such
as metabolite and ion binding sites, protein interfaces, and catalytic pockets. This
suggests that evolutionarily-important nucleotides in structured ncRNA molecules
evolve in a manner that is detectable, and that functional regions in these ncRNA
molecules can be predicted based on sequence and phylogenetic information.
Contributors: Wilkins, Angela; Lichtarge, Olivier
Derek Steven O’Neil
Program in Translational Biology & Molecular Medicine
Advisor: Kjersti Aagaard, M.D./Ph.D.-Department of Obstetrics & Gynecology
William Gibbons, M.D.-Department of Obstetrics & Gynecology
Objective: We have previously demonstrated that a maternal high fat diet is
associated with the disruption of the circadian pathway in the fetal liver. Npas2, a
circadian gene, is associated with metabolic regulation in fetal development. It is
unknown how circadian rhythms are established in fetal development with the lack of
light/dark cues through the central clock. We have developed a novel Npas2 conditional
knock out (cKO) mouse model of the peripheral clock (liver) to investigate the role of the
peripheral clock in establishing metabolic homeostasis.
Study Design: Npas2 cKO were generated by targeting the deletion of exon 3
through the cre-lox system of conditional gene deletion. Mice with the Albumin Cre
transgene and heterozygous for the loxp flanked (Fl) region of the Npas2 gene
(Albcre;Fl/+) were mated to control (Fl/Fl) mice to generate Npas2 cKO mice
(Albcre;Fl/Fl) and control mice (Albcre;Fl/+ ). Mice are genotyped between postnatal
day 14-20 (p14-20) by PCR amplification of tail genomic DNA. 149 mice were weaned
at p21 onto control (CD, Harlan Teklad TD.08485) or high fat diet (HFD,
Harlan Teklad TD.88137) and weighed biweekly.
Results: Loss of Npas2 in the liver does not alter weight gain of the mice. As
expected, both the male cKO mice and the Albcre;Fl/+ control mice placed on a HFD
gained significantly more weight than the mice placed on the CD by 5 weeks post
weaning (p=.02 and .04 respectively). However, male Fl/Fl mice on both CD and HFD
were not significantly different at 5 weeks (p=.29) and 15 weeks (p=.27) post weaning.
We observed that among the mice placed on the HFD the Albcre;Fl/+ mice weighed
more than the mice of the cKO (2.1±.90g) and Fl/Fl (1.99±.59g) groups (15 weeks post
Conclusion: We successfully generated a novel mouse model to study to role of
the peripheral clock, regulated by Npas2. The initial phenotype indicates that Npas2 in
the peripheral clock (liver) is necessary for maintaining metabolic homeostasis in
response to a high fat diet.
Contributors: Derek O’Neil, Danielle Goodspeed, Laura Krannich, Kjersti Aagaard
Ninad Ramesh Oak
Department of Molecular & Human Genetics
Advisor: Sharon Plon, M.D./Ph.D.-Department of Pediatrics
Identification of cancer susceptibility gene mutations can impact patient risk
stratification, surveillance for second primary malignancies and risk in family members.
Germline mutations in genes related to telomere maintenance are associated with bone
marrow failure and hematologic malignancies.
To explore genetic predisposition to pediatric cancers, we performed whole
exome sequencing of constitutive DNA from childhood cancer patients including seven
kindreds with familial leukemia/lymphoma in at least two first degree relatives. We
filtered for rare variants (<1% in dbSNP or in another non-cancer database,
Atherosclerosis Risk in Communities (ARIC)) that are shared between affected
individuals within a family and were predicted to exert deleterious effects on the protein.
We limited our analysis to truncating (nonsense and frameshift) mutations. In one of the
families, we found 7 frameshift and 4 nonsense mutations. The most interesting
candidate among these was the novel variant p.R314X in TEP1. Loss-of-function
mutations in TEP1 are very rare in sequence databases including the NHLBI exome
sequencing project. This rare mutation was shared by a parent-child pair affected by
acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML) respectively.
We further explored whether germline mutations in TEP1 are found in larger
cohort of apparently sporadic AML patients. AmpliSeq™ sequencing of 43 genes
related to telomere maintenance and DNA repair pathways was performed on DNA
samples from 82 pediatric AML patients. This analysis revealed 10 rare missense
germline mutations in TEP1. Four of these 10 mutations predicted to have damaging
effect on protein function according to Combined Annotation Dependent Depletion
(CADD) tool (CADD scaled score of 15 or more).
The TEP1 familial p.R314X truncating mutation in addition to germline TEP1
missense mutations in sporadic AML patients warrant further functional studies. These
studies will include assessing the role of these TEP1 mutations on the telomere length
phenotype of cells from the patients carrying the mutation. The results of these studies
are designed to determine whether TEP1 represents another member of the telomere
maintenance genes to be associated with AML predisposition.
This work was supported by research grant RP10189 from the Cancer
Prevention and Research Institute of Texas and R01-CA138836 to Plon, SE and
training grant T32 GM007526 to Powell, BC.
Contributors: Oak, Ninad; Ritter, Deborah; Powell, Bradford; Cheung, Hannah; Gramatges,
Monica; Wheeler, David; Plon, Sharon
Joshua Michael Oakes
Program in Cardiovascular Sciences
Advisor: Susan Hamilton, Ph.D.-Department of Molecular Physiology & Biophysics
Two immunophilins, FKBP12 and FKBP12.6, regulate the activity of cardiac
Ca2+ release channels (RyR2). Mice with a cardiac-specific deficiency in FKBP12
display an early onset dilated cardiomyopathy. To define the mechanisms that drive
this myopathy, we assessed changes in Ca2+ handling in isolated cardiomyocytes.
Ca2+ spark frequency and amplitude, but not duration, are increased by FKBP12
deficiency. We found that this increase in SR leak is due to enhanced SR stores
alongside an increase in cytoplasmic Ca2+ levels. The source of this global Ca2+
increase in unclear, but could be due to enhanced Ca2+ influx, reduced efflux, or
lowered Ca2+ buffering capacity
Female FKBP12 deficient mice also displayed dilated cardiomyopathy but
following birth have an increased risk of dying due to heart failure. Performing
echocardiography, we found that between E14.5 and birth FKBP12 deficient females
transition from beneficial to deleterious hypertrophy. In summary, these results indicate
that FKBP12 plays a vital role in cardiac function at both baseline and during
pregnancy, and Ca2+ handling in cardiomyocytes.
Contributors: Oakes, Joshua; Hanna, Amy; Dagnino-Acosta, Adan; Lee, Chang Seok; Hamilton,
Vitor Onuchic
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Aleksandar Milosavljevic, Ph.D.-Department of Molecular & Human Genetics
A known issue with DNA methylation profiling in complex tissues, such as
breast, is the fact that such datasets are generated from a heterogeneous cell
population, and that each of the constituting cell types has a distinct methylation profile.
Since methylation profiling techniques only measure the average level of methylation for
a cell population, changes in cell type proportions between different samples will lead to
differences in methylation between those samples. Distinguishing between methylation
differences that are due to changes in proportions of cell types and those that are due to
changes in the methylation status of a particular region of the genome in a specific cell
type is essential for improving the sensitivity and specificity of case-control studies
focused on changes in methylation.
Further, determining the proportions of different cell types in each complex
tissue sample will also allow one to detect changes in proportions of particular cell types
between different sets of samples. Examples of the importance of detecting such shifts
in cell type compositions are that changes in proportions of cell types in blood have
been shown to associate with aging, and higher levels of immune cells in breast cancer
have been shown to associate with better prognosis.
We selected a set of 1000 target regions that are known to be involved in
breast cancer biology. Using a set of primers designed to specifically amplify these
regions in bisulfite treated genomic DNA, we have applied microdroplet-based targeted
sequencing approach to 64 bisulfite-converted samples including breast cancer cell
lines, a range of ER positive and negative tumors, and a set of breast tumor samples in
different stages of development coming from the same individual.
This dataset will be used to test a few different approaches for estimating
both cell type composition of breast cancer samples, as well as methylation profiles of
individual constituent cell types of such samples. The same approaches will then be
applied to the 450K and 27K array breast cancer datasets available in TCGA.
Contributors: Onuchic, Vitor; Hartmaier, Ryan; Boone, David; Roth, Matt; Samuels, Michael;
Lee, Adrian; Milosavljevic, Aleksandar;
Mario G Oyola
Department of Neuroscience
Advisor: Shailaja Mani, Ph.D.-Department of Molecular & Cellular Biology
Mariella De Biasi, Ph.D.-Department of Neuroscience
In addition to androgenic properties mediated via androgen receptors, DHT also
regulates estrogenic functions via an alternate pathway. These estrogenic functions of
DHT are mediated by its metabolite 5α- androstane-3β, 17β-diol (3β-diol) binding to
estrogen receptor β (ERβ). CYP7B1 enzyme, involved in conversion of 3β-diol to
inactive 6α- or 7α-triols, could play an important role as a regulator of estrogenic
functions mediated by 3β-diol. Using a mutant mouse carrying a null mutation for the
CYP7B1 gene (CYP7B1KO), we examined the contribution of CYP7B1 on physiology
and behavior. Male, gonadectomized CYP7B1KO and their wild type (WT) littermates
were assessed for their behavioral phenotype, anxiety-related behavioral measures and
hypothalamic pituitary adrenal (HPA) axis reactivity. No significant effects of genotype
were evident in anxiety-like behaviors in open field (OFA), light-dark (LD) exploration,
and elevated plus maze (EPM). Testosterone (T) significantly reduced open arm time
on the EPM, while not affecting LD exploratory and OFA behaviors in CYP7B1KO and
WT littermates. T also attenuated the enhanced corticosterone response, following
EPM, in both genotypes. T was able to reinstate male-specific reproductive behaviors
(number of mounts, intromission, and ejaculation, and their respective latencies) in the
WT, but not in the CYP7B1KO mice. The defect in male reproductive behaviors appears
due to their inability of CYP7B1KO to distinguish olfactory cues from an estrous female.
CYP7B1KO mice also showed a reduction in androgen receptor (AR) mRNA expression
in the olfactory bulb. Our findings suggest a novel role for the CYP7B1 enzyme in the
regulation of male reproductive behaviors.
Contributors: Oyola, Mario G; Zuloaga, Damian G; Carbone, David; Malysz, Anna M; AcevedoRodriguez, Alexandra; Handa, Robert J; Mani, Shaila K.
Ayse-Sena Ozseker
Program in Developmental Biology
Advisor: Meng Wang, Ph.D.-Department of Molecular & Human Genetics
Lipid metabolism is under control of environmental and genetic factors. The
nervous system receives environmental and sensory inputs, and consequently
communicates with adipose tissue to control lipid metabolism. To date, the molecular
mechanisms underlying the neuroendocrine regulation of lipid metabolism remain poorly
understood. My project aims to dissect the regulation of lipid storage and metabolism by
sensory perception using Caenorhabditis elegans as a model organism. Based on the
preliminary studies, my hypothesis is that signaling in sensory neurons modulates the
release of neuroendocrine factors, which transduce signals to systemically regulate lipid
To find the neurons and neuropeptides that are involved in the regulation of lipid
storage, I have examined lipid storage in 30 chemosensation mutants using stimulated
Raman scattering (SRS) microscopy. SRS microscopy is a quantitative chemical
imaging technology that does not require labeling lipids or fixing worms and assesses
the levels of triacylglycerides, the main lipid storage form in the worm adipose tissue,
the intestine. Our best hit from this screen is the daf-11 mutant that lack a guanylyl
cyclase expressed in five different head sensory neurons all of which show defective
neuronal signaling in the mutant worms. The intestine of C. elegans is not innervated
and daf-11 mutants display normal food intake, suggesting that secreted factors from
these sensory neurons regulate intestinal lipid storage in a cell non-autonomous
manner. Consistent with this hypothesis, EGL-21, a carboxypeptidase essential for
neuropeptide processing, is required for the lipid storage increase in daf-11 mutants.
Additionally, inactivation of the worm homolog of FoxO transcription factor, daf16, suppresses the increased lipid storage phenotype of daf-11 mutants. DAF-16 is the
major effector of insulin/insulin-like growth factor signaling (IIS). daf-2 mutants that lack
the insulin receptor also have high intestinal lipid storage in a daf-16 dependent
manner. To test whether IIS is downstream of daf-11, we knocked-down the insulin
receptor daf-2 in daf-11 mutants by RNAi and found that this can further increase the
lipid storage suggesting daf-11 regulates daf-16 in a parallel pathway to IIS.
Currently, we are analyzing which neuropeptides are differentially expressed in
daf-11 mutants in comparison to wild-type. After identifying such neuropeptides, we will
determine their necessity and sufficiency for increased lipid storage. In parallel, we are
studying the tissue specificity of daf-16 in lipid storage regulation.
Contributors: Ozseker, Ayse Sena; Yu, Yong; Wang, Meng (PI)
Francis Pankowicz
Department of Molecular & Cellular Biology
Advisor: Karl-Dimiter Bissig, M.D./Ph.D.-Department of Molecular & Cellular Biology
While gene therapy holds great potential for treating a wide variety of diseases,
vector delivery remains a significant obstacle to overcome. Viral vector delivery
systems are among the most promising, but are hindered by varied tissue-specificity
and immunogenic effects. Adeno-Associated Virus (AAV) is a non-integrating virus that
can infect both dividing and non-dividing cells, and is currently being evaluated for gene
therapy potential due to its diminished immune response in humans. Tissue-specificity
for AAV is mediated by its capsid serotype, some of which have been studied and
tested in a clinical trial setting. Our work focuses on identifying the most suitable AAV
serotype for gene transfer in the liver. Using AAV with known serotypes as well as
recently isolated novel serotypes, we have highlighted the dramatic differences between
in vitro and in vivo transduction efficiency. Furthermore, by utilizing a human liver
chimeric mouse, we have demonstrated the preferential infection of hepatocytes from a
species-specific perspective. Finally, we have identified AAV9 as the serotype most
suitable for transduction of human hepatocytes in vivo (human liver chimeric mouse).
This novel use of human liver chimeric mice therefore uncovers major transduction
variability between the AAV serotypes. Moreover, our data suggest that AAV9 may
produce superior hepatic transduction in clinical trials than the currently used AAV8.
Contributors: Pankowicz, Francis; Wang, Lili; Bell, Peter; Bissig- Choisat, Beatrice; Barzi
Dieguez, Mercedes;
Kruse, Robert; Legras, Xavier; Wieland, Stefan; Bissig, Karl- Dimiter
Jiyoung Park
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Stelios Smirnakis, M.D./Ph.D.-Department of Neurology
Although slice recordings and model studies provide a lot of information about
how neurons integrate their inputs to overall responses, it is still not clear how neurons
in the brain integrate their synaptic inputs to derive their functional properties in vivo.
Pyramidal neurons in layer 2/3 of mouse V1 receive input from LGN via layer 4 and
feedback from higher visual areas. Here, we focus on the function of apical dendrites,
which play an important role in communicating with higher visual areas. We ablate
apical dendrites of layer 2/3 pyramidal neurons in area V1, in vivo, and monitor their
responses to visual stimuli by two photon microscopy. We compare orientation
selectivity, contrast sensitivity and other receptive field properties of L2/3 pyramidal
neurons before and after selective apical dendritic ablation. This approach provides
information on how different dendritic inputs contribute to neuronal receptive field
properties of L2/3 pyramidal neurons in mouse V1.
Contributors: Park, Jiyoung ; Smirnakis, Stelios M
Maha Praful Patel
Program in Translational Biology & Molecular Medicine
Advisor: Timothy Palzkill, Ph.D.-Department of Pharmacology
Robert Atmar, M.D.-Department of Medicine
The Centers for Disease Control and Prevention reports that 2 million people
acquire antibiotic-resistant infections and 23,000 people die as a result each year in the
United States. β-lactam antibiotics are the most commonly prescribed antibiotics for
Gram-negative bacterial infections. The most common mode of resistance to β-lactam
antibiotics in Gram-negative bacteria is the production of β-lactamases. In particular, the
CTX-M family of β-lactamases is the most widespread and frequently isolated in clinical
samples around the world. CTX-M β-lactamases continually evolve in their ability to
hydrolyze extended spectrum β-lactam antibiotics under selective pressure through
point mutations in and around their active site. Studying the effect that naturally
occurring mutations have on the structure, function and specificity of the CTX-M family
of β-lactamases will provide insight into the evolution of these enzymes.
The CTX-M family of β-lactamases is divided into five subfamilies based on
amino acid sequence homology. The P167S and D240G substitutions were identified
individually in the active site of CTX-M enzymes in several subfamilies upon the
introduction of ceftazidime, a third generation cephalosporin antibiotic, into the clinic.
These substitutions allow CTX-M β-lactamases to effectively hydrolyze ceftazidime but
also result in an overall loss in stability to the enzyme. Stability defects can be offset
through the acquisition of secondary mutations. The A77V mutation has been identified
in combination with the P167S and D240G substitutions in numerous clinical isolates.
The prevalence of the A77V mutation among clinical isolates emphasizes its probable
importance in the evolution of CTX-M β-lactamase enzymes. Here we characterize the
A77V mutation in the CTX-M-14 model enzyme using kinetic analysis, competition
assays and thermostability assays. We hypothesize that the A77V substitution acts as a
stabilizing mutation to offset the defects caused by both the P167S and D240G
substitutions, allowing the enzyme to retain stability while acquiring the ability to
hydrolyze ceftazidime.
Contributors: Patel, Meha; Fryszczyn, Bartlomiej; Palzkill, Timothy
Sasha McKai Pejerrey
Department of Molecular & Cellular Biology
Advisor: Suzanne Fuqua, Ph.D.-Department of Medicine
Tamoxifen (Tam) has been used for many years to treat women with estrogenreceptor alpha (ER()-positive breast cancer. However, 40% of women who receive Tam
eventually relapse with Tam-resistant tumors. Aromatase inhibitors (AIs) are another
type of hormone treatment used in patients, but resistance can also develop to these
drugs. The mechanisms behind endocrine therapy resistance remain largely undefined,
severely limiting treatment options.
We have discovered that a novel potential driver of endocrine therapy
resistance, called PRKRIR (protein-kinase, interferon-inducible double stranded RNA
dependent inhibitor, repressor of (P58 repressor)) was overexpressed in resistant
tumors by comparing Tam-sensitive primary human breast tumors to Tam-resistant
metastatic tumors. Preliminary data indicates that PRKRIR probably interacts with
PAK1 (p21 protein (Cdc42/Rac)-activated kinase 1), another protein known to be
involved in resistance. PRKRIR and PAK1 are both co-amplified and co-overexpressed
in breast tumors. Additionally, initial studies suggest that PRKRIR overexpression (OE)
in vitro promotes Tam resistant growth. I hypothesize that PRKRIR cooperates with
PAK1 to promote endocrine therapy resistance in ER-positive breast cancer.
Our overall objective is to define how PRKRIR OE promotes endocrine therapy
resistance in breast tumors, and to determine if that resistance in turn promotes
metastasis and altered cell signaling. Our goal is to restore hormone sensitivity in
resistant tumors. We predict that PRKRIR is a novel driver of endocrine therapy
resistance. By binding to and activating PAK1, we anticipate that PRKRIR confers
growth advantage to cells under endocrine therapy treatment, causing them to become
resistant. Elucidating the mechanisms by which cells evolve into resistant populations
will introduce a novel biomarker and uncover new clinical targets for personalized
Contributors: Gu, Guowei; Rechoum, Yassine; Beyer, Amanda R., Wang, Xiaosong; Fuqua,
Suzanne A. W.
Braden Kyle Pew
Department of Molecular & Cellular Biology
Advisor: Kjersti Aagaard, M.D./Ph.D.-Department of Obstetrics & Gynecology
Dennis Bier, M.D.-Department of Pediatrics
The mechanism by which prenatal glucocorticoid treatment promotes fetal lung
maturation remains poorly understood despite decades of clinical use. In an Erk3-/murine model of neonatal respiratory distress syndrome (RDS), we showed partial
abrogation of the pulmonary immaturity phenotype through antenatal dexamethasone
(dex) treatment. Using transcriptomic analysis, we identified key molecular pathways in
lung maturation and sought to determine the interaction of glucocorticoid treatment with
these genes.
Dexamethasone (0.4 mg/kg) or saline was administered on E16.5 and 17.5, and
lungs were harvested at E18.5. RNA-seq was performed and analyzed for differential
expression, then tested by Ingenuity Pathway Analysis (IPA) to determine gene/dex
interaction. CRH and SFTPB protein expression was determined by
immunohistochemistry of fetal lungs (n=5) examined by two reviewers. Statistical
analysis was performed using Student's t test.
Of 596 differentially expressed genes, IPA revealed 36 genes that interact with
dex, including several with roles in lung development, including corticotropin releasing
hormone (Crh) and surfactant protein B (Sftpb). Antenatal dex was associated with
significantly attenuated CRH levels at E18.5 in both wildtype (WT) and Erk3-/- lungs
(0.56-fold and 0.67-fold, p<0.001). As in humans, lungs of WT mice demonstrated
increased SFTPB production in response to dex (p=0.003). However, Erk3-/- mice
exhibited decreased pulmonary SFTPB when treated with dexamethasone (p=0.012).
Using transcriptomics in this neonatal RDS model, we identified molecular
pathways altered in response to glucocorticoid treatment. Our findings suggest an Erk3
independent and dependent mediation of CRH and SFTPB, respectively, in their roles
as modulators of fetal lung maturation. These discoveries broaden our understanding of
the underlying mechanism of treatment for neonatal pulmonary immaturity in preterm
Contributors: Pew, Braden; Harris, R. Alan; Sbrana, Elena; Cuevas Guaman, Milenka;
Klinger, Sonia; Shope, Cindy; Wang, Hui; Chen, Rui; Meloche, Sylvain; Aagaard, Kjersti
Victor George Piazza
Program in Cardiovascular Sciences
Advisor: Mary Dickinson, Ph.D.-Department of Molecular Physiology & Biophysics
Arteriovenous malformations that can arise during development affect an
estimated 300,000 Americans and can have severe impacts on the central nervous
system. However, much remains to be understood about how arteries and veins form,
including the signaling pathways involved and the consequences of disrupting these
pathways. It is known that specification of arterial and venous vessels from a primitive
capillary plexus is a critical event that begins prior to remodeling of that plexus into
mature arteries and veins. Using a mouse model of endothelial FoxO1 transcription
factor deletion (Tie2-Cre; FoxO1flox/flox), we have found embryonic lethality associated
with defective vessel remodeling. Additionally, a significant reduction was observed in
expression of genes associated with arterial fate specification at the pre-blood flow
stage (E8.25). Erk activity levels, which we have determined are temporally required for
arterial specification at this stage, are reduced in Tie2-Cre; FoxO1flox/flox embryos.
Additionally, decreased arterial marker expression was associated with a significant upregulation of the Sprouty2 (Spry2) gene, the product of which is a known Erk antagonist.
We have now determined that the Spry2 genomic locus is a target of direct FoxO1
binding in yolk sac endothelial cells. Collectively, this suggests FoxO1 transcriptionally
regulates Sprouty2 expression to mediate arterial specification in the mouse embryo.
We hypothesize that FoxO1 mediates Erk signaling in the pre-flow mouse
embryo by transcriptionally repressing Sprouty2, therefore modulating arterial
specification. We are testing this hypothesis as follows: 1) By determining whether
Sprouty2 over-expression in the pre-flow mouse embryo recapitulates the arterial
phenotype seen in the Tie2-Cre; FoxO1flox/flox FoxO1 mutant and Erk-inhibited
embryos, and 2) by determining whether Sprouty2 knockout or knockdown in the Tie2Cre; FoxO1flox/flox mutant is necessary and sufficient to rescue arterial marker
expression and vascular remodeling. Parallel experiments in vitro will support the causal
relationship between FoxO1 and Sprouty2 in the context of arterial specification.
Through this work we will elucidate a novel mechanism governing arterial specification
and subsequently embryonic vascular remodeling. The applicability of this knowledge
extends to new approaches to both promote vessel growth, as in tissue engineering of
vascular constructs, and inhibit neo-vessel formation that accompanies tumor growth.
Contributors: Piazza, Victor G; Garcia, Monica D; Wong, Leeyean; Udan, Ryan S; Dickinson
Mary E
Meagan Rochelle Pitcher
Program in Translational Biology & Molecular Medicine
Advisor: Jeffrey Neul, M.D./Ph.D.-Department of Pediatrics
Daniel Glaze, M.D.-Department of Pediatrics
Over thirty percent of Rett Syndrome (RTT) cases are due to nonsense
mutations in MECP2, where a change in nucleotide sequence leads to a premature stop
codon in the mRNA transcript. One strategy to overcome disease-causing stop
mutations is treatment with nonsense suppressing read-through compounds, such as
gentamicin, which reduce the stringency and fidelity of ribosomes translating mRNA
messages to allow expression of full length proteins from a mutated gene. To
determine if this strategy may be useful in RTT we characterized a new mouse model of
RTT that has a knock-in nonsense mutation (p.R255X) in the Mecp2 locus
(Mecp2tm1.1Irsf/J). Mecp2 is a four exon gene that encodes two functional domains:
the methyl binding domain from exons 3 and 4 and the transcription repression domain
in exon 4. Because the R255X mutation is located in the transcription repression
domain of Mecp2, it is possible that a dominant negative DNA binding truncation
product could be produced from the disease allele.
To determine if the truncated gene product acts as a dominant negative
allele, we genetically introduced an extra copy of MECP2 via a MECP2 transgene. This
allows us to determine whether adding a wild-type version of MeCP2 is sufficient to
rescue phenotypic abnormalities in Mecp2tm1.1Irsf/J mice, or whether the truncated
allele has a dominant negative effect insurmountable by a wild-type copy.
Mecp2tm1.1Irsf/J mice have phenotypes nearly identical to complete null animals:
decreased weight early in life, decreased heart rate late in life, abnormal breathing
phenotypes, poor motor coordination, and decreased survival time. The addition of the
MECP2 transgene to Mecp2tm1.1Irsf/J mice abolished the phenotypic abnormalities
and resulted in near complete rescue. This provides a proof of concept that this
mutation is amenable to read-through therapy. Future studies will include
pharmacokinetic and efficacy preclinical trials in the R255X model using read-through
compounds that are currently in clinical trials for peripheral indications. We hope to
demonstrate that read-through therapy is a viable treatment option for neurological
disease caused by nonsense mutations.
Contributors: Pitcher, Meagan R; Herrera, Jose A; Fisher, Amanda R; Schanen, N. Carolyn;
Neul, Jeffrey L
Amy E Pohodich
Department of Neuroscience/M.D.-Ph.D. Program
Advisor: Huda Zoghbi, M.D.-Department of Pediatrics
Methyl-CpG-Binding Protein 2 (MeCP2) was first described as a transcriptional
repressor that binds to methylated DNA and garnered much research interest after the
discovery that mutations in MECP2 cause Rett syndrome (RTT). RTT is a postnatal
neurodevelopmental disorder characterized by a period of seemingly normal
development followed by a progressive loss of acquired speech, cognitive abilities, and
motor coordination. Stereotyped hand movements replace purposeful hand use, and
seizures and autonomic dysfunction develop within a few years of disease onset. The
importance of having the correct level of MeCP2 in the brain is underscored by the fact
that duplications and triplications spanning MECP2 produce syndromic intellectual
disability, progressive motor dysfunction, and seizures. In RTT patients, the delayed
onset of disease coincides with the timing of peak stimulus-driven synaptic
reorganization required for appropriate circuit formation and neuronal maturation, which
led to the hypothesis that MeCP2 is important for establishing, refining, and/or
maintaining synapses. Consistent with this hypothesis are the numerous abnormalities
observed in MeCP2-deficient mice, including changes in basal transmission,
presynaptic function, and alterations in the balance between excitatory and inhibitory
signaling. Currently, however, the precise mechanisms by which MeCP2 can contribute
to neuronal plasticity are unclear. To gain insight into the role of MeCP2 during neuronal
activity in the mature brain, this project utilizes deep brain stimulation (DBS) to elicit
robust activation of the dentate gyrus (DG) in awake, freely moving mice. This paradigm
will enable assessment of the genome-wide transcriptional requirements for MeCP2
upon activation, and these data will be paired with analysis of changes in both MeCP2
localization and protein interactions to yield insight into MeCP2 function in neurons.
Contributors: Pohodich, Amy; Zoghbi, Huda
Erica Jean Polleys
Integrative Program in Molecular and Biomedical Sciences
Advisor: Alison Bertuch, M.D./Ph.D.-Department of Pediatrics
The Saccharomyces cerevisiae Iml1 complex inhibits TORC1 signaling and
SEACAT antagonizes the Iml1 complex. Whereas conditions in which the Iml1 complex
inhibits TORC1 have been described, those in which SEACAT functions remain largely
unknown. The SEACAT member Sea3 was linked to telomere maintenance and DNA
repair via physical and genetic interactions reported in genome-wide studies. Therefore,
we questioned whether Sea3 functioned through TORC1 to influence these pathways.
Deletion of SEA3 delayed emergence of telomerase independent survivors that utilize
break-induced replication (BIR) to maintain their telomeres. Similarly, sea3∆ mutants
exhibited a delay in colony formation in a BIR assay strain following double strand break
(DSB) induction as well as on the DNA damaging agent bleomycin. The delay in colony
formation was not due to a delay in the repair of the DSB or termination of the DNA
damage checkpoint, but due to tryptophan auxotrophy. High levels of tryptophan in
yeast peptone dextrose media did not rescue the delay in colony formation, suggesting
a defect in tryptophan import. Consistent with this, the tryptophan permease Tat2
prematurely declined in sea3∆ mutants compared to wildtype. Deletion of IML1 rescued
the delay in colony formation, consistent with Sea3 functioning as a regulator of TORC1
signaling. Together, these findings highlight the importance of enforcement of TORC1
signaling and internal tryptophan in the recovery of growth post DNA damage.
Contributors: Polleys, Erica; Bertuch, Alison
Geetali Pradhan
Program in Translational Biology & Molecular Medicine
Advisor: Yuxiang Sun, M.D./Ph.D.-Department of Pediatrics
Susan Samson, M.D.-Department of Medicine
Introduction: Obestatin, a 23 amino acid peptide derived from the ghrelin gene, is
expressed in various tissues including stomach and pancreas. Obestatin is known to
reduce food intake and body weight, improve memory and regulate sleep, but has no
effect on secretion of growth hormone and corticosterone. Obestatin is also known to
increase mass and survival of pancreatic β cells but it’s effect on insulin secretion
remains unclear.
Methods: We studied the effect of obestatin on insulin secretion under glucosestimulated condition both in vitro and ex vivo using rat insulinoma INS-1 cells and
mouse pancreatic islets. To determine whether the effect of obestatin on insulin
secretion is mediated through the ghrelin receptor, Growth Hormone Secretagogue
Receptor (GHS-R), we used islets from our GHS-R knockout mouse model (ex vivo).
We also transiently knocked down GHS-R in INS-1 cells and pharmacologically blocked
GHS-R using antagonists YIL 781 and JMV2959.
Results: Treatment of INS1 cells and pancreatic islets with different doses of
Obestatin, significantly increased glucose stimulated insulin secretion (GSIS) compared
to no obestatin treatment. However, static incubation of pancreatic islets from GHS-R
KO mice showed no response to obestatin treatment. Further, treatment of obestatin in
GHS-R knockdown INS-1 cells also showed similar effect on insulin secretion under
glucose-stimulated condition. Similarly, on blocking GHS-R in INS-1 cells
pharmacologically using antagonists also reduced obestatin’s effect on GSIS. Our
results indicate that obestatin has profound stimulator effect on insulin secretion in both
INS-1 cells and mouse pancreatic islets and this effect is potentially mediated via GHSR in β cells.
Conclusions: In conclusion, our studies indicate that obestatin is a potent insulin
secretagogue under glucose-stimulated condition. This effect of obestatin is likely
mediated via its receptor GHS-R in pancreatic islets. Obestatin’s stimulatory effect on
insulin secretion and promoting effect on β cell survival together make obestatin a
powerful therapeutic candidate for Type 2 diabetes.
Contributors: Pradhan, Geetali; Lee, Jong Han; Sun, Yuxiang
Muralidhar Hebbur Premkumar
Clinical Scientist Training Program
Advisor: Brendan Lee, M.D./Ph.D.-Department of Molecular & Human Genetics
Background: Nitric oxide (NO) is a mediator in the pathogenesis of necrotizing
enterocolitis (NEC). Argininosuccinate lyase (ASL) is the only enzyme in the body
capable of generating arginine, the substrate for NO. Previously, we have shown that
ASL is required for the assembly of the NO synthesis complex and, loss of ASL results
in decreased NO production. Enterocytes are the principal sites expressing ASL during
the neonatal period. Understanding the contribution of cell specific NO production to the
development of NEC has been limited by redundancies of the nitric oxide synthase
(NOS) isoforms and the temporal and spatial regulation of NO production. Hence, we
have generated an enterocyte-specific Asl CKO mouse, a novel, and better model to
study the role of NO in causation of NEC in a cell-specific manner.
Objective: We hypothesize that the deficiency of enterocyte-derived NO
secondary to loss of ASL contributes to the development of NEC.
Design/Methods: Utilizing Cre-Lox technology, we generated an enterocyte-specific
knockout of Asl (CKO). NEC was established in these mutant and control mice by
subjecting premature mouse pups to exclusive formula feed, hypoxia, and hypothermia.
The severity of NEC was graded based on histological changes. Characterization of the
model was performed by intestinal histomorphometry, immunostaining, nitrosylation
studies, cytokine expression and microarray analysis. In-vitro assays were performed
on IEC-6 cells.
Results: Significant loss of ASL in the enterocytes was demonstrated by RTPCR, western blot and immunostaining. The expression of eNOS, IL-6, and BAX were
significantly elevated in the CKO. The incidence of NEC was significantly higher
(p=0.003) in the CKO [29/46(63%)] as compared to controls [32/84 (38%)]. CKO mice
demonstrated elevated levels of IL-6, and neutrophilic infiltration. CKO also
demonstrated significantly increased apoptosis as assessed by TUNEL staining and
levels of pro-apoptotic proteins (BAX, AIF). IEC-6 cells with knockdown for Asl (shAsl)
showed decreased migration in response to LPS.
Conclusions: Loss of ASL in the enterocytes resulted in increased incidence of
NEC. This enterocyte-specific loss of ASL was associated with increased states of
inflammation and apoptosis. In-vitro studies suggested that loss of Asl in presence of
stress impairs the migration of enterocytes in light of stress. Thus, our studies suggest
that enterocyte-derived ASL is protective against the development of NEC. Hence,
manipulation of enterocyte ASL may be of translational value in prevention or treatment
of NEC. Contributors: Muralidhar H. Premkumar1, Brendan Lee2.
1Division of Neonatology, Baylor College of Medicine, Houston
2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston
Liying Qin
Department of Biochemistry & Molecular Biology
Advisor: Choel Kim, Ph.D.-Department of Pharmacology
Type Iα cGMP-dependent protein kinase (PKG Iα) regulates physiological
processes such as vasorelaxation and smooth muscle tone, thus plays a crucial role in
heart protection and blood pressure regulation. Because PKG Iα is a broad specificity
kinase, its interaction with specific substrates is essential for proper substrate
phosphorylation and signal transduction. As one of the functionally important substrates
of PKG Iα, RhoA is recognized by the N-terminal leucine zipper (LZ) of the kinase.
Phosphorylation of RhoA at serine188 by PKG Iα inhibits RhoA from activating Rho
kinase, resulting in smooth muscle relaxation. While it is well established that PKG Iα
can be activated by either cGMP or autophosphorylation, recent reports suggest that
residue cysteine42 (C42) may serve as a redox sensor where its oxidation and the
resultant interchain disulfide bond (C42-C42’) formation directly activates PKG Iα
independent of cGMP. However, the underlying mechanism of this redox sensing
remains unknown.
We hypothesize that the formation of C42-C42 disulfide bond does not directly
activate PKG Iα independent of cGMP, but indirectly by increasing the stability of the LZ
domain, resulting in a higher affinity for RhoA. Since most of oxidizing regents suffer
from partial oxidation and non-specific effects, we constructed two LZ mutants, C42S
and C42L, representing the reduced and oxidized states of the wild type LZ,
respectively. We then solved the crystal structures of both wild type and C42L LZ
domains. The structure of the C42L LZ is highly similar to that of the wild type
containing the C42-C42 disulfide with an RMSD of 0.49 angstrom. The C42L LZ also
has a lower temperature factor than wild type, indicating a higher thermal stability. To
further investigate the stability of the LZ domains, we are in the process of obtaining
their melting temperatures using circular dichroism. Ongoing studies include a pull-down
assay to test the interaction between wild type and mutant PKG Iα and RhoA and a
kinase assay to investigate the phosphorylation of RhoA by PKG Iα in vitro.
Contributors: Qin, Liying; Reger, Albert S.; Guo, Elaine; Yang, Matthew P.; Zwart, Peter; Kim,
Ann Pepper Quick
Program in Cardiovascular Sciences
Advisor: Xander Wehrens, M.D./Ph.D.-Department of Molecular Physiology & Biophysics
BACKGROUND. Heart failure is one of the leading causes of hospitalization, with
a median survival rate of only 5 years,1 yet there remains a lack of targeted therapeutic
options. Calcium dysregulation through disruption of calcium release proteins including
ryanodine receptor type 2 (RyR2) and junctophilin-2 (JPH2) has been implicated in
heart failure. SPEG, a novel prospective regulator of calcium release was found by coimmunoprecipitation (co-IP) from mouse heart lysate for RyR2 and JPH2 followed by
mass spectrometry. SPEG is a kinase necessary for cardiac development.2 However, it
remains unknown whether SPEG is essential for adult cardiac function. Therefore, we
obtained SPEG tamoxifen-inducible conditional knockout (cKO) mice in order to study
the physiological role of SPEG in the adult mouse heart.
HYPOTHESIS. SPEG maintains proper adult cardiac function by regulating RyR2
calcium release.
METHODS. Transthoracic echocardiography was performed on anesthetized
mice at baseline and 4 and 8 weeks post tamoxifen injection. Echocardiograms were
analyzed using Vevo 2100 to determine cardiac function. At 8 weeks post tamoxifen
injection, mice were euthanized and cardiomyocytes were isolated for either 1) calcium
imaging with fluo-4AM 2) T-tubule staining with di-8-anneps, or 3) co-localization studies
of JPH2 and RyR2 by immunofluorescence. Cells were imaged by confocal microscopy
and analyzed using Image J.
RESULTS. 8 weeks post tamoxifen injection, cKO mice developed heart failure
with an average ejection fraction of 27.1%+/-17.7% compared to 66.3% in the control.
SPEG cKO cells exhibited > 2-fold increased Ca spark frequency and deceased
transient amplitude. T-tubule power was decreased by 50% in SPEG cKO cells and
JPH2-RyR2 co-localization was decreased in SPEG cKO cardiomyocytes (R= 0.19)
CONCLUSIONS. SPEG is necessary for adult cardiac function in mice. Physiological
and cellular studies confirm that SPEG cKO mice develop heart failure associated with
calcium dysregulation, T-tubule disruption, and decreased JPH2-RyR2 co-localization.
Future studies prior to the onset of heart failure are needed to determine whether the
loss of SPEG is the cause or effect of disrupted calcium homeostasis.
1. Go AS et al. Circulation.129:e28-e292
2. Liu X et al. Circulation. 2009;119:261-268
Contributors: Quick, Ann P.1; Wang, Qiongling1; Reynolds, Julia O.1; Chiang, David Y.2;
Beavers, David L.2; Wehrens, Xander H.T.1.
Joel Patrick Quiros
Integrative Program in Molecular and Biomedical Sciences
Advisor: Olivier Lichtarge, M.D./Ph.D.-Department of Molecular & Human Genetics
The malaria parasite Plasmodium falciparum causes the most deadly form of
malaria in humans. According to the World Health Organization, an estimated 225
million cases of malaria infection were reported in 2009 with 781,000 resulting in death.
Before the discovery of artemisinin, widespread use of the antimalarial chloroquine has
lead to the emergence of drug resistant P. falciparum strains. In these resistant strains,
one up-regulated and important, yet functionally unknown gene is the exported antigen
1 (EXP1), a human antigen and vaccine candidate that is exported to the parasital
vacuole membrane and food vacuole. EXP1 is essential and may play an important role
in malaria and in its human immune response. Here, we functionally predict and
biochemically characterize EXP1 as a membrane glutathione S-transferase using a
computational network method. EXP1 efficiently degrades cytotoxic hematin, is potently
inhibited by artesunate, and is associated with artesunate metabolism and susceptibility
in drug-pressured malaria parasites. These data implicate EXP1 in the mode of action
of a frontline antimalarial drug.
Contributors: Quiros, Joel*; Lisewski, Andreas*; Ng, Caroline; Adikesavan, Anbu; Miura,
Kazutoyo; Putluri, Nagireddy; Eastman, Richard; Scanfeld, Daniel; Regenbogen, Sam;
Altenhofen, Lindsey; Llinás, Manuel; Sreekumar, Arun; Long, Carole; Fidock, David; Lichtarge,
Adithya Raghavan
Department of Molecular & Human Genetics
Advisor: William Craigen, M.D./Ph.D.-Department of Molecular & Human Genetics
Voltage-dependant anion channels (VDACs) are pore-forming ion-channel
proteins located in the mitochondrial outer membrane (MOM). We are interested in the
role of the mammalian VDAC isoform VDAC2 in apoptosis, since we have previously
shown that VDAC2 directly interacts with and anchors the pro-apoptotic protein BAK to
the MOM. Here, we report that in the absence of VDAC2, in addition to BAK being
reduced in mitochondria, BAK levels in the endoplasmic reticulum (ER) are significantly
increased. We further identified that upon prolonged treatment with ER stressors, there
is a significantly reduced rate of ER stress-induced apoptosis in Vdac2-/- MEFs. A
double knockout of Vdac2/Bak in MEFs restores ER-stress induced rate of cell death to
wildtype (WT) levels, demonstrating a dominant negative role for ER-localized BAK in
the process. In mice, while Vdac2-/- whole-body knockouts exhibit embryonic lethality,
heart-specific conditional knock-outs develop cardiac fibrosis and cardiomyopathy. A
double knockout of Bak and heart-specific Vdac2 rescues the aforementioned
phenotypes. In order to further identify the potential mechanisms, we performed a
microarray expression analysis between Wt and Vdac2-/- MEFs. We observed a
consistent downregulation in the expression levels of key players in the ER stress
response pathway in Vdac2-/- MEFs, confirmed by quantitative RT-PCR and
immunoblotting. A double knockout of Vdac2/Bak increases expression of ER stress
genes to levels comparable to Wt MEFs. Preliminary results from Vdac2-/- MEFs
indicate impaired function of ER-stress response protein IRE1alpha, a known interactor
of BAK in the ER. We propose that in the absence of VDAC2 there is loss of BAK in the
MOM, accumulation of BAK in the ER, interactions of ER-localized BAK with ER stress
response proteins, resulting in blunted transcription of ER stress response genes and a
reduced rate of ER-stress induced apoptosis.
Contributors: Raghavan, Adithya; Sheiko, Tatiana; Graham, Brett H.; Craigen, William C.
Vivek Rajasekharan
Department of Molecular & Cellular Biology
Advisor: Frederick Pereira, Ph.D.-Department of Molecular & Cellular Biology
During local invasion in metastasis, cancer cells form migratory structures like
filopodia, lamellipodia and invadopodia via actin remodeling, enabling them to produce
protrusive forces required to breach the basement membrane and/or invade
surrounding stroma. These forces are produced by actin networks through the growth of
individual filaments against the cell membrane. There are two types of actin networks,
bundled and branched. One of the migratory structures is a filopodium – a finger like
projection that protrudes in all directions beyond the cell's edge; filopodia are filled with
parallel rods of actin filaments bundled by cross-linking proteins. The specific role of
filopodia in relation to carcinoma is not understood. We developed a force assay to
measure the change in the axial membrane force that results from activity of actin
polymerization and depolymerization at the tip of a filopodium-like structure formed from
a cancer cell. We see force fluctuations, and we hypothesize that along with pulls
(inverse sawtooths) due to actin polymerization, pushes (sawtooths) due to actin
depolymerization, are also observed at the tip. We determine the force (F) and its
direction (θ) and extract the stationary states (dF/dt=0), their dwell-times, the change in
force between adjacent stationary states and the time it takes to go from one state to
another, for all the transient stationary states in the system. Analysis of this information
reveals characteristics of the actin biochemical network.
We form the structure from HN31, head and neck squamous cell carcinoma cells,
because they exhibit an H-Ras mutation that results in constitutively active RhoGTPases which activate the actin remodeling pathway. We will compare the
characteristics of the networks for untreated cells, with cells treated with drugs that
inhibit motor proteins like actin, microtubules and myosin, and by H-Ras knockdown as
a control, to assess the role of actin remodeling in force generation.
Contributors: Rajasekharan, Vivek; Sreenivasan, Varun; Yuan, Tao; Patel, Ameeta A; Myers,
Jeffrey N;
Pereira, Fred A; Farrell, Brenda;
Prasanna Ramachandran
Department of Molecular & Human Genetics/M.D.-Ph.D. Program
Advisor: Meng Wang, Ph.D.-Department of Molecular & Human Genetics
Neurodegenerative disorders are debilitating conditions characterized by the
progressive degeneration or death of neurons. Although degeneration is limited to cells
of the nervous system, manifestations of the disease often afflict distant peripheral
tissues, and non-neuronal pathology contributes significantly to the morbidity and
mortality of these disorders. The molecular mechanisms underlying these seemingly
disparate findings remain unknown. Our research focuses on the neuronal regulation of
peripheral metabolic processes, using the evolutionarily conserved Tubby protein as a
model. Mutations in the Tub gene, which is expressed exclusively in neurons, result not
only in retinal degeneration and neurosensory hearing loss, but also in morbid adult
onset peripheral obesity and insulin resistance. Loss of function of the Caenorhabditis
elegans homolog of Tub, tub-1, also results in analogous phenotypes of neurosensory
deficit and lipid accumulation. In preliminary studies, we identified that neuronal
deficiency of tub-1 gene leads to abnormal lysosomal biogenesis in the fat storage
tissue. Interestingly, lysosomal dysfunction has been linked to neurodegeneration and
metabolic morbidity. I hypothesize that tub-1 modulates lipid metabolism through a
neuroendocrine regulatory axis of lysosomal function. To elucidate this novel regulatory
axis, we will first identify the specific subset of neurons involved in modulating peripheral
lysosomal content, using single-neuron ablation as well as optogenetic activation. We
will also delineate the neuroendocrine signaling pathway that mediates the cell-nonautonomous effect on lysosomal function, through genetic screens, proteomic analysis
and transcriptome profiling. Finally, we will identify lysosomal metabolic changes that
are responsible for the neurodegenerative and lipid storage phenotypes in tub-1
mutants, using high- throughput metabolomic profiling of lysosomes.
Sam Julian Regenbogen
Department of Pharmacology
Advisor: Olivier Lichtarge, M.D./Ph.D.-Department of Molecular & Human Genetics
Discerning the relationship between protein structure and function is one of
the major goals of modern biology, and many methods have been devised to both
predict and observe this relationship. We are using two methods to investigate the
structure-function relationship of the E. coli enzyme orotidine-5’-monophosphate
decarboxylase (ODCase): a computational method to predict the impact of specific
mutations on protein function, and large-scale randomization to experimentally assay
the functional effects of mutations.
ODCase is an essential enzyme in the pyrimidine biosynthesis pathway and
is found in all domains of life; it catalyzes the conversion of orotidine-5’-monophosphate
(OMP) into uridine-5’-monophosphate (UMP). It is one of the most proficient enzymes
known, catalyzing the decarboxylation of OMP at a rate 1017-fold faster than the
uncatalyzed reaction. This has made ODCase an attractive target for structure-function
studies, and yet, despite catalytic residues and overall structure that are virtually
invariant among species, its mechanism is still not fully understood.
We have used a computational method of predicting the functional effects of
amino acid substitutions in ODCase and are currently undertaking an experimental
method of investigating the real outcomes of the same mutations. We are designing
mutagenic primers for each of the 245 codons in the pyrF gene (which encodes for E.
coli ODCase), which replace the three nucleotides of each codon with NNS – N
meaning A,T,G, or C; S meaning C or G. By performing site-directed mutagenesis with
each of these NNS primer pairs, we will produce, for each codon, a library containing all
possible permutations (all 20 amino acids have at least one possible codon represented
by NNS). These libraries will then be transformed into an E. coli strain lacking pyrF, and
selected on minimal media, which will only allow the growth of mutants that can
synthesize UMP. These selected libraries will then be pooled and submitted for deep
sequencing, allowing us to identify all single-residue substitutions that still maintain
ODCase function.
For each position in the ODCase protein sequence, we will be able to
determine the relative effect on ODCase function of all 20 possible amino acids using
their relative frequencies in the pooled sequence data. This information will provide a
strong test for the predictions made computationally, and will provide further insight to
the mechanistic requirements of this highly proficient enzyme.
Contributors: Atri, Benu; Katsonis, Panagiotis; Palzkill, Timothy; Lichtarge, Olivier
Alexander Renwick
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Chad Shaw, Ph.D.-Department of Molecular & Human Genetics
Although genome wide gene expression data has become common in biomedical
science, fully exploiting the multivariate character of the resource has remained a
challenge. The data are frequently used as a massively parallel screening tool to
identify individual gene candidates. When more systems level analyses are performed
on data they are often introduced sequentially, where, for example, a list of differentially
expressed genes is generated and then interrogated for pathway enrichment. These
approaches fail to exploit the dependence structure in gene expression that can help to
detect differential expression. Computational tools for more sophisticated analyses
have recently been developed. One of these is the Gaussian Lasso, which applies the
tools of sparse modeling to infer an undirected Bayesian Network. Such a network can
be decomposed into expression modules, each of which can be analyzed as an
ensemble. We have applied this method to the transcriptome response to influenza
vaccine in human subjects. A systems-level approach is especially appropriate for this
study because of the complexity of the process of immune response. We compare
result of the network-based analysis to that of a univariate, gene-by-gene analysis. Our
analysis verifies that interferon signaling system is important in the first day after
vaccination. Our results highlight the similarities and differences as well as the
additional power of the multivariate content analysis approach.
Contributors: Renwick, Alexander; Belmont, John; Shaw, Chad
Natalie Michelle Reyes
Integrative Program in Molecular and Biomedical Sciences
Advisor: Suzanne Fuqua, Ph.D.-Department of Medicine
Background: Although 75% of breast tumors are ER(-postive, there are no ERpositive preclinical models which reliably metastasize to study the effects of hormones
on invasion and metastasis. We have studied the role of estrogen in metastasis in an
ER(-positive MCF-7 sub-line (MCF-7 Met) which has spontaneously acquired the ability
to metastasize in vivo.
Experimental design and methods:
Microarray analysis was performed
comparing MCF-7 Met to parental, non-metastatic MCF-7 cells. The differential gene
expression was compared to genes differentially expressed when cells expressed
K303R ER alpha. We performed soft agar growth assays, mammosphere assays,
signal transduction assays and ERE luciferase assays. We injected MCF-7 Met and
MCF-7 cells into athymic nude mice to study in vivo growth properties.
Results: We found that the MCF-7 Met line shared 66% gene expression
similarity with cells expressing K303R ER alpha. We also found that upon hormone
treatment the MCF-7 Met cells had higher levels of pS118 and pS305 ER alpha, which
was observed in cells expressing K303R ER alpha. The K303R ER alpha model also
had an increased in anchorage independent growth under estrogen treatment and we
observed similar results in the MCF-7 met line. The MCF-7 Met line was able to form
more spheres during a mammosphere assay when compared to MCF-7. In preliminary
results we also observed estrogen hypersentivity in the MCF-7 Met line during ERE
luciferase assay, which is another phenotype that was observed in cells expressing the
K303R ER alpha mutation. 94% of the mice injected with the MCF-7 Met line
metastasized to distant sites regardless of hormonal treatment but their was no
difference in primary tumor growth when treated with Tam or estrogen withdrawal.
Conclusion: I have determined that many of the genes differentially expressed in
MCF-7 Met cells are related to estrogen signaling and tamoxifen resistance using
bioinformatic approaches. This suggests that the processes of invasion and metastasis
may be influenced by the hormonal mileau. I have also determined that the MCF-7 Met
line may share a similar phenotype to cells expressing the K303R ER alpha mutation.
Although MCF-7 Met cells remain hormone-dependent in vivo, they have acquired the
ability to metastasize, potentially suggesting that hormone resistance is not a
prerequisite for ER-positive breast cancer cells to metastazise to distant sites.
Contributors: Corona, Arnoldo; Covington, Kyle; Gu, Guowei
Christopher J Rhee
Clinical Scientist Training Program
Advisor: Kenneth Brady, M.D.-Department of Anesthesiology
Premature infants are at risk of vascular neurologic insults. Hypotension and
hypertension are considered injurious, but neither condition is defined with consensus.
Critical closing pressure (CrCP) is the arterial blood pressure (ABP) at which cerebral
blood flow ceases. CrCP may serve to define subject-specific low or high ABP.
To quantify CrCP as a function of gestational age (GA).
Premature infants (n=179) with GA from 23–31 weeks had recordings of ABP
and middle cerebral artery flow velocity twice daily for 3 days and then daily for the next
4 days during the first week of life. CrCP was calculated using an impedance-model
derivation with Doppler-based estimations of cerebrovascular resistance and
compliance. The association between GA and CrCP was determined in a multivariate
analysis adjusting for hour of life (HOL), use of vasopressors, 5-minute Apgar score,
and arterial carbon dioxide tension.
The median [interquartile range] CrCP for the cohort was 22 mm Hg [19-25].
CrCP increased significantly with GA (r=0.6; slope = 1.4 mm Hg/week gestation), an
association that persisted with multivariate analysis (p<0.0001). Higher GA was
associated with increased CrCP.
CrCP increases significantly at the end of the second and beginning of the third
trimester at a rate of 1.4 mm Hg per week of gestation. The low CrCP observed in very
premature infants may explain their ability to tolerate low ABP without global cerebral
infarct or hemorrhage
Contributors: Rhee, Christopher; Fraser III, Charles; Kibler, Kathleen; Easley, Ronald;
Czosnyka, Marek; Varsos, Georgios; Smielewski, Peter; Rusin, Craig; Brady, Ken; Kaiser,
Alex David Ridgeway
Department of Molecular & Cellular Biology
Advisor: Dolores Lamb, Ph.D.-Department of Urology
Non-obstructive azoospermia (NOA) is a severe form of male infertility that
results in a complete absence of sperm in the ejaculate. While the molecular
mechanisms underlying NOA remain incompletely understood, pathways crucial for
spermatogenesis are believed to play an important role. MSH5 is a meiosis-associated
gene implicated in the repair of double-stranded breaks and in the resolution of the
Holliday junction - both key events during the meiotic division of spermatogenesis.
MSH5 deficiencies in mice result in an infertile, NOA-like phenotype. Aberrant DNA
methylation is a potential epigenetic cause for deficient MSH5 expression in humans. As
such, we sought to identify and investigate the role of altered DNA methylation in men
with NOA.
We examined the DNA methylation profile of NOA men (n=31) and fertile
controls (n=20) using the Illumina HumanMethylation450 array and bisulfite sequencing.
A cohort of 6/31 NOA men were identified with significantly increased DNA methylation
at 5 specific CpG-sites within exon 2 of MSH5. This cohort also exhibited decreased
MSH5 mRNA and MSH5 protein levels. Removal of the aberrant hypermethylation, in
vitro, using the demethylating agent 5-aza-2-deoxycytidine (5-Aza) resulted in an
increase of MSH5 expression. Discontinuing 5-Aza treatment led to the reemergence of
aberrant MSH5 methylation, suggesting a potential driving cis element/trans factor. In
opposite effect, the targeted methylation of the identified CpG-sites using guide RNA’s,
resulted in a reduction of MSH5 expression in a control cell line. Reduced expression of
MSH5 also led to the loss of cellular proliferation and the ability to repair double
stranded DNA breaks in response to treatment with the radiomimetic drug,
Taken together, our data shows that the hypermethylation of CpG sites within
exon 2 of MSH5 deleteriously affects its expression. The presence of this
hypermethylation in NOA patients and the resulting loss of MSH5 expression may be a
significant contributor to their infertility.
Justin Michael Roberts
Department of Molecular & Cellular Biology
Advisor: Nancy Weigel, Ph.D.-Department of Molecular & Cellular Biology
Epidemiological data suggests vitamin D may play a role in prostate cancer
(PCa) prevention and data from our lab and others have demonstrated the active
metabolite 1,25-dihydroxyvitamin D3 (1,25D) inhibits the growth of PCa both in vivo and
in vitro. The main circulating form of vitamin D is 25-hydroxyvitamin D3 (25-OHD); it is
converted to 1,25D, by 1α-hydroxylase (CYP27B1) in the kidney and other tissues like
prostate. Normal prostate epithelial cells express CYP27B1 and are capable of
converting 25-OHD into active 1,25D allowing pro-differentiating and growth regulatory
functions to occur. Conversely, some PCa cell lines exhibit very low CYP27B1 activity
resulting in low intracellular 1,25D synthesis insufficient to observe growth inhibitory
effects in these cells. Preliminary data from our lab using RWPE-1 cells, an immortal
non-transformed prostate cell line, confirms CYP27B1 expression and growth inhibition
after 25-OHD treatment. In contrast, the VCaP PCa cell line expresses minimal
CYP27B1 and is not growth inhibited after treatment with 25-OHD suggesting CYP27B1
is necessary for 25-OHD mediated growth inhibition. Loss of CYP27B1 expression has
been suggested to be an early event in PCa progression that confers a growth
advantage by reducing the ability of prostate cells to respond to circulating 25-OHD.
This may also cooperate with the early formation of TMPRSS2:ETS fusions in PCa
development. The enzyme 24-hydroxylase (CYP24) tightly regulates 1,25D by
metabolizing it to a less active form. Our lab has shown ERG and 1,25D can cooperate
to hyper-induce CYP24 in VCaP cells. Comparatively, VCaP cells, which contain the
TMPRSS2:ERG fusion, express higher CYP24 levels than LNCaP PCa cells lacking this
fusion. Preliminary data shows a time dependent decrease in 1,25D mediated gene
induction in VCaP cells suggesting metabolism of 1,25D by CYP24. Clinical trials using
1,25D (calcitriol) have thus far been unsuccessful. Two reasons for this may be
inactivation by CYP24 and hypercalcemia at high doses of calcitriol. We are testing nonsecosteroidal VDR agonists, which should be resistant to metabolism by CYP24 and
have a larger safety range for hypercalcemia than calcitriol. Early data shows these
VDR agonists inhibit the growth of both LNCaP and VCaP PCa cells in vitro and are
resistant to metabolism by CYP24. Future studies will include assessing the ability of
these VDR agonists to inhibit tumor growth in vivo. The loss of CYP27B1 activity
coupled with increased CYP24 expression provides a model of early escape from
vitamin D regulation by developing PCa cells. We aim to gain insight into the roles of
CYP27B1 and CYP24 in cancer progression and how sensitivity to vitamin D
metabolites is altered through deregulation of these enzymes in PCa.
Contributors: Roberts, Justin; Kim, Jung-Sun; Weigel, Nancy
Amanda Rodriguez
Department of Molecular & Cellular Biology
Advisor: Stephanie Pangas, Ph.D.-Department of Pathology & Immunology
Sumoylation is a dynamic post-translational modification process in which a
SUMO (small-ubiquitin related modifier) moiety is conjugated to specific proteins
involved in transcriptional regulation, protein transport, chromosome segregation and
signal transduction via an enzymatic cascade and affects their protein subcellular
localization, interactions, stability and activity. Although research has suggested that the
SUMO pathway may play a crucial role during meiotic maturation in mice, the role of
sumoylation in the oocyte has not been well characterized. A central component of the
sumoylation cascade is the SUMO- conjugating enzyme UBC9, which is highly
conserved across species and is essential for several developmental processes such as
cell cycle regulation, chromosome segregation and cell viability. However, the
connection between UBC9 and its potential function during oocyte development remains
largely unknown. The overall goal of our project is to investigate the role of Ubc9 during
oocyte development and to identify novel SUMO-target proteins in oocytes. We
hypothesize that Ubc9 plays an essential role during oocyte development and loss of
Ubc9 leads to ovarian defects and infertility. As conventional knockout Ubc9 leads to
embryonic lethality, we generated conditional knockout mice with oocyte- specific
deletion of Ubc9 (OoUbc9 cKO) by using the growth differentiation factor 9 (Gdf-9) iCre
driver. Our preliminary fertility analysis revealed that targeted deletion of Ubc9 in
oocytes leads to sterility in female mice. To investigate how the loss of UBC9 causes
sterility, folliculogenesis, oocyte maturation and postnatal ovarian development will be
assessed. In addition, we will identify target proteins sumoylated by UBC9 in oocytes
and characterize their functions in fertility regulation. By identifying the causes of
infertility in OoUbc9 cKO mice we will gain insight to the role of sumoylation in oocyte
development and ovarian function.
Contributors: Rodriguez, Amanda; Tripurani Swamy; Pangas, Stephanie
Perla Janet Rodriguez
Integrative Program in Molecular and Biomedical Sciences
Advisor: Henry Pownall, Ph.D.-Department of Medicine
Serum opacity factor (SOF), a protein produced by S. pyogenes causes clouding
of human plasma. In vitro, an active truncated recombinant (r) SOF catalyzes the
disproportionation of plasma high density lipoproteins (HDL), its sole target, giving lipidfree apo A-I, a small neo HDL that is apo A-II-rich and cholesterol poor, and a
cholesteryl ester rich microemulsion (CERM). In vitro tests showed that the neo HDL
supports several reverse cholesterol transport (RCT) steps and has better
atheroprotective qualities than the HDL from which it was derived: neo HDL is better as
a substrate for lecithin:cholesterol acyltransferase (LCAT), as an acceptor of
macrophage cholesterol efflux, and as an anti-inflammatory lipoprotein. Mice injected a
low dose (4 g) rSOF had reduced plasma cholesterol ~50% in three hours. Size
exclusion chromatography reveals that the CERM is prominent in the plasma profile in
vivo. However, lipid-free apo A-I and neo HDL in vivo occur at <1% of their in vitro
levels. The in vivo fate of neo HDL is not yet known. Our current hypothesis is that neo
HDL fuses to HDL and is subsequently taken up by the HDL receptor SR-BI.
To determine the catabolism of neo HDL, its nontransferable cholesteryl ester
(CE) was labeled with tritium. Neo HDL-CE [3H] was incubated with human plasma and
each isolated lipoprotein, with or without lipoprotein deficient serum (LPDS) which
contains lipoprotein modifying enzymes cholesteryl ester transfer protein (CETP),
phospholipid transfer protein (PLTP), and LCAT. Over time, CE transferred to all
lipoproteins in the presence of LPDS. Neo HDL-CE transferred to HDL in the absence
of LPDS but not to the other lipoproteins. In mouse plasma, the CE transferred to HDL
at a much faster rate. These results suggest a potential fusion between neo HDL and
HDL. Torcetrapib, a CETP inhibitor, was added to similar experiments which resulted in
partial inhibition of neo HDL-CE transfer to all lipoproteins suggesting its involvement in
neo HDL catabolism.
Neo HDL-CE [3H] uptake studies were performed using three CHO cell lines, two
of which express low endogenous levels of SR-BI and one overexpressing murine SRBI. CE uptake from neo HDL only occurred when SR-BI was over expressed. CE
uptake experiments will be repeated in human hepatocytes using radiolabeled forms of
Neo HDL.
Together these results suggest that neo HDL may fuse to HDL and that neo HDL
may potentially interact with the SR-BI receptor. To further investigate the role of
plasma factors in neo HDL catabolism inhibitors targeting LCAT and PLTP will be used
similarly. Future studies include injecting mice with radiolabeled neo HDL to effectively
track its destination in vivo.
Contributors: Rodriguez, Perla; Gillard, Baiba; Rosales, Corina; Pownall, Henry
Soung Hun Roh
Department of Biochemistry & Molecular Biology
Advisor: Wah Chiu, Ph.D.-Department of Biochemistry & Molecular Biology
David Tweardy, M.D.-Department of Medicine
Acute Myeloid Leukemia (AML) is the most severe type with 40% frequency in
total leukemia and characterized by non-random chromosomal translocation giving rise
to oncogenic fusion genes. One of the most frequent oncogenic fusion protein is AML1ETO (12%) resulting from the translocation at (8;21)(q22;q22). AML1-ETO is hard to be
managed by conventional drug development strategies since it does not mediate its
function by ligand-binding or enzyme activity, where are eligible drug-targeting sites by
small molecules. In order to control this undruggable oncoprotein, a novel approach of
altering the oncoprotein proteostasis network through type II eukaryotic chaperonin,
TCP-1 Ring Complex (TRiC/CCT), has been suggested. Our study focuses on the
functional relationship between TRiC and AML1-ETO and exploring the discrete
interaction mechanism between oncoproteins and chaperonin. In this study, Co-IP
results showed AML1-ETO is a TRiC client and its folding intermediate directly
associates to TRiC through the DNA binding domain (AML_1~175). Chemical crosslinking and cryo-EM study also revealed that multiple TRiC subunits (CCT4, 6) bind
cooperatively to AML1-ETO. More interestingly, total expression level of AML1-ETO is
dramatically decreased in vitro translational system when endogenous TRiC was
immune-depleted, which suggests not only TRiC is required for AML1-ETO’s biogenesis
but also translational AML1-ETO-TRiC interaction is a potential drug target to control
Acute Myeloid Leukemia.
Contributors: Soung-Hun, Roh; Tweardy, David J; Chiu, Wah
Ramon Roman-Sanchez
Integrative Program in Molecular and Biomedical Sciences
Advisor: John Wilson, Ph.D.-Department of Biochemistry & Molecular Biology
Retinitis pigmentosa (RP) is an inherited degenerative disease of the retina that
results in rod and cone photoreceptor death. Initially as rods degenerate, subjects lose
peripheral and night vision; this is followed by cone photoreceptor degeneration leaving
the patients completely blind in some instances. RP affects 1 in every 4000 individuals
worldwide and around 30-40% of these are the result of autosomal-dominant (ADRP)
mutations. Rhodopsin (Rho) is a G protein-coupled receptor of the rods and it initiates
the phototransduction cascade upon photon capture. Mutations in Rho, which account
for about 25% of all ADRP cases, affect many processes, including Rho posttranslational modifications, transport, folding and signaling capabilities.
More than 150 mutations that result in RP have been identified within Rho.
Seven of these are nonsense mutations of which five cause dominant RP (dRP) and the
two cause recessive RP (rRP). One possible explanation for this difference is that rRP
mutants undergo nonsense-mediated mRNA decay (NMD) whereas dRP do not,
thereby producing a toxic Rho truncation. The NMD pathway is the cell’s quality control
mechanism for the detection and degradation of aberrant mRNA transcripts. If an
mRNA molecule contains a premature termination codon (PTC), defined as a stop
codon ~50nt upstream of an exon-exon junction, then degradation will ensue by NMD.
Our lab has found that the transcript of Rho-Q64X, which causes dRP in
humans, is present a similar levels to wild type Rho in a mouse model. The same
observation was made in a mouse model with a duplicated exon 2 that results in a PTC
at the exon-exon junction. One study in cells detected degradation by NMD of the Rho
Q249X nonsense mutant that causes rRP, but in vivo studies are absent. These
observations raise the question of whether Rho is insensitive to NMD in photoreceptors.
I am studying Rho nonsense transcripts and NMD in the context of the retina so as to
have a better understanding of RP, its mechanisms and aid in the development of gene
therapy strategies based on suppression and replacement of the mutant Rho.
Contributors: Roman-Sanchez, Ramon; Wilson, John H
Amanda Nicole Rosewell Shaw
Department of Molecular & Human Genetics
Advisor: Philip Ng, Ph.D.-Department of Molecular & Human Genetics
Viral-based Cystic Fibrosis (CF) gene therapy has not, to date, been successful
because the receptors for the most widely used vectors are not accessible via the apical
surface of the airway epithelium. Indeed, the major receptor for adenovirus serotype 5
(Ad5), CAR (coxackie-adenovirus receptor), is sequestered to the basolateral surface
by tight junctions. Ad5 can efficiently transduce the airway epithelium only if the tight
junctions are transiently disrupted by agents such as LPC or EGTA. We have found that
administration of these agents into nonhuman primate lungs cause temporary but
significant increases in respiratory resistance and decreases in compliance
measurements. Thus, opening tight junctions may be unsuitable for CF gene therapy
considering the compromised pulmonary status of CF patients with advanced disease.
Consequently, identification of vectors that transduce via an apical surface receptor is
needed to obviate the need for opening tight junctions. Recently, it was reported that
adenovirus serotype 3 (Ad3) could mediate apical infection of various polarized
epithelial cells using Desmoglein-2 as its receptor. This observation suggested that Ad3
might be useful for apical infection of airway epithelial cells for CF gene therapy. To
evaluate this potential, we compared the transduction efficiency of Ad5 versus Ad3 in
polarized human bronchiolar epithelium (HBE). We found that Ad3 transduces polarized
HBE at least 10-fold more efficiently than Ad5. Indeed, transduction of polarized HBE by
Ad3 alone was comparable to transduction by Ad5 only if the Ad5 infection was
preceded by opening tight junctions with EGTA. These results suggest that vectors
based on Ad3 may be useful for CF gene therapy because highly efficient transduction
of the airway epithelium may be achieved without the need to open tight junctions. We
have constructed a novel Helper-Dependent Ad3 vector by creating a replication
incompetent Ad3 helper virus.
Contributors: Rosewell Shaw, Amanda; Palmer, Donna; Ing, Jordan; Grove, Nathan; Ng, Philip
Matthew Clayton Ross
Department of Molecular Virology & Microbiology
Advisor: Joseph Petrosino, Ph.D.-Department of Molecular Virology & Microbiology
Comprising an estimated 1031 total particles, viruses are the most diverse and
numerous biological entities on Earth. However, estimates of the number of viruses
inhabiting the human body, the human virome, are shots in the dark at best. But we can
be certain we have explored little of the virome by sequence, and even less by function.
Fortunately, the field of viral metagenomics has evolved rapidly over the past few years
owing mainly to breakthroughs in next generation sequencing. We now have the ability
to probe viral communities from virtually any source, without the need for culture.
Due to high interindividual variation, studies of the human microbiome,
including the virome, require large numbers of samples to accomplish significant
statistical power. Large-scale projects often require special considerations from a
sample-processing standpoint. For example, concentration of large volumes of primary
sample is not feasible for projects containing tens-of-thousands of samples, and often,
specimens collected in the clinic as part of routine visits are very low biomass.
Additionally, high-level multiplexing is a requirement to keep large projects within
reasonable timelines and budgets.
Our aim was to develop a high-throughput method to probe the human virome
suitable for projects with thousands or tens-of-thousands of clinical samples, that is
highly-multiplexable, without sacrificing significant sensitivity compared to whole
genome shotgun (WGS) sequencing. Here we report a viral metagenomics sample
preparation method that is highly sensitive, highly scalable and delivers significant cost
cutting at the stage of sequencing library preparation. Through a combination of semirandom primer amplification and molecular barcoding, we are able to pool tens to
hundreds of samples prior to sequencing, allowing for a single library to be created for a
pool of samples instead of one library per sample. We demonstrate sensitivity through
the use of a contrived mock viral community, and show that we do not introduce
significant bias through pooling. Ultimately, this technique affords the ability to
interrogate the human virome in a manner highly conducive to large scale microbiome
Contributors: Ross, Matthew; Wong, Matthew; Holder, Michael; Metcalf, Ginger; Gibbs, Richard;
Petrosino, Joseph
Michelle Jenny Rubin
Integrative Program in Molecular and Biomedical Sciences
Advisor: Gad Shaulsky, Ph.D.-Department of Molecular & Human Genetics
Dictyostelium discoideum is a eukaryotic amoeba that consumes bacteria during
growth. Upon starvation, Dictyostelium cells cooperate with one another while
developing into multicellular structures in which some of the cells sacrifice themselves
while supporting the sporulation of the others. Currently, sociality is defined as
intercellular interactions during Dictyostelium development, but we propose that social
interactions occur during vegetative growth as well. We have generated many
Dictyostelium mutants that do not grow on Gram-negative bacteria and we tested the
cell-autonomous properties of the phenotype. Interestingly, when we mixed just 10%
wild type Dictyostelium cells with one of the mutants, we observed that the mutant grew
on Gram-negative bacteria. Additionally, when we plated the mutant at different cell
densities on Gram-negative bacteria, the cells were able to grow at high cell densities.
Additionally, we found that Dictyostelium cells secret a soluble factor, most likely a
polypeptide that kills bacteria. Therefore, we hypothesize that Dictyostelium growth on
bacteria is a social process that is mediated by cooperative predation. We will
determine whether social interactions take place during growth on Gram-negative
bacteria and we will use biochemical and genetic tools to determine the identity and the
roles of the secreted factors in the process.
Contributors: Rubin, Michelle; Shaulsky, Gad
Ingrid Sophie Runquist
Department of Molecular & Cellular Biology
Advisor: Michael Lewis, Ph.D.-Department of Molecular & Cellular Biology
The Terminal End Bud (TEB) at the growing tip of mammary ducts is one of the
fastest growing structures in mammals. TEBs drive ductal elongation during puberty and
regress once development of the ductal tree is complete. Successful modeling of this
structure may yield insight into breast development as well as cancer progression.
Previous mathematical models have focused on branching morphogenesis, but there
are currently no models that address ductal elongation itself. Our model exploits the
constrained geometry of the TEB in vivo which provided the framework for an initial
mathematical model. Parameters in this model were then informed with measureable
data (morphological characteristics, proliferation rate, cell cycle duration, and
apoptosis). These data were then used to calculate a value representing the movement
of cells from the TEB into the mature duct (termed the flux) and this calculated flux was
then used to predict a linear elongation rate. The prediction was compared to an
experimentally measured TEB displacement rate and then underwent rounds of revision
and validation. Our initial measurements of proliferation, apoptosis, and cell sizes,
predicted a linear displacement rate of 1.39 mm per day, which was faster than our
experimentally measured displacement rate of 0.54mm per day. We then refined our
model by incorporating changes in the direction of growth due to bifurcation, a cost
function for bifurcation (which describes duplication of the TEB), as well as an additional
flux term to account for a migration of cap cells into the body cell layer. Iteration of the
revised mathematical model yielded an estimate significantly closer to the measured
displacement rate, thus indicating that the most relevant biological parameters have
been accounted for. In addition, our data overturned a long held belief that cap cells
contribute to the body cell lineage. We are now poised for in silico experiments that may
yield predictions consistent with cancer phenotypes, as well as predictions that
recapitulate known mutation phenotypes.
Contributors: Chauviere, Arnaud; Landua, John; Sreekumar,Amulya; Cristini, Vittorio; Rosen,
Jeffrey; Lewis, Michael
Eric Benjamin Rutledge
Program in Structural and Computational Biology and Molecular Biophysics/M.D.-Ph.D.
Advisor: Zhandong Liu, Ph.D.-Department of Pediatrics
As next-generation sequencing (NGS) technology continues to improve, our
ability to explore new parts of the genome increases. In order to study complex,
genomic elements, new bioinformatic approaches must be developed to accurately and
reliably analyze fast-growing NGS databases.
Here we focus on profiling small, non-coding RNA elements microRNA and shorthairpin RNA (shRNA) from high-throughput RNA sequencing. Both microRNA and
shRNA are short, non-coding RNAs that mediate translational suppression of target
mRNAs through imperfect sequence homology. Profiling microRNA expression levels is
of widespread interest because of their pivotal roles in all biological processes.
Synthetic microRNAs can be introduced via shRNA infection and play a pivotal role in
developing drugs and understanding pathological disease pathways from RNA
interference (RNAi) screens. The biology of microRNAs and shRNAs is challenging to
expression profiling with NGS microRNA-sequencing because it requires experimental
modifications that must be accounted for in downstream data analysis.
We hypothesize that minimizing contamination from adapter sequences will
enhance the mappabiltiy of short reads generated from high-throughput microRNA
sequencing and improve accuracy in measuring changes in miRNA expression levels.
Developing a pipeline to reliably can increase the accuracy of microRNA expression
profiling and accurately measure miRNA expression level changes from microRNA-seq
data. We have identified sources of variability by comparing the performance of
algorithms with different approaches to adapter sequence removal, mapping, and
annotation using a control set with known levels of spiked-on microRNA. We intend to
standardize a pipeline and demonstrate applicability to biologically complex conditions
by applying it to TCGA Ovarian cancer datasets and evaluating correlation with the wellestablished Agilent microarray expression profiling.
Furthermore, we intend to improve short read data analysis by developing a
pipeline to increase accuracy in identifying targets in RNAi screens using local
alignment mapping algorithms. Previously, the intractable computational time required
for local alignment hindered its use short read data analysis. We have utilized the
accuracy and precision of the Smith-Waterman algorithm to develop a robust pipeline in
for shRNA expression profiling.
Contributors: Rutledge, Eric
Jasdeep Singh Sabharwal
Department of Neuroscience/M.D.-Ph.D. Program
Advisor: Samuel Wu, Ph.D.-Department of Ophthalmology
The retina must encode visual activity in dim starlight as well as bright sunlight. In
order to accomplish this feat the retinal circuitry is organized into two parallel pathways
originating from rods or cones for dim or bright light conditions, respectively. Each of
these pathways has unique properties that are passed onto the retinal ganglion cells
(RGC), the output cells of the retina. By using a multielectrode array (MEA) to record
from many RGCs and probing with stimuli at different light intensities we find that RGCs
slow their temporal tuning and widen their spatial tuning in dim light. These changes
allow the RGC to encode light over longer time and wider space which is critical in low
light settings because fewer photons are present. Identifying how RGC modify their
properties will shed light on normal visual encoding and also improve our understanding
of diseases that damage RGCs and cause deficit in dim light settings, like glaucoma.
Contributors: Sabharwal, Jasdeep; Cowan, Cameron; Wu, Sam
Zeenat Safdar
Clinical Scientist Training Program
Advisor: Mark Entman, M.D.-Department of Medicine
Objectives. The goal of this study was to investigate the relationship between
peripherally measurable collagen metabolism biomarkers and health related quality of
life (HRQoL) in PAH patients.
Methods. We prospectively enrolled 68 stable idiopathic, anorexigen-associated,
and hereditary PAH subjects and 37 healthy controls with similar distributions of age
and gender. Serum samples were analyzed for N-terminal propeptide of type III
procollagen (PIIINP), c-terminal telopeptide of collagen type I (CITP), matrix
metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1). The
Minnesota Living with Heart Failure (MLWHF), EQ-5D-3L (EQ-5D) and Short Form (SF36) general health survey were administered at the time of blood draw. Univariate and
multivariable linear regression models were used to assess associations between
Results. Mean age of PAH patients and controls were similar (45±15 and 49±14
years respectively, mean±SD, p=0.18). The six-minute walk distance, Borg dyspnea
score, WHO functional class, presence of edema, brain naturiectic peptide were all
significantly different between controls and PAH patients (p<0.01 for all). CITP, PIIINP,
MMP9, and TIMP1 levels, and all HRQoL domains were significantly different between
controls and PAH patients (p<0.001 for each). Several of the CAMPHOR scores
strongly linearly associated with PIIINP. PIIINP levels were significantly associated with
MLWHF physical (coef=1.63, and p=0.02), SF-36 physical (coef=-2.62, p=0.007), and
EQ-5D aggregate (coef=0.21, p=0.03) scores also correlated well with PIIINP. An ROC
curve analysis was conducted for six-minute walk test distance with a threshold of 330
meters, using PIIINP as the predictor variable. Our result show that for each unit
increase in PIIINP the odds of a patient obtaining a distance of ≥330 decreases by 38%
(OR=0.62; 95% CI=0.43, 0.90; p=0.011). The area under the curve was 0.8142 and the
PIIINP cutoff that gave the highest sensitivity and specificity was 4.63 ug/ml.
Conclusions. Of all biomarkers we tested, PIIINP is the best predictor of disease
severity, and is strongly related to HRQoL scores in PAH patients. These relationships
suggest PIIINP as a promising tool for PAH clinicians to determine or confirm the level
of disease severity.
Contributors: Safdar, Zeenat; Tamez Emilio; Frost, Adaani; Guffey, Danielle; Minard, Charles;
Entman Mark
Jason Saliba
Department of Molecular & Human Genetics
Advisor: Sharon Plon, M.D./Ph.D.-Department of Pediatrics
We use whole exome sequencing, bioinformatics analysis, and functional assays to
discover and assess potential cancer susceptibility mutations in families segregating risk for
pediatric lymphocytic leukemia and lymphoma. In one kindred with four individuals diagnosed
during childhood with lymphocytic leukemia or lymphoma transmitted in an autosomal dominant
pattern of inheritance, exome sequencing on the constitutional DNA of three of the affected
individuals and an intra-familial control was performed. Exclusion of common variants seen in
population databases and variants in the control, along with systematic bioinformatics, narrowed
our list to eight heterozygous missense variants shared among all three patients predicted to be
functionally important. Within this list, the L254P variant occurs at a highly conserved residue of
Human Cytosolic 5’ Nucleotidase 1A (NT5C1A), which is involved in the adenosine metabolic
pathway. NT5C1A’s primary function is to catalyze the production of adenosine by the
dephosphorylation of AMP. In addition, NT5C1A can dephosphorylate chemotherapeutic
nucleoside analogs used in the treatment of leukemia. Prior studies have shown overexpression
of wild type NT5C1A in HEK293 cells improved cell survival after treatment with purine and
pyrimidine analogs. To test the functional effect of the L254P variant, HEK293 cell lines that
stably overexpress wild type or L254P NT5C1A were created. These cells underwent
cytotoxicity assays after treatment with various nucleoside analogs. We demonstrated that cells
overexpressing wildtype, but not L254P mutant NT5C1A are at least ten fold more resistant to
nucleoside analog chemotherapy agents indicating L254P interferes with enzymatic activity
without evidence for change in substrate specificity. In our search for a cancer susceptibility
gene, we found a gene with potentially impactful pharmacogenetic effects. Recently, another 5’
nucleotidase, NT5C2, was shown to contain somatic mutations that activated the protein and
ultimately resulted in tumor resistance to nucleoside analogs in ALL relapse patients. Oncomine
contains data showing NT5C1A is overexpressed in a variety of tumor types. According to the
NHLBI exome variant server, very few common mutations of NT5C1A occur within or near the
protein’s three functional domains. We hypothesized that other variants seen within NT5C1A
could lead to altered drug metabolism. We created multiple stable cell lines expressing 8
different NT5C1A variants reported in the NHLBI exome variant server by using Gateway®
technology and a lenitviral vector delivery system. These overexpression cell lines are being
assessed by cytotoxicity assays for their response to nucleoside analogs. This is the first
systematic analysis of naturally occurring variants of any 5’ nucleotidase with regard to their
ability to process chemotherapy drugs. NT5C1A could be another 5’ nucleotidase whose
expression and sequence should be assessed in tumors in order to determine impact on
chemotherapeutic drug responses.
Contributors: Saliba, Jason; Zabriskie, Ryan; Powell, Bradford; Hicks, Stephanie; Kimmel,
Marek; Cheung, Hannah; Ritter, Deborah; Muzny, Donna M; Reid, Jeffrey G; Wheeler, David A;
Gibbs, Richard A; Plon, Sharon E
Jason Scott Salvo
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Rui Chen, Ph.D.-Department of Molecular & Human Genetics
Familial exudative vitreoretinopathy (FEVR) is a developmental disease that can
cause visual impairment and retinal detachment at a young age. Four genes involved in
the Wnt signaling pathway were previously linked to this disease: NDP, FDZ4, LRP5,
and TSPAN12. Identification of novel disease causing alleles, allows for a deeper
understanding of the disease, better molecular diagnosis, and improved treatment.
Sequencing libraries from 92 FEVR patients were generated using a custom capture
panel to enrich for 163 known retinal disease causing genes in humans. Samples were
processed using next generation sequencing (NGS) techniques followed by data
analysis to identify and classify single nucleotide variants and small insertions and
deletions. Sanger validation and segregation testing were used to verify suspected
variants. Of the cohort of 92, 45 patients were potentially solved (48.9%). Solved cases
resulted from the determination of 49 unique mutations, 41 of which are novel. 13 of the
novel variants discovered were highly likely to cause FEVR due to the nature of these
variants (frameshifting indels, splicing mutations, and nonsense variants types). This is
the largest study of a FEVR cohort utilizing NGS that we are aware of. We were able to
determine probable disease causing variants in a large number of FEVR patients, the
majority of which were novel. Knowledge of these variants will help to further
characterize and diagnose FEVR.
Contributors: Salvo, Jason; Xu, Mingchu; Lyubasyuk, Vera; Wang, Hui; Wang, Feng; Nguyen,
Duy; Wang, Keqing; Luo, Hongrong; Wen, Cindy; Shi, Catherine; Lin, Dannin; Zhang, Kang;
Chen, Rui
Rebeca San Martin
Department of Molecular & Cellular Biology
Advisor: David Rowley, Ph.D.-Department of Molecular & Cellular Biology
Reactive stroma co-evolves with cancer, exhibiting tumor-promoting properties. It
is also evident at sites of wound repair and fibrosis, playing a key role in tissue
homeostasis. The specific cell types of origin and the spatial/temporal patterns of
reactive stroma initiation are poorly understood. In this study, we evaluated human
tumor tissue arrays by using multiple labeled, quantitative, spectral deconvolution
microscopy. We report here a novel CD34/vimentin dual-positive reactive fibroblast that
is observed in the cancer microenvironment of human breast, colon, lung, pancreas,
thyroid, prostate, and astrocytoma. Recruitment of these cells occurred in xenograft
tumors and Matrigel plugs in vivo and was also observed in stromal nodules associated
with human benign prostatic hyperplasia. Because spatial and temporal data suggested
the microvasculature as a common site of origin for these cells, we analyzed
microvasculature fragments in organ culture. Interestingly, fibroblasts with identical
phenotypic properties and markers expanded radially from microvasculature
explants. We propose the concept of reactive microvasculature for the evolution of
reactive stroma at sites of epithelial disruption common in both benign and malignant
disorders. Data suggest that the reactive stroma response is conserved among tissues,
in normal repair, and in different human cancers. A more clear understanding of the
nature and origin of reactive stroma is needed to identify novel therapeutic targets in
cancer and fibrosis.
(Am J Pathol 2014, 184: 1860e1870)
Contributors: Rebeca San Martin, David A. Barron,* Jennifer A. Tuxhorn, Steven J. Ressler,
Simon W. Hayward, Xiaoyun Shen,Rodolfo Laucirica, Thomas M. Wheeler, Carolina Gutierrez,
Gustavo E. Ayala, Michael Ittmann, and David R. Rowley
Balaji Santhanam
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Gad Shaulsky, Ph.D.-Department of Molecular & Human Genetics
Multicellular development is achieved by the coordinated action of many
transcriptional regulators and their associated regulatory modules. The master regulator
GtaC, a ‘GATA’-family transcription factor is essential for morphological progression in
the social amoeba Dictyostelium discoideum. Despite its obvious importance, little is
known about the network modules regulated by GtaC. We characterized its function
during development using genetic perturbation experiments and quantitatively
measured the physiological states of these different strains through transcriptional
profiling. Our results indicate that the strain carrying a mutated DNA-binding GtaC
phenocopies gtaC-null, both morphologically and transcriptionally. We assayed for the
developmental genomic occupancy of both the wild-type and mutant forms of GtaC and
our results indicate that the mutant form has decreased propensity to bind DNA.
Further, our results allude to positional but not temporal concurrence of their binding
events. Finally, we adopted an integrative approach to combine both binding and
transcriptome data to uncover the developmental targetome of this master transcription
factor. Our findings shed novel insight in to GtaC-controlled components underlying the
gene regulatory network orchestrating Dictyostelium development.
Contributors: Katoh-Kurasawa, Mariko; Cai, Huaqing; Devreotes, Peter N; Kuspa, Adam;
Shaulsky, Gad
Laura Louise Satterfield
Integrative Program in Molecular and Biomedical Sciences
Advisor: Jason Yustein, M.D./Ph.D.-Department of Pediatrics
Osteosarcoma (OS) and Ewing’s sarcoma (EWS) are the most common bone tumors in
the pediatric population. While significant progress has been made with towards increasing
patient outcomes, our success in eradicating these two malignances is significantly less than
most other pediatric malignancies. One reason for continued treatment failure is due to high risk
disease states such as the presence of metastatic lesions. Metastasis is of the utmost clinical
relevance as it accounts for more than 90% of solid tumor deaths. Improvements in treatment
regimens have been stagnant over the past 20-30 years most likely due to our lack of
understanding their molecular pathogenesis. Overall survival rates are approximately 65-70%
for localized disease and less than 20% for aggressive, disseminated states. Thus, the clinical
need to understand high risk disease, such as metastasis is significant in order to develop novel
miRNAs are highly conserved short 18-22 single stranded non-coding RNAs that have
the ability to repress target translation and affect key cell signaling pathways often deregulated
in cancer. Recently, miRNAs have been implicated in numerous cancers; however the
functional role of miRNAs in sarcomas is limited. In order to identify novel alterations in miRNAs,
we have utilized our analysis of microRNA expression from localized and metastatic tumors
derived from novel genetically engineered mouse models of osteosarcoma. Among the miRNAs
most significantly dysregulated, miR-130b was shown to be significantly upregulated in the
metastatic lesions. Furthermore, it has been shown that miR-130b is overexpressed in OS and
EWS patient samples, and this event is significantly associated with poor patient outcome in the
latter sarcoma. Furthermore, we have verified miR-130b overexpression in both mouse and
human sarcoma cell lines and in metastatic EWS tumor samples obtained from Texas
Children’s Hospital. Functional analysis shows overexpression of miR-130b increases migration
and invasion in vitro
In order to identify novel targets of miR-130b contributing to metastasis, we performed
gene expression analysis. We identified Cdc42GAP as a novel target of miR-130b. Cdc42GAP
is a negative regulator of Cdc42 which hydrolyzes the active GTPase. ELISA analysis of Cdc42
activity reveals that Cdc42 activity is increased with overexpression of miR-130b.Further
microarray and pathway analysis suggest a mechanism by which activation of Cdc42 leads to
positive regulation of the MAPK cascade. Subsequent western blot analysis reveals that
overexpression of miR-130b leads to the activation of SAPK/JNK and subsequent
phosphorylation of c-JUN suggesting activation of AP-1 transcription factor. Taken together,
these findings suggest that overexpression of miR-130b promotes sarcoma cell migration and
invasion through targeting of Cdc42GAP and modulation of the Cdc42 and SAPK pathways.
Contributors: Satterfield Laura, Kurenbekova Lyazat, Donehower Lawrence, Yustein Jason
Jessica Diane Scott
Integrative Program in Molecular and Biomedical Sciences
Advisor: David Moore, Ph.D.-Department of Molecular & Cellular Biology
Hepatocellular carcinoma is a devastating liver cancer that is on the rise in the
developed world. Constitutive Androstane Receptor (CAR) is a nuclear receptor which is
located primarily in the liver and mediates xenobiotic response. CAR is also essential to
mouse liver tumor development induced by phenobarbital, a CAR agonist. Recently, our
lab has demonstrated that the administration of the CAR inverse agonist androstanol
causes reduction in tumor size and number. In 80% of tumors induced by phenobarbital
and the genotoxic carcinogen diethylnitrosamine (DEN), β-catenin is mutated, while
tumors induced by DEN alone do not contain β-catenin mutations. This suggests that βcatenin and CAR may cooperate to induce liver hyperproliferation and tumor formation.
To test this hypothesis, we activated CAR and β-catenin simultaneously in mice. Doing
so results in hepatomegaly in the short term, and tumor development in the long term.
Activating either component alone results in transient, relatively mild liver growth. In
CAR-activated liver cells, high polyploidy serves as a proliferation checkpoint. In livers
with CAR and β-catenin dual activation, ploidy is abnormally high and the normal
senescence response is bypassed, allowing cells to proliferate unchecked. The
PI3K/Akt pathway has been implicated in insulin-mediated liver polyploidy and is a
commonly disrupted pathway in many types of cancer, including hepatocellular
carcinoma. I propose that CAR and β-catenin act together through the PI3K/Akt
pathway to overcome the polyploidy block to proliferation. Further studies will focus on
elucidating this mechanism.
Contributors: Scott, Jessica; Dong, Bingning; Moore, David
Robert Lionel Seilheimer
Program in Structural and Computational Biology and Molecular Biophysics/M.D.-Ph.D.
Advisor: Samuel Wu, Ph.D.-Department of Ophthalmology
Photoreceptor cells in the retina (rods and cones) convert light into electrical
impulses that are ultimately sent to the brain for interpretation and integration, allowing
us to see. These cells not only synapse with second-order cells in the retina to transmit
visual information downstream to the brain, but also connect laterally with each other.
While the physiology of individual photoreceptors has been well studied, the effect of
electrical coupling on the action of photoreceptors is poorly understood. To investigate
this coupling, we have recorded electrical responses from connected photoreceptors
using dual cell patch techniques. These experiments demonstrate symmetric, linear
coupling between pairs of photoreceptors. However, these methods are not sufficient to
understand how photoreceptors interact on a large scale because of the extensive
coupling in the network. Thus, we present a computational model of photoreceptor
interaction that incorporates the biophysical properties of individual cells as well as the
connections between cells. With this model, we will investigate how individual ion
currents and electrical coupling affects the function of photoreceptors in a network.
Contributors: Seilheimer, Robert; Gao, Fan; Wu, Samuel
Fatih Semerci
Program in Developmental Biology
Advisor: Mirjana Maletic-Savatic, M.D./Ph.D.-Department of Pediatrics
Formation of new neurons in the adult hippocampus represents an adaptive
response of the brain to our environment and/or internal needs. The balance between
underproduction and overproduction of newborn neurons is achieved by whole-cell
means of altering functional circuitry based on the demand. This whole-cell plasticity
suggests that some form of feedback signaling from the progeny to the primary neural
stem cell (NSC) may exist. A known mechanism for cell-cell communication involves the
Notch pathway. Thus, we hypothesized that amplifying neuroprogenitors (ANPs), which
are in direct contact with the primary NSCs, may participate in regulating the NSC
quiescence vs. active proliferation. Indeed, the key components of the Notch pathway
are expressed in the neurogenic niche in a cell type specific manner. Notch receptor is
expressed in the NSCs, while Notch ligands, Jag1and Delta1, are expressed in ANPs
and granule cells, respectively. Moreover, utilizing a comprehensive bioinformatics
approach, we have discovered that Lunatic Fringe (Lfng), a key modifier of Notch
receptor, is selectively expressed in NSC. Further characterization of the Lfng-eGFP
expressing cells confirmed that they are NSCs, as they gave rise to neurogenic
progeny, decreased in number with increased age, and responded to the electrical
stimuli known to increase NSC proliferation. Functionally, removal of Lfng resulted in
increased NSC proliferation followed by decrease in NSC number and neuron
production, suggesting the necessity of Lfng for proper NSC function. On the other
hand, constitutive heterozygote deletion of Jag1 resulted in increased NSC cell cycle
duration with no change in their absolute numbers. These results strongly implicate
Notch signaling in the control of NSCs and their progeny, and suggest a potential
communication and feedback mechanism between the NSCs and their progeny to fine
tune adult neurogenesis. Our data are very significant as we present Lfng-eGFP as a
new mouse model that permits comprehensive and specific studies of NSC properties
and propose a novel mechanism that may operate in the hippocampal neurogenic
Contributors: Semerci F,1 Choi WT, 1,2 Thakkar A, 1 Encinas JM, 1,3 Maletic-Savatic M1*
Mumine Senturk
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M.-Department of Molecular & Human Genetics
Integrins form strong adhesive junctions between tissue layers, a process that is
required in the wing to keep the dorsal and ventral cells attached to each other. A loss
of adhesion between these two epithelial layers causes wing blisters. Our lab generated
a collection of Drosophila X chromosome mutants to identify essential genes involved in
wing development. To identify new players in the integrin signaling pathway, we
screened these EMS induced mutations for wing blisters in mutant clones using a UbxFLP. We identified 30 new alleles of known integrin pathway genes and 25 additional
mutants that exhibit similar wing blistering phenotypes. We mapped many of these
mutants and one corresponds to ubiquilin (ubqn), the Drosophila homologue of UBQLN.
Ubqn functions in the ubiquitin-proteasome system-mediated degradation by
recruiting ubiquitinated proteins to the proteasome. Mutations in UBQLN2 have been
shown to cause dominant X-linked ALS and mutations in UBQLN1 have been linked to
Alzheimer’s disease (AD) by genetic linkage analysis and family-based association
studies. We found that loss of ubqn in fly eye causes a neurodegeneration, glial death,
and an accumulation of small mitochondria upon aging. Moreover, young retina of the
ubqn mutants displays an ER expansion which is one of the distinguished features of
several mutants that cause ALS. Interestingly, several components of the integrin
signaling pathway have recently been associated in GWAS for AD. The link between
Ubqn, integrin signaling, and neuronal maintenance is intriguing and I am therefore
exploring this link using Drosophila as a model system.
Contributors: Mumine Senturk, Shinya Yamamoto, Manish Jaiswal, Lita Duraine, and Hugo
Michelle L. Seymour
Department of Molecular & Cellular Biology
Advisor: Frederick Pereira, Ph.D.-Department of Molecular & Cellular Biology
Animal models and human studies support a link between altered cholesterol
homeostasis and sensorineural hearing loss. However, mechanistic understanding of
how circulating lipids modulate cochlear cellular activities is lacking. The rising
prevalence of obesity and hypercholesterolemia demands a clearer model of how
systemic cholesterol homeostasis influences cochlear cholesterol homeostasis and
hearing function. We seek to define the relationships between systemic and cochlear
cholesterol homeostasis during hearing development and adulthood, as well as how
elevations of systemic cholesterol affect cochlear and hearing function.
Serum and cochlear cholesterol levels change significantly during normal
development of cochlear and auditory function. Interestingly, the sensory epithelium
(SE) contains higher levels of cholesterol than the vascular compartment. Low-density
lipoprotein receptor knock-out (LDLR KO) mice display elevated hearing thresholds,
indicative of hearing loss, concomitant with elevations in serum cholesterol by 1 month
of age. However, cochlear cholesterol levels do not increase in LDLR KO mice until 812 months of age. Vascular organization within the stria vascularis (SV), the metabolic
engine of the cochlea, is also altered in LDLR KO mice. Perivascular-resident
macrophage-like melanocytes (PVM/Ms) contribute to maintenance of the structural
integrity of the cochlear blood-labyrinth barrier (cBLB) in the SV vessels and are
decreased in LDLR KO mice. Current studies focus on quantifying changes in SV
vasculature organization and directly assessing the permeability of the cBLB during
acute and chronic hypercholesterolemia.
In summary, cochlear cholesterol homeostasis is influenced by serum cholesterol
levels during development, but is relatively isolated from serum fluctuations after cBLB
maturation. Mechanisms may also exist for concentrating cholesterol in the SE or for
endogenous synthesis of cholesterol within the organ of Corti. LDLR KO mice have
impaired auditory function that presents simultaneously with hypercholesterolemia. Loss
of PVM/Ms and alterations in SV vascular organization occur prior to elevations in
cochlear cholesterol, suggesting that cholesterol-induced hearing loss may initially result
from strial damage and compromise of cBLB integrity.
Contributors: Seymour, Michelle L.; Pereira, Fred A.
Ayesha Shafi
Department of Molecular & Cellular Biology
Advisor: Nancy Weigel, Ph.D.-Department of Molecular & Cellular Biology
Prostate cancer (PCa) is the second leading cause of cancer-related death in
American men. Androgen ablation therapy is the most common therapy for advanced
PCa. Within two years tumors become resistant to therapy and develop castrationresistant prostate cancer (CRPC). There is no effective treatment for CRPC, which is
androgen-depletion resistant, but androgen receptor (AR)-dependent. AR is a hormoneactivated transcription factor that mediates androgen action. Recent studies have
shown that constitutively active AR splice variants that lack hormone-binding domains
are expressed in CRPC. Reports of the actions and contributions of variants to CRPC
relative to full-length AR are conflicting. To address this question, we have generated
derivatives of androgen-responsive LNCaP and VCaP cell lines with inducible
expression of AR-V7, a variant containing exons 1, 2, and 3 of AR and a small amount
of unique sequence from exon 3b. AR-V7 stimulates expression of some AR target
genes, induces cell growth, and increases cell migration. We performed RNA-Seq to
compare the transcriptomes of AR and AR-V7. RNA-Seq analysis reveals a common
subset of genes regulated by both receptors and more intriguingly genes uniquely
regulated either by AR or by AR-V7 suggesting isoform specific actions. Pathway
analysis showed significant regulation of metabolic pathways. Cancer cells frequently
become more dependent on the glycolytic pathway than are normal cells. Using liquid
chromatography-mass spectrometry (LC-MS), we examined the effect of AR or AR-V7
activation on the levels of several metabolites involved in energy metabolism and
proliferation. The changes in metabolite levels suggest that AR-V7 does not induce lipid
accumulation, while it does preferentially enhance glycolysis and increase rates of
glutamine metabolism (i.e. glutaminolysis). Consistent with this, a Seahorse metabolic
flux assay showed that AR-V7 preferentially increases extracellular acidification rates
(ECAR), a measure of glycolytic activity. In addition, AR-V7 induces changes in the kreb
cycle metabolites including decreased citrate, which is consistent with aggressive PCa.
We have confirmed that AR-V7 regulates genes that regulate these metabolites not only
in our inducible LNCaP-V7, but in the VCaP-V7 cell model. The previously reported
metabolic profile in 22Rv1 cells, which endogenously express full-length AR and AR-V7,
is generally consistent with our findings. This study suggests that AR-V7 does not
simply substitute for AR, but exhibits gains of function that may include the ability to
grow more efficiently in an oxygen poor environment.
Contributors: Shafi, Ayesha; Arnold, James; Putluri, Vasanta; Krause, William C; Zheng, Xia; Li,
Wei; Putluri, Nagireddy; Sreekumar, Arun; Weigel, Nancy L
Shan Shen
Department of Neuroscience
Advisor: Andreas Tolias, Ph.D.-Department of Neuroscience
The mammalian visual system is composed of multiple hierarchically organized
cortical areas that extract progressively more complex features of the visual scene as
information from the retina is fed forward from one cortical area to the next. These
multiple visual areas are also extensively connected via feedback pathways, which
allows the information extracted by higher areas in the visual pathway to influence more
primitive visual responses in earlier areas such as primary visual cortex (V1). Compared
to feedforward pathways, we know very little about the functions of feedback
projections. Lateral-medial area (LM) project to V1 with abundant axon terminal, but the
retinotopical organization and function of these feedback projections are largely
unknown. Using optogenetics and eletrophysiological methods, we were able to study
the wiring diagram of LM to V1 connections. Anatomically, we found that the LM to V1
feedback projections mainly target the retinotopically related area. Functionally, we
found that L2/3 parvalbumin (PV) neurons and somatostatin (SST) neurons receive
strongest excitatory input from feedback projections, while pyramidal cells receive both
excitatory and inhibitory input. The balance of excitation and inhibition on pyramidal
cells creates a sharp temporal excitatory window. Moreover, the feedback projections
sharpen the spike timing of pyramidal cells in V1. These results suggest that the
modulation of feedback pathway from LM to V1 is to induce temporally precise spiking.
Contributors: Shen, Shan; Jiang, Xiaolong; Reimer, Jacob; Tolias, Andreas
Ye Shen
Integrative Program in Molecular and Biomedical Sciences
Advisor: H Lacorazza, Ph.D.-Department of Pathology & Immunology
Acute lymphoblastic leukemia is the most common hematological malignancy in
children. Even though more than 50% of T-cell ALL patients, a subset of ALL, exhibits
activating mutations of NOTCH1, the use of gamma-secretase inhibitors has not been
successful in patients due to limited response and toxicity. Therefore, identification of
genetic factors that cooperate with T-ALL leukemogenesis is needed for the
development of alternative therapies.
KLF4 is a transcription factor that functions as a tumor suppressor or an
oncogene depending on cellular context. Our data showed significant reduction of KLF4
transcripts in lymphoblasts from T-ALL patients compared to blood and bone marrow
cells from healthy individuals. From these findings we hypothesized that KLF4 has
tumor suppressor function in T-ALL leukemogenesis. To test our hypothesis, we
transduced 5-FU treated bone marrow (BM) cells from control (Klf4fl/fl) and Klf4 null
(Klf4fl/fl; Vav-iCre) mice with retrovirus carrying a NOTCH1 activating mutant (L1601P∆P) and then transplanted these BM cells into irradiated recipient mice. In contrast to
controls, mice transplanted with transduced Klf4-null BM cells developed T-ALL with
significantly higher penetrance and shorter latency. On the cellular level, loss of KLF4
led to increased proliferation of leukemia cells as assessed by in vivo BrdU
incorporation, which correlated with decreased levels of p21 protein. Limited dilution
transplantation of primary leukemia cells into secondary recipients showed a 9-fold
increase of leukemia initiating cells (LIC) frequency in Klf4 null leukemia cells compared
to controls.
To elucidate molecular mechanism we performed microarray and ChIP-Seq in
control and Klf4 null CD4+CD8+ leukemia cells. Combined analyses revealed 202
genes as KLF4 direct targets, of which 11 genes are also deregulated in human T-ALL
cells by comparing with published microarray datasets. One of the top upregulated
genes is Map2k7, which encodes a kinase upstream of the JNK pathway. Immunoblots
in leukemia cells confirmed increased expression of MAP2K7 protein and enhanced
phosphorylation of its downstream targets JNK and ATF2. JNK inhibitor SP600125
showed dose-dependent cytotoxicity in all human T-ALL cell lines tested regardless of
their NOTCH1 status.
Overall our results showed that KLF4 functions as a tumor suppressor in T-ALL
by regulating proliferation of leukemia cells and frequency of LIC. Additional study
elucidated that KLF4 suppresses the JNK pathway via direct transcriptional regulation of
MAP2K7. Moreover, the vulnerability of human T-ALL cell lines to JNK inhibition
provides a novel target for future therapy in T-ALL patients.Contributors: Ye Shen, Koramit
Suppipat, Chun Shik Park, Takeshi Yamada, Toni-Ann Mistretta, and H. Daniel Lacorazza
Jonathan Shepherd
Department of Molecular & Cellular Biology
Advisor: Powel Brown, M.D./Ph.D.-Department of Molecular & Cellular Biology
Intrinsic differences in gene expression between basal-like breast cancer (BLBC) and
other breast cancer subtypes imply that transcriptional regulators are differentially activated in
breast cancer subtypes and may be promising therapeutic targets. We hypothesized that
genomic comparisons between BLBC and non-BLBC will identify transcription factors (TFs)
critical for BLBC growth. We identified TFs using an integrative analysis comparing mRNA
expression, frequency of TF response elements in differentially expressed genes, and DNAbinding activity of nuclear proteins in BLBC and non-BLBC. We then tested whether inhibition of
these specific TFs suppresses the growth of BLBC.
Analysis of mRNA expression identified 132 TFs significantly more highly expressed in
TNBC tumors compared to non-TNBC samples across 15 breast tumor datasets. Examining
promoter sequences of 117 BLBC genes, we identified 94 TF motifs over-represented among
BLBC promoters versus ~1500 non-BLBC gene promoters. Binding of DNA motifs by nuclear
protein was measured by protein/DNA Arrays (Affymetrix, Santa Clara, CA) using nuclear
protein collected from BLBC and non-BLBC cells. This analysis identified 11 TF motifs which
were more highly bound by BLBC nuclear lysate.
To integrate the results of the three individual screens, we selected the set of TFs
identified by at least 2/3 assays. This resulted in a set of 33 candidate BLBC TFs. We then
performed a secondary growth screen using 2-dimensional growth of BLBC and non-BLBC cell
lines transfected with siRNA to each candidate TF to determine which TFs are critical for BLBC
growth. We found that inhibition of SOX11 reduced growth selectively in the BLBC cell lines
tested, while growth in the non-BLBC was not affected. Further investigation of SOX11
demonstrated that SOX11 is also critical for other BLBC phenotypes. SOX11 inhibition resulted
in reduced migration and invasion, and reduced expression of several genes which are
characteristically high in BLBC.
Further investigation of the SOX11 will improve our understanding BLBC and may
provide a novel target for the treatment and prevention of BLBC.
Contributors: Shepherd, Jonathan; Uray, Ivan; Mazumdar, Abhijit; Tsimelzon, Anna; Hilsenbeck, Susan
G; Brown, Powel H
Hongsup Shin
Department of Neuroscience
Advisor: Whee Ma, Ph.D.-Department of Neuroscience
Michael Beauchamp, Ph.D.-Department of Neuroscience
Visual working memory of items with multiple features has been one of the main
interests in cognitive psychology and neuroscience. When more than one feature exists
in an item, it brings several issues which do not exist in memorizing a single-feature
item. First, memory resource can be shared among features or not. Second, if present,
irrelevant feature can be encoded or not. And if irrelevant feature is encoded, it can
either affect decision-making or not. These three factors give us six different models. In
the current study we used a same experimental paradigm to examine the difference
among the models to find the best candidate to explain human working memory
behavior. We found that memory resource is not shared by orientation and color, and if
present, irrelevant feature is automatically encoded, but can be ruled out during the
decision process.
Contributors: Shin, Hongsup; Ma, Wei Ji
Binoy Shivanna
Clinical Scientist Training Program
Advisor: Bhagavatula Moorthy, Ph.D.-Department of Pediatrics
Background: Newborn aryl hydrocarbon receptor (AhR) dysfunctional mice are
more susceptible to hyperoxia-induced lung inflammation and alveolar simplification.
However, the molecular mechanism(s) by which AhR mitigates hyperoxic injury is poorly
understood. The NFkB family member, Rel B, is known to exert anti-inflammatory
effects in the lungs of adult mice. Interestingly, AhR is known to cross talk with Rel B to
modulate inflammation in mouse fibroblasts exposed to cigarette smoke. Whether AhR
interacts with Rel B to modulate hyperoxic injury in newborn human lung cells is
unknown. Therefore, we tested the hypothesis that AhR attenuates hyperoxia-induced
oxidative stress, inflammation, and cell death in HPMEC via Rel B.
Objective: To determine the mechanism(s) by which AhR mitigates hyperoxic
injury in HPMEC.
Methods: AhR gene in HPMEC was knocked down by small interfering RNA
(siRNA) transfection and the results were validated by western blotting and real-time
RT-PCR analysis. HPMEC transfected with siRNA or treated with an AhR agonist,
indole-3-carbinol, were exposed to room air (95% air and 5% CO2 at 370C) or
hyperoxia (95% O2 and 5% CO2 at 370C) for upto 48 h. Following exposure, the cells
were harvested to determine viability (3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) assay), necrosis and apoptosis (Annexin V staining
and flow cytometry), reactive oxygen species (ROS) generation (2’-7’dichorodihyrofluorecein diacetate staining and flow cytometry), inflammation (milliplex
cytokine/chemokine assay), cleaved PARP and nuclear Rel B expression (western
blotting), and AhR activation (nuclear AhR protein expression by western blotting).
Results: Hyperoxia decreased AhR expression and activation in HPMEC. AhR
deficient HPMEC had significantly increased hyperoxia-induced ROS generation,
cleavage of PARP, and cell death compared to AhR sufficient HPMEC. Additionally,
AhR deficient HPMEC culture supernatants had significantly increased macrophage
inflammatory protein 1α and 1β, indicating a heightened inflammatory state.
Interestingly, loss of AhR was associated with a significantly decreased nuclear Rel B
protein expression. AhR agonist, indole-3-carbinol, increased nuclear Rel B expression,
decreased hyperoxia-induced ROS generation, and transiently improved the viability of
Conclusions: AhR protects HPMEC against hyperoxia-induced oxidative stress,
inflammation, and cell death via Rel B. Our data suggest that strategies directed
towards increasing AhR activation would be effective in the prevention and treatment of
BPD in preterm infants.Contributors: Shivanna Binoy, Shaojie Zhang, Chu Chun, Jiang
Weiwu, Wang Lihua, Welty Stephen, and Moorthy Bhagavatula
Thomas Shum
Program in Translational Biology & Molecular Medicine/M.D.-Ph.D. Program
Advisor: Cliona Rooney, Ph.D.-Department of Pediatrics
Stephen Gottschalk, M.D.-Department of Pediatrics
Adoptively transferred chimeric antigen receptor (CAR) T-cells can specifically
home to tumors, proliferate extensively and attack tumor cells with highly effective
cytotoxic mechanisms, while producing significantly less adverse effects compared to
conventional chemotherapy. However, the lack of sustained T-cell activity after adoptive
transfer remains a major challenge for solid tumor immunotherapy. One mechanism of
this phenomenon is from T-cell anergy after encountering inhibitory cytokines in tumor
microenvironments, with adenosine emerging as a key immunosuppressant.
Physiologically, adenosine receptors predominantly activate the cyclic AMP/protein
kinase A (PKA) axis to inhibit T-cell activation, serving as a feedback mechanism to
prevent T-cells from damaging bystander tissue during injury and inflammation.
However, adenosine’s normally protective signaling axis becomes an instrument of
tumor growth when adenosine concentrations are elevated in hypoxic tumor
While adenosine has been shown to be immunosuppresive for lymphocytes
applicable for adoptive immunotherapy strategies such as tumor-infiltrating Tlymphocytes (TILs), natural killer cells, natural killer T-cells, and tumor-specific T-cells, it
has yet to be demonstrated that adenosine is immunosuppressive for CAR T-cells.
Using the stable adenosine analog, 2-chloro-adenosine (CADO), we have established
that CADO inhibits cytokine production and proliferation of OKT3/CD28 activated T-cells
(ATCs) and in ATCs expressing a GD2-specific CAR, in-vitro. Based on our preliminary
data, we hypothesize that genetic modification of CAR T-cells to resist the adenosine
axis of immunosuppression can boost CAR T-cell efficacy against tumor cells in hypoxic
co-cultures in-vitro and against established solid tumors in-vivo.
Contributors: Shum, Thomas; Omer, Bilal; Tashiro, Haruko; Gottschalk, Stephen; Rooney,
Meagan Siehr
Department of Molecular & Human Genetics
Advisor: Jeffrey Noebels, M.D./Ph.D.-Department of Neurology
X-linked Infantile Spasms Syndrome (ISSX) is a catastrophic childhood epilepsy
disorder characterized by motor spasms that typically arise in the first year of life. Children
diagnosed with ISSX often develop seizures later in life that are difficult to clinically manage.
Currently, there are few effective drugs available to treat infantile spasms as well as prevent
epilepsy later in life and improve developmental outcomes [1]. The most common genetic cause
of ISSX is a triplet repeat expansion in the Aristaless-related homeobox (ARX) gene. Our lab
generated a mouse model of this mutation (Arx(GCG)10+7) that recapitulates many of the
phenotypic features seen in patients with ISSX including epilepsy and motor spasms in early
life. In several brain regions, the Arx(GCG)10+7 shows significantly reduced numbers of
GABAergic interneurons, a neuronal type that produces the main inhibitory signals of the CNS
important for maintaining excitation/inhibition balance in the brain [2].
Recently, our lab found that 17β-Estradiol (E2) (40ng/g/day) administered to neonatal
Arx(GCG)10+7 mice from P3-P10 reduced spasms and seizures in this model. Interestingly, E2
treatment in neonatal Arx(GCG)10+7 mice also increased numbers of GABAergic interneurons.
However, this effect appeared to be age-dependent, since treatment of adult mice from P33P40 with E2 had no effect on these phenotypes [3]. These results indicate that only early
administration of E2 may have an antiepileptogenic effect in the Arx(GCG)10+7 model and
there may be a critical, developmental window for effective E2 treatment.
In this work, we aimed to define the temporal boundaries of the developmental
window for E2 treatment by delaying treatment initiation from P3 to P7, and we have found that
delaying E2 treatment initiation until P7, at both 40ng/g/day and 80ng/g/day, renders treatment
ineffective and does not reduce seizures and interictal spikes Arx(GCG)10+7 mice. This
suggests a critical, developmental window for E2 antiepileptogenic effect in a model of ISSX.
Understanding the biological mechanism underlying this developmental window is crucial for
understanding how E2 treatment can be utilized in patients with ISSX.
Swann, J.W. and S.L. Moshe, On the Basic Mechanisms of Infantile Spasms. Jasper's
Basic Mechanisms of the Epilepsies, 2012.
Price, M., et al., A triplet repeat expansion genetic mouse model of infantile spasms
syndrome, Arx(GCG)10+7, with interneuronopathy, spasms in infancy, persistent seizures, and
adult cognitive and behavioral impairment. J. Neurosci, 2009. 29(27): p. 8752-8763.
Olivetti, P., A. Maheshwari, and J. Noebels, Neonatal estradiol stimulation prevents
epilepsy in Arx model of x-linked infantile spasms syndrome. Science translational medicine,
2014. 6(220).
Contributors: Siehr, Meagan; Lucero, Rocco; Noebels, Jeffrey
Lukas Simon
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Chad Shaw, Ph.D.-Department of Molecular & Human Genetics
Blood platelets are essential for normal hemostasis and play an important role in
inflammation, tissue repair and regeneration, endothelial cell stability and cancer
metastasis. Reduced platelet function is a common cause of pathologic bleeding,
whereas increased platelet numbers and reactivity are believed to contribute to
pathologic thrombosis, such as myocardial infarction and stroke. Human platelets
express and translate mRNAs, and contain a diverse repertoire of long and short noncoding RNAs. Thus, platelets present a unique opportunity to correlate transcriptomic
information with cell function and other biologic parameters. The Platelet RNA and
eXpression-1 study was designed to investigate significant heritable inter-individual
variation in platelet functional properties. This study profiled platelet mRNA expression
and genotype in a multi-ethnic cohort of 154 healthy humans. To broaden our
understanding of the genetic basis underlying human platelet gene expression, we
conducted expression quantitative trait locus (eQTL) analysis. Among 5911 genes
expressed in platelets, we identified 439 genes with at least one significant cis-eQTLs
(P<10e-6). Using data from the Genotype-Tissue Expression project we discovered that
108 of these genes also contained an eQTL in at least one different tissue and that the
majority cis-eQTL genes are platelet-specific, including platelet genes PCTP, PDK1 and
VAMP8. When analyzing data from a collection of genome wide association studies
(GWAS), we observed that platelet genes containing cis-eQTLs are more likely to be
disease associated compared to platelet genes without cis-eQTLs (P=4.7e-6). Plateletspecific cis-eQTL genes were significantly enriched among genes annotated to the
MESH term “Cardiovascular Diseases” (P<0.05). Among GWAS traits significantly
enriched for platelet-specific eQTL genes are platelet relevant terms such as
“Apolipoproteins B”, “Carotid Arteries”, “Intracranial Aneurysm” and “Platelet Count”
(P<0.05). The comprehensive catalog of all platelet cis-eQTLs can be a useful resource
for future studies, such as in the prioritization and interpretation of GWAS implicated
genes and polymorphisms.
Contributors: Simon, Lukas; Chen, Edward; Edelstein, Leonard; Bray, Paul; Shaw, Chad
Papiya Sinha
Department of Pathology & Immunology
Advisor: Jordan Orange, M.D.-Department of Pediatrics
Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system
that kill virally infected and malignant cells. They contain specialized secretory
lysosomes called lytic granules, which mediate target cell death by the secretion of
perforin and granzymes onto a diseased cell for host defense. This process of
cytotoxicity depends upon the ability of NK cells to transport their granules to the
immunologic synapse (IS) with the help of microtubules. This is followed by granules
approaching the NK cell plasma membrane by the attachment of actin-based motor
protein myosin IIA present on granules to the F-actin network at the IS. Then, granule
docking and fusion with the cell membrane and subsequent release of granule contents
takes place. Until recently, it was suggested that immediately after granule-MTOC
polarization to the IS, granules docked/ fused with the plasma membrane. However,
using advanced high-resolution 2D-Total Internal Reflection Fluorescence microscopy
(TIRFm), we recently defined an unexpected multidirectional movement of granules at
the IS prior to docking and fusion. We first characterized the highly dynamic lytic
granule motility at the IS through previously unapplied complex quantitative and
statistical analyses. Initial results showed that granules are sub-diffusive (constrained
motility due to the crowded environment) at the IS. Furthermore, depolymerization of
microtubules using nocodazole inhibitor revealed lesser granule displacement, while
inhibition of F-actin with latrunculin A treatment displayed more granule displacement at
the IS. Inhibition of both microtubules and F-actin together led to decreased granule
motility. Surprisingly, inhibition of myosin IIA with ML-9 and blebbistatin inhibitors led to
lower granule displacement within the first few minutes post-treatment, followed by
increased motility thereafter. In order to compare the sub-diffusive granule motility at the
IS with an object that is freely diffusing, we calculated the free diffusion coefficient of
granules. Since free diffusion motility is dependent on the viscosity of the local
environment, it was important to determine the viscosity of NK cells which is currently
unknown We microinjected NK cells with fluorescent beads of similar size to granules,
tracked their motility at the IS using TIRFm, and determined the synaptic viscosity on
NK cells for the first time. This study is important because it underscores a new and
unforeseen step that facilitates NK cell killing. In addition to filling the gap in knowledge
of granule motility at the IS, these results identified previously unknown requirements for
effective cytotoxicity. Our results broaden the understanding of NK cell biology with the
use of advanced microscopy and multifaceted quantitative analysis, which will be
beneficial for therapeutic intervention of NK cell function to treat immunological
Contributors: Sinha, Papiya; Tsao, David; Diehl Michael; Kolomeisky Anatoly; Orange, Jordan.
Priya Sivaramakrishnan
Department of Molecular & Human Genetics
Advisor: Christophe Herman, Ph.D.-Department of Molecular & Human Genetics
For accurate function and viability, all organisms possess pathways that protect
their nucleic acids. One of the most devastating forms of DNA damage is a doublestrand DNA break (DSB) caused by both endogenous and exogenous carcinogens.
Improper processing of these breaks can lead to chromosomal rearrangements that
trigger genomic instability. Transcription elongation can affect DSB repair as both these
phenomena occur on the same DNA template. When an elongating RNA polymerase
(RNAP) encounters a DSB, the cell has to choose between aborting transcription to fix
the break, and attempting to continue transcription in order to preserve RNA fidelity.
Although it would seem most likely that the cell would prefer to repair the break and
safeguard the genome while compromising transcription, the fact that transcription
errors occur more frequently than DNA replication errors and that mRNA mutations can
have heritable consequences suggest that there may be situations when maintaining
RNA fidelity outweighs protection of the genome. In Escherichia Coli, transcription
factors that directly bind RNAP modulate its processivity. These include the transcription
elongation factor DksA and the transcript cleavage factor GreA (Eukaryotic TFIIS). GreA
rescues stalled RNAP complexes, ensuring the production of full-length transcripts and
hence preserves RNA fidelity. We have found that loss of GreA results in greatly
increased survival when cells are exposed to DSB inducing agents. Further, these
mutants also show enhanced viability in an assay that is dependent on the ability to
prevent or fix DSBs created by the I-SceI endonuclease. This result in conjunction with
analysis of breaks through pulsed-field gel electrophoresis suggests that loss of GreA
causes improved repair of DSBs. This ameliorated repair is through the canonical
homologous recombination pathway. The absence of DksA on the other hand, displays
an opposite phenotype, rendering cells highly sensitive to induced DSBs.
Overexpression of DksA can rescue wild-type survival on exposure to DSBs, suggesting
that a competition between GreA and DksA for RNAP binding ultimately affects repair
outcome. We hypothesize that a transcribing RNAP can run into repair proteins that are
responsible for processing DSBs to promote homologous recombination. In wild-type
cells, RNAP acts as a physical blockade to DSB processing. In the absence of GreA,
RNAP is a less formidable obstacle for these proteins, allowing for enhanced DSB
repair. This idea is supported by our in vitro experiments showing that RNAP
dissociates at a faster rate without GreA, when proteins are bound to the end of a DSB.
Thus, these transcription factors modulate DSB repair and may play a role in the cellular
decision between maintaining genome integrity and preserving transcription fidelity.
Contributors: Sivaramakrishnan, Priya; Liu, Zhaokun; Halliday, Jennifer A.; Shee, Chandan; Artsimovitch,
Irena; Rosenberg, Susan M.; and Herman, Christophe
Zachry Tore Soens
Department of Molecular & Human Genetics
Advisor: Rui Chen, Ph.D.-Department of Molecular & Human Genetics
Currently the identification of human disease-causing mutations has been largely
limited to protein coding regions due to our limited knowledge of the rest of the genome.
The purpose of this study is to assess the contribution to disease of identified novel
sequence variations in non-protein coding regions in a cohort of more than 1000 human
retinal disease patients.
Our approach combines computational and experimental methods. Based on the
exon sequence of all known retinal disease genes in over 1000 patients, a statistical
method was developed to estimate the contribution of noncoding mutations in each
gene to each disease. Noncoding variants of the top ranked disease genes were
identified using a combination of next generation sequencing and array comparative
genomic hybridization. Identified variants were analyzed using an integrative approach
for their potential to disrupt the function of genes required for proper retinal function.
Candidate pathogenic variants were Sanger verified and functionally validated to alter
gene function or expression.
16 genes that were likely to contain a significant number of noncoding
pathogenic mutations, such as USH2A and ABCA4, were identified. Characterization of
the genomic region of these genes in our patient cohort identified several novel
candidate pathogenic noncoding variants in multiple disease cohorts including Leber
congenital amaurosis, Usher syndrome, and Stargardt disease. Identified variants are
predicted to disrupt gene splicing causing a proportion of transcripts to lose a typical
exon or gain a cryptic exon.
We have developed a new statistical method to estimate the mutation load in the
noncoding region for each known retinal disease gene. Follow up studies of top ranked
genes identified multiple types of non-protein coding mutations including SNVs and
indels. As a significant proportion of patients’ disease can be attributed to mutation in
noncoding regions, further improving our ability to identify and interpret these types of
mutations is crucial.
Contributors: Soens, Zachry; Zaneveld, Jacques; Gelowani, Violet; Jiang, Lichun; Sui, Ruifang;
Koenekoop, Robert; Chen, Rui
Jessica Nichole Sowa
Department of Molecular & Human Genetics
Advisor: Meng Wang, Ph.D.-Department of Molecular & Human Genetics
As women age, they experience both a decline in fertility and an increased risk of
miscarriage and birth defects. In addition to being a significant public health concern, this ageassociated reproductive decline is one of the first aging phenotypes to manifest in humans.
Although predicted to be a regulated process, little is known about the molecular mechanisms
regulating the onset and progression of reproductive aging. In addition to purely genetic
contributions, reproductive aging is thought to be regulated by complex interactions of
environment-sensitive genetic signaling pathways and environmental conditions. However, the
molecular mechanisms integrating environmental and genetic signals to regulate reproductive
aging remain unknown.
As soil-dwelling nematodes, Caenorhabditis elegans experience their diet of bacteria
as a source of both metabolic and sensory input. We took advantage of this relationship as a
convenient method of introducing environmental variation by raising C. elegans on different
bacterial diets. We found that C. elegans exposed to different bacterial environments show
significant differences in the duration of their reproductive span. To further dissect the molecular
mechanisms underlying these reproductive span differences we chose to focus on two strains of
Escherichia coli that gave drastically different effects on C. elegans reproduction. OP50 E. coli
is the standard lab diet for C. elegans, and we found that worms raised on this strain reproduce
longer and maintain their fertility later than worms raised on the alternate E. coli strain HB101.
This effect is mediated by a pair of olfactory neurons which perceive a volatile odorant signal
from the HB101 E.coli. The presence or absence of this environmental cue affects germline
proliferation and maintenance in C. elegans, ultimately contributing to the timing of reproductive
senescence. We have identified a pair of olfactory neurons, the AWB neurons, that are
specifically required for this olfaction-mediated reproductive adaptation as well as for
chemotaxis of C. elegans to the smell of the HB101 bacterial diet. Furthermore, we found that
AWB neuronal photoactivation is sufficient to promote this reproductive adaptation. Finally, we
identified neuropeptide release as the mechanism of AWB neuronal signaling. Together, our
results reveal a novel pathway for the regulation of reproductive aging in C. elegans, and
suggest the relevance of environment-sensitive signaling mechanisms in regulating the onset
and progression of reproductive aging.
Contributors: Sowa, Jessica; Ozseker, Ayse Sena; Wang, Meng
Aaron Jonathon Spike
Department of Molecular & Cellular Biology
Advisor: Jeffrey Rosen, Ph.D.-Department of Molecular & Cellular Biology
The goal of these studies is to elucidate the mechanisms by which the
transcription factor C/EBPβ regulates mammary stem cells (MaSC) and tumor initiating
cells (TICs). There are three C/EBPß isoforms, LAP1, LAP2, and LIP. C/EBPβ is known
as a master regulator of mammary gland (MG) development however, its precise
function in MaSCs is unclear. C/EBPß-/- mammary epithelial cell (MEC) transplantation
results in severe defects and a significant reduction in MaSCs. Expression of LIP in
human patients correlates with poor prognosis in ER/PR-/- breast cancer. Gene
expression analysis of C/EBPß-/- MECs revealed a set of genes whose expression was
modulated by C/EBPß which includes: Eya1, Notch3, ∆Np63, and Stat3. Eya1 is a
negative regulator of Notch, and is also necessary for MaSCs asymmetric cell division.
Expression of Notch3 in the MG is a marker for luminal progenitor cells. ∆Np63 is
known to be a master regulator of stemness and contains C/EBPß binding sites in its
promoter. Deletion of Stat3 results in a reduction of MaSCs and leads to developmental
defects in regenerated MGs. C/EBPβ binds the Stat3 promoter to drive its expression.
Preliminary data shows that Stat3 activation is evident in a small pool of MECs
suggesting that Stat3 may be activated in MaSCs. These observations lead to my
hypothesis that C/EBPβ regulates the expression of a set of genes necessary for
stemness in an isoform specific manner in MaSCs. I will test this with the following aims:
Aim 1: I will determine that C/EBPβ regulates MG development in an isoform specific
manner. Aim 2: I will dissect the relationship between C/EBPβ isoforms and the
expression of the above mentioned gene set in MaSCs. Aim 3: I will determine if these
interactions are also evident in TICs, and if so, what the consequence is. These studies
will broaden our knowledge of MG development, MaSC function, and breast cancer and
may also provide new insights into discovering novel interactions important for stem cell
Contributors: Spike, Aaron
Amulya Sreekumar
Department of Molecular & Cellular Biology
Advisor: Jeffrey Rosen, Ph.D.-Department of Molecular & Cellular Biology
Asymmetric cell division (ASCD) typifies stem cells and results in a stem cell and
a differentiated cell via mitosis, thus maintaining a steady stem cell pool. ASCD is
critical for organ growth and homeostasis. Tumours often display increased symmetric
cell divisions (SCD), that exponentially expands the stem cell pool. Studies in embryos
and in vitro have revealed the p53 and Wnt pathways as possible mediators of cell fate
decision. We asked if this holds in adult organisms in vivo. We propose to study stem
cells in the mammary gland and the aggressive, cancer stem cell (CSC) driven basallike breast cancer (BLBC) subtype. We hypothesize that p53 and Wnt are critical
modulators of ASCD, perturbation of which skews towards SCD, and in tumours results
in an expanded CSC pool. To test this, we propose the following aims. (1) To elucidate
the role of Wnt and p53 in ASCD of mammary stem cells Pubertal mammary glands
show a restricted Wnt signalling niche, which is the potential site of ASCD. We will
modulate levels of Wnt +/- p53 to study the role of these pathways in ASCD. (2) To
demonstrate that perturbation of Wnt and p53 are both required to increase SCD The
MMTV-Wnt1 overexpression model, p53-/- transplanted mammary gland model and the
MMTV-Wnt1;p53-/- model will be used to delineate the role of Wnt and p53 in precancerous lesions (3) To establish the dependence of tumour growth on the pool of
symmetrically dividing CSCs A panel of p53-/- BLBC mouse tumours with varying
proportions of CSCs will be used to study the effect of novel pharmacological inhibition
of the Wnt pathway. This will yield a titrable system to study the effects of targetting
SCD in the CSC population and will be tested with chemotherapy to effect complete
BLBC regression. This study is significant as it could shed light on the mechanism
underlying a core developmental process and generates a rationale for pharmacological
targeting of modulatory components of stem cell biology in BLBC patients.
Contributors: Sreekumar, Amulya; Rosen, Jeffrey
Trace L Stay
Department of Neuroscience
Advisor: Dora Angelaki, Ph.D.-Department of Neuroscience
The vestibular system provides information about one’s movement through the
world. This is a critical component in sensorimotor integration, which underlies accurate
movements. One complication in sensory processing is the inherent ambiguity in the
signals sent by the otolith organs, which encode inertial and gravitational accelerations
identically. Theory has predicted that the brain can differentiate inertial and gravitational
components of self-motion centrally by combining otolith and semicircular canal
information. Cells in the macaque monkey cerebellum have responses to vestibular
stimulation which match these predictions. However, the exact functions of individual
synapses in the circuit underlying this central computation are not known. We have
pursued this question directly with single-unit electrophysiological recordings of
cerebellar lobules 9c and 10 in alert mice. We chose mice because of the opportunity
to use powerful genetic methods to precisely manipulate the vestibular circuit. Using
multiple stimulation methods, we find that mice do have significant populations of cells
which selectively encode inertial or gravitational accelerations. This suggests that the
fundamental mechanisms of vestibular processing are common between mice and
macaques, and establishes mice as a relevant model for investigating the mammalian
vestibular circuit. We can now use a novel genetic strategy to silence individual cell
types in the cerebellum and determine their specific computational roles. Our findings
thus establish an important framework for future studies on the role of the cerebellum in
sensory processing.
Contributors: Stay, Trace; Shinder, Michael; Sillitoe, Roy*; Angelaki, Dora*
Gary R. Stinnett
Department of Molecular Physiology & Biophysics
Advisor: Robia Pautler, Ph.D.-Department of Molecular Physiology & Biophysics
Cell labeling with MR contrast agents has been a major focus of in vivo imaging.
Labeling of cells with T2 agents has allowed the visualization of cell populations in vivo,
but precise concentration and location measurements remains challenging. T1 labeling
of cells is preferable to T2 labeling because T1 contrast exudes positive contrast.
Quality labeling of cells with T1 agents has been difficult due to difficult synthesis of
agents, toxicity, poor stability at physiological pH, and no assay to easily determine
novel targeted T1 agent efficacy in vivo. The Colvin group has synthesized gadolinium
oxide nanoparticles that are coated with PAMPS-LA. These nanoparticles are stable
down to a pH of 3 and are non-toxic to cells at a labeling concentration of up to 250 uM.
These nanoparticles also provide about 9000 times more contrast per molecule than
Gd-DTPA in water. We have electroporated PAMPS nanoparticles into developing
chick neural tube cells to track neural development in vivo. Imaging cells electroporated
with PAMPS nanoparticles highlights the growth and development of the neural
structure in chick embryos.
Contributors: Stinnett, G.R. ; Cho, M. ; Zhu H. ; Pedersen, S.E. ; Deneen, B. ; Colvin, V.L. ;
Pautler, R.G.
Loredana Georgiana Stoica
Department of Molecular & Cellular Biology
Advisor: Mauro Costa-Mattioli, Ph.D.-Department of Neuroscience
Eukaryotic cells have complex post-transcriptional quality control
mechanisms that ensure correct gene expression in normal, physiological settings as
well as under stress conditions. One of these mechanisms is nonsense-mediated decay
(NMD), a surveillance pathway that targets for degradation mRNA transcripts carrying
premature translation termination codons (PTC). Thus, NMD blocks translation of
potentially harmful truncated proteins that might have dominant-negative functions.
Given that 1) a growing list of neuronal genes are reported to be regulated by nonsense
mediated decay during brain development and synaptic plasticity and 2) mutations in
several core components of the NMD pathway have been described in patients
suffering from mental retardation and autism, we decided to explore the consequences
of dysfunctional NMD in the adult brain. Therefore, we generated a new mouse model in
which UPF2, one of the key components of NMD machinery, is deleted in the forebrain
in a αCaMKII Cre-dependent manner. Similar to patients carrying UPF2 mutations,
UPF2-deficient mice display behavioral alterations including impaired social interaction,
behavioral inflexibility, and learning & memory deficits. Currently, we are investigating
the molecular mechanisms underlying these neurocognitive defects. Interestingly, we
discovered that, in these mutant mice, the behavioral abnormalities are accompanied by
reactive astrogliosis, recruitment of a variety of immune cells to the brain, and increased
production of chemokines and proinflammatory cytokines.
In summary, we found that dysregulation of UPF2-mediated NMD could
contribute to cognitive dysfunction and propose that UPF2-deficient mice can serve as a
useful tool to study the function of nonsense mediated decay in brain processes.
Ultimately, understanding how impaired NMD mediates cognitive dysfunction and
autism-like behaviors will lead to drug discovery and therapeutic intervention for patients
with NMD-related intellectual disability.
Contributors: Zhu, Ping Jun; Johnson, Jennifer; Buffington, Shelly; Bhattacharya, Abhisek
;Stinett, Gary; Broussard, John; Eissa, Tony; Pautler, Robia; Pedersen, Steen; Nakada,
Daisuke; Porse, Bo; Costa-Mattioli, Mauro;
Vlatko Stojanoski
Department of Biochemistry & Molecular Biology
Advisor: Timothy Palzkill, Ph.D.-Department of Pharmacology
Serine β-lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics.
Mechanistically, serine β-lactamases are very similar to serine proteases such as
chymotrypsin. They both utilize acylation and deacylation of an active site serine in their
mechanism of catalysis. TEM, a common plasmid-encoded serine β lactamase,
catalyzes the hydrolysis of early penicillins and cephalosporins. Here we examine a
previously identified triple mutant of TEM 165-TyrTyrGly-167 (wild type165-TrpGluPro167) with switched substrate specificity from ampicillin to ceftazidime. When compared
to chymotrypsin, the Glu166Tyr substitution in the TEM triple mutant is analogous to a
substitution of the general base His57 in chymotrypsin that results in an enzyme that
maintains function. Our findings agree with previous observations of altered substrate
specificity of the triple mutant, which displays increased hydrolysis of ceftazidime.
Additionally, enzyme kinetic analysis shows that the hydrolysis of ceftazidime follows a
branched pathway characteristic of substrate-induced reversible inactivation. Structural
analysis of the triple mutant at 1.39 Å resolution reveals enlargement of the active site
making it more accessible for larger substrates. Furthermore, in the crystal structure of
the triple mutant Tyr166 is within hydrogen bond distance to Ser70 suggesting it acts as
a general base to activate the serine in the catalytic mechanism of the enzyme. These
findings change the current view that Glu166 is indispensible in the mechanism of
serine β-lactamases. More generally, this provides insights into alternate mechanisms
for hydrolysis reactions catalyzed by enzymes.
Contributors: Vlatko Stojanoski, Liya Hu, BV Prasad, Dar-Chone Chow, Banumathi Sankaran,
and Timothy G. Palzkill
Adrianne Elayne Stone
Program in Translational Biology & Molecular Medicine
Advisor: Brendan Lee, M.D./Ph.D.-Department of Molecular & Human Genetics
Carlos Bacino, M.D.-Department of Molecular & Human Genetics
Osteoarthritis (OA) is a progressively debilitating condition involving joint
degeneration that causes substantial joint stiffness and pain, decreased mobility, and
increased healthcare costs. Despite approximately 27 million Americans being afflicted
with this disease, there are no medical treatments that significantly alter its progression.
Currently, an individual suffering from OA will only be proscribed pain management
medication, physical therapy, and lifestyle modification, and in end-stage disease a joint
replacement surgery will be advised. Injections of cytokines and proteoglycans have
shown some promise in clinical and pre-clinical trials, yet due to the rapid degeneration
and turnover of these compounds, repeated injections are necessary to obtain any longlasting results, potentially leading to an increase in adverse events. Major obstacles to
the development of new treatments for OA include both the difficulty of identifying
quantifiable endpoints for progression in animal models, and the complex cell
autonomous and non-cell autonomous components of the pathophysiology of the joint.
To overcome these obstacles, our lab developed a novel imaging technique combining
staining, phase-contrast optics, and microCT analysis of the joint, which allows for the
robust quantification of murine cartilage volume, surface area, and osteophyte
formation. To address the issue of the complex etiology of OA development, our lab has
identified secreted molecules as potential therapeutic targets – Proteoglycan 4 (PRG4),
Interleukin 1 Receptor antagonist (IL1Ra), and the Insulin-like Growth Factor 1 (IGF1).
PRG4 is a major component of the cartilage extracellular matrix and mediates
mechanical stress by providing lubrication. Inflammation has also been attributed to
disease progression, and IL1Ra has been proven to function as an anti-inflammatory
through the inhibition of Interleukin 1 (IL1). IGF1 is an anabolic agent that can help
maintain cartilage by promoting chondrocyte proliferation and inhibition of apoptosis.
Both published work and preliminary data from our lab and others shows that
individually each provides some protection from OA progression. We hypothesize that
combinatorial therapy with PRG4, IL1Ra, and IGF1 will provide increased protection
from OA development and progression through addressing cartilage matrix loss and
inflammation. This will be tested by comparing the effects of combinatorial therapy to
duo- and mono-therapy in a post-traumatic model which involves generation of joint
injury through the Cruciate Ligament Transection surgery – a procedure which mimics
the human condition of knee injury by ACL tear. We will also assess the effects of the
combinatorial therapy in an age-related model of OA development, as the contribution
of factors leading to disease progression is different in each model.
Contributors: Stone, Adriane; Ruan, Merry Z; Dawson, Brian; Lee, Brendan
Danielle Dee Stuhlsatz
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: B. Pettitt, Ph.D.-Biochemistry
Misfolded and unstructured domains of proteins represent important examples of
disease states where the understanding of the recognition, or folding process has
important potential therapeutic implications. The mechanisms governing molecular
recognition and the transition of proteins from their unfolded state to their native state
remain related but unanswered fundamental biophysical questions. Refolding of protein
domains often occurs during DNA binding. Specifically, LacI contains a hinge region
that is disordered without DNA or when nonspecifically bound, but becomes ordered
when bound to the correct DNA sequence. Experimentally, the hinge region has a
measurable disorder to order transition when binding its specific sequence. This system
has a nontrivial sequence and allows us to test the importance of the protein folding
transition versus that of the contributions of DNA bending and DNA protein interactions
separately. The hinge-helix sequence of the LacI system will be computationally tested
in various multicomponent aqueous solutions. Our recent theoretical work suggests the
solvent effect on the initial collapse toward folding and the early processes of molecular
recognition are related.
Contributors: Pettitt, B.M.
Qingtai Su
Integrative Program in Molecular and Biomedical Sciences
Advisor: Li Xin, Ph.D.-Department of Molecular & Cellular Biology
Prostate cancer is the second leading cause of cancer-related deaths in western
countries’ males. Within that metastasis contributes to most of mortality and morbidity.
Tissue microarray assay had revealed that Jag1 protein was elevated in tumor samples
derived from patients who developed metastatic recurrent diseases; however, the roles
of Jag1 in prostate cancer development are inadequately defined.
We sought to use transgenic mouse models and human prostate cancer cell
lines to investigate the role of Jag1 in prostate cancer progression. Firstly, we evaluated
the effects of Jag1 deregulation on the proliferation and migration of the prostate cancer
cell lines. Up-regulation of Jag1 in Du145 did not affect their proliferation. However,
Jag1 positively regulates the migratory capacities of Du145 in an in vitro wound-healing
assay. To validate the role of Jag1 in metastasis of prostate cancer in vivo, we injected
the control Du145 cells and the Du145 cells over-expressing Jag1 into NOD/SCID mice
via tail veins. Our result showed that Jag1 up-regulation promoted distal colonization of
tumor cells in lung, reduced latency of metastasis and shortened life span. We also
generated a transgenic mouse model that allows conditional over-expression of Jag1 in
prostatic epithelial cells. We found that over-expression of Jag1 alone did not
transformed epithelial cells by the age of 26week. Growth rate and resistance to death
of prostate epithelial cells were not altered by the presence of higher Jag1 expression.
Nevertheless, we discovered that Cyclins levels were up-regulated in the epithelium,
which might cause some effects in older mice. On the other hand, we are currently
inducing Jag1 expression in the well-defined Pten-null prostate cancer model which
otherwise is not aggressively metastatic in quest of functions of Jag1 in the prostate
cancer metastasis.
Contributors: Su, Qingtai; Kwon, Oh-Joon; Zhang, Li; Valdez, Joseph; Zhang, Boyu; Wei, Xing;
Xin, Li
Ann Tabitha Sukumar
Department of Molecular & Human Genetics
Advisor: Alison Bertuch, M.D./Ph.D.-Department of Pediatrics
Telomeres, the nucleoprotein structures found at chromosome ends, consist of
tandem repeats of TTAGGG sequence bound by proteins collectively known as the
shelterin complex. The higher order DNA-protein structure of telomeres shields the
chromosome ends from appearing as DNA double strand breaks. Without such
protection, end-to-end fusion of chromosomes takes place via non-homologous end
joining (NHEJ), which leads to cell growth arrest. Ku, a crucial upstream factor in the
NHEJ pathway, binds to DNA ends at double strand breaks to initiate NHEJ. However,
Ku also associates with telomeres and contributes to telomere function. How Ku is
regulated to inhibit its NHEJ function at the telomeres and to perform its telomere
protection function is not well understood.
Ku interacts with shelterin proteins TRF1 and TRF2 at the telomeres. Previous
data from our lab suggests that Ku-TRF2 interaction might prevent Ku from participating
in NHEJ at telomeres. To investigate this idea, we aim to understand how Ku associates
with telomeres. We hypothesize that Ku associates with telomeres via protein-protein
interaction as opposed to end binding, which could result in deleterious fusions. We will
perform in vitro studies using purified Ku and TRF2 proteins and DNA substrates with
and without telomeric repeats or access to the ends to assess if Ku associates with
telomeres via TRF2. In addition, we will use Ku mutants that are impaired for TRF2
binding or end binding to explore Ku’s mode of association with telomeres via chromatin
immunoprecipitation assay. By studying how Ku associates with telomeres, we hope to
gain a better understanding of Ku’s conflicting roles at telomeres.
Contributors: Sukumar, Ann; Bertuch, Alison
Sujita Sukumaran
Program in Translational Biology & Molecular Medicine
Advisor: Ann Leen, Ph.D.-Department of Pediatrics
William Fisher, M.D.-Department of Surgery
The promising clinical results achieved using adoptively transferred chimeric
antigen receptors (CAR)-modified T cells in patients with B cell malignancies has prompted
investigators to explore this strategy for the treatment of solid tumors. However, while the
infusion of CAR T cells may provide therapeutic benefit, solid tumors use a variety of immuneevasion mechanisms, including the production of immunosuppressive cytokines (IL4, IL10,
TGF ) that can inhibit effector T cells. To protect our CAR T cells from the suppressive effects
of IL4, we generated a chimeric cytokine receptor (4/7R) that comprises the ectodomain of the
IL4 receptor fused to the endodomain of the immunostimulatory IL7 receptor, with mOrange coexpressed to allow transgene detection. Thus, transgenic expression of the 4/7R should enable
T cells to utilize tumor-produced IL4 to promote CAR T cell expansion and cytotoxic activity.
To implement this approach for pancreatic cancer, a highly aggressive disease
with a five-year survival of <1%, we generated a CAR targeting the tumor-expressed prostate
stem cell antigen (PSCA). When expressed on T cells (78±20%, n=5) transgenic cells were able
to specifically kill PSCA-expressing cell lines (CAPAN1 and K562-PSCA) but not 293T (PSCAtarget) (74±4%, 73±6% and 9±3% specific lysis, respectively, 10:1 E:T, n=3). To next evaluate
the protective effects of the 4/7R we double transduced T cells to express both CAR-PSCA and
the 4/7R (30-72% double positive cells; n=4) and assessed the expansion of both control (CARPSCA) and 4/7R/CAR-PSCA T cells in the presence of antigen and IL4. Although CAR-PSCA T
cells were unable to expand in the presence of IL4 (from 2x106 cells on day 0 to 6.1±3.8 x 107
on day 28), 4/7R/CAR-PSCA T cells expanded exponentially (from 2x106 cells to 5.1±3.6 x 109)
(n=3). Moreover, culture in IL4 for 4 weeks enriched the 4/7R (mOrange+) transgenic cells
(mean 45.9±15% - day 0 to 86±11% - day 28, n=3). We next assessed the cytokine profile of
4/7R/CAR-PSCA T cells exposed to IL4- a prototypic Th2 cytokine, while IL7 induces a Th1polarized cytokine profile. Exposure of CAR-PSCA T cells to IL2 induced prototypic Th1
cytokines including IFN- and TNF23±14 ng/mL and 2.7±2 ng/mL respectively), which
was diminished in the presence of IL4 (1.8±1.7 ng/mL and 0.4±0.1 ng/mL). However, the 4/7R
restored the Th1 cytokine profile of transgenic cells exposed to IL4 (IFN- - 30.2±20 ng/mL and
TNF3.2±2 ng/mL). Importantly, the selected 4/7R CAR-PSCA T cells continue expanding
only in presence of both antigen and cytokine confirming their safety for in vivo translation.
Thus, we have demonstrated the feasibility of the 4/7R in protecting CAR T cells
from inhibitory IL4 by inverting a tumor-derived suppressive signal to enhance anti-tumor
Contributors: Sukumaran, S; Mohammed, S; Watanabe, N; Bajgain, P; Anurathapan, U; Leen,
A.M; Vera, J.F.
Jenny J Sun
Department of Neuroscience
Advisor: Russell Ray, Ph.D.-Department of Neuroscience
The perpetual rhythm of breathing is essential to survival and emerges through
the interactions of a complex array of neuronal networks. Noradrenergic (NA) neurons
have been implicated as a critical participant in the circuitry of this homeostatic
behavior, and past studies point to a high molecular and functional complexity within the
small system of only 4000 neurons. However, the relationship between the system’s
genetic heterogeneity and its underlying functional organization remains unknown.
Because early gene expression is critical to the development of neural circuits and cell
identity, we hypothesize that subpopulations of NA neurons originating from different
rhombomeres, differential gene expression domains in the developing brainstem,
regulate distinct and specific aspects of respiratory physiology. To test this hypothesis,
we will take an intersectional genetic approach that subdivides the NA system into
narrowly defined, genetically distinct cohorts that can be functionally characterized.
Preliminary studies show that NA neurons are required for the respiratory response to
hypercapnic (high CO2) and hypoxic (low O2) conditions, as pharmaco-genetic
perturbation of NA-expressing neurons results in reduced ventilatory responses. We
also see a decrease in room air ventilation and the hypoxic response when
rhombomeres 3&5-derived neurons are perturbed. These data present a working model
to query the function of a genetically defined neuron population and the observed
phenotypes offer a potential mechanistic understanding for homeostatic disorders
associated with noradrenergic abnormalities. Linking these developmentally distinct
subtypes of neurons in respiratory homeostasis will give us greater insight into the
functional organization of neuromodulatory networks and improve potential therapeutics
for life-threatening respiratory disorders.
Contributors: Sun, Jenny; Ray, Russell
Jiayi Monika Sun
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Ching Lau, M.D./Ph.D.-Department of Pediatrics
Osteosarcoma is the most common type of malignant bone cancer in children
and young adults. Little improvement to the long term survival of osteosarcoma patients
has been seen in the past 20 years, especially for high-risk patients who develop
metastasis or respond poorly to standard therapy. Genetic aberrations associated with
osteosarcoma pathogenesis include aberrations of well-known tumor suppressors such
as TP53 and RB1 however, very little is known about the epigenetic landscape. DNA
methylation is a type of epigenetic regulation by which a methyl group is added to the
cytosine of a cytosine-guanine (CpG) dinucleotide. Aberrant DNA methylation has been
known to play a major role in the progression of cancer. As part of the Therapeutically
Applicable Research to Generate Effective Treatments (TARGET) initiative, we have
comprehensively profiled approximately 100 osteosarcoma samples on several genomic
DNA methylation was processed using Illumina’s Infinium
HumanMethylation450 BeadChip. This array interrogates more than 480,000 CpG
sites, covering 99% of all RefSeq genes and 96% of known CpG islands. In addition to
including a majority of the probes from the previous version of the array (HM27) this
array also includes an additional type of assay chemistry which is designed to
interrogate regions of low CpG density. We evaluated the array’s control probes as well
as probes located on sex chromosome to check for quality control and sample
annotation integrity. For pre-processing, we performed background subtraction and dyebias correction through an R package called Lumi. A final methylation level called a
‘beta-value’ is calculated from a ratio of the unmethylated(U) and methylated(M)
intensities (M/(M+U)) for each site which ranges between 0 and 1, where 0 represents
complete demethylation and 1 represents complete methylation. The beta-values are
corrected for probe design bias due to the different probe chemistries using the Beta
Mixture Quantile Dilation method (BMIQ). Finally, we filtered out any probes with
detection p-values greater than 0.05, probes overlapping repeat regions, probes
containing SNPs located within 10 bp or at the CpG site and any probes located on the
sex chromosomes to correct for gender and potential technical artifacts. Through nonnegative matrix factorization and recursively partitioned mixture modeling we identified
two groups within the data with significantly different overall survival. We observed that
patients with lower survival were characterized by hypermethylated probes. A very high
correlation was observed between DNA methylation and gene expression for FAS, a
gene whose expression has previously been shown to correlate with metastatic
potential of osteosarcoma patients. We are currently in the process of pursuing this
study in functional models.
Contributors: Sun, Jiayi; Yu, Alex; Voicu, Horatiu; Lin, Howard; Taylor, Aaron; Shen, Jianhe;
Guerra, Rudy; Man, Chris; Lau, Ching
Yu Sun
Integrative Program in Molecular and Biomedical Sciences
Advisor: Weiwei Dang, Ph.D.-Department of Molecular & Human Genetics
Sir2 is a class III histone deacetylase that is a key regulator of life span. Sir2
(Silencing Information Regulator) belongs to Sirtuin protein family. Overexpression of
Sir2 extends life span and this is conserved among higher eukaryotes. Sir2 protein is
lost during aging, leading to high levels of H4K16 acetylation at telomere region that
affects yeast replicative life span 1. Previous study has shown that the transcriptional
level of Sir2 is not significantly reduced in old cells. We raise the question that what
mediates Sir2 loss during aging. We found that neither Ubiquitin-Proteasome System
nor macroautophagy is responsible for Sir2 degradation in old cells. From a proteomic
study comparing protein profile in old and young cells, Pep4p stands out as one of the
most up-regulated proteins in old cells. Pep4p is the vacuolar protease A, required for
precursor maturation of most vacuolar proteases and also the homolog of Cathepsin D
in mammalian cells. When Pep4 is deleted, Sir2 protein is preserved and even
increased in old cells, thus Pep4 is a Sir2 regulator during aging. We tracked Sir2-GFP
fusion protein in wild type and pep4∆ old cells and found that Sir2 is largely preserved in
the vacuoles in pep4∆ strains, indicating the translocation of Sir2 from nucleus to
vacuoles during aging. In pep4∆ strains, the conformation of Sir2 is changed, as shown
by limited proteolysis test, while the enzymatic activity of Sir2 is not affected.
Immunoprecipitation-Mass Spectrometry study showed that Ssa2 and Ssb2, which
belongs to HSP70 protein chaperone family, are potential Sir2 binding factors that are
differently regulated in wild type and pep4∆ strains. Co-IP experiment confirmed that
Ssa2 and Ssb2 interact with Sir2. Future study will be focused on elucidating the
interacting patterns of Ssa2, Ssb2, Sir2 and Pep4, and how this interplay will cause the
degradation of Sir2 during aging.
Dang, W. et al. Histone H4 lysine 16 acetylation regulates cellular lifespan.
Nature 459, 802–7 (2009).
Contributors: Sun, Yu; Berger, Shelley L.; Dang, Weiwei.
Yun-Min Sung
Department of Biochemistry & Molecular Biology
Advisor: Theodore Wensel, Ph.D.-Department of Biochemistry & Molecular Biology
Previous studies of amino acid co-variation in G protein-coupled receptors
(GPCRs) mainly focused on predicting structural proximity of residue pairs in order to
construct protein 3D structures. However, whether evolutionary amino acid co-variation
can be used to detect functional coupling between residue pairs has not been tested.
We investigated the functional relationships between evolutionarily correlated residue
pairs in GPCR, focusing on dopamine D2 receptors (D2Rs). The D2R ligand binding
pocket is predicted to be structurally similar to that of 5-HT2A serotonin receptors (5HT2ARs); however, D2R responds and binds to dopamine better than serotonin,
indicating the ability of D2R to discriminate between the neurotransmitters dopamine
and serotonin. We hypothesized that residues may work in pairs or larger groups to
maintain functional specificity of D2R. By applying the Evolutionary Trace (ET) algorithm
to pairs of residues, we first predicted residue pairs in D2R that were likely to co-vary in
evolution and replaced the co-varying ET-residues with the corresponding residues in 5HT2AR. By comparing the double swaps with individual swaps, we observed nonadditive effect on the efficacy of G i activation induced by dopamine-stimulated D2R,
indicating functional coupling between the ET-residue pairs. We also found dramatic
rescue effect on serotonin responses in some of the co-varying ET-residue pairs,
suggesting their functional role in discriminating between dopamine and serotonin.
Furthermore, we observed that one combined ET-residue swap showed higher binding
affinity for serotonin compared to the wild-type, while the individual swaps had no effect
on serotonin affinity. The non-additive free energy change upon binding revealed the
functional coupling in ligand recognition. Taken together, these findings indicate that key
co-varying ET-residue pairs have worked together during evolution to help fine-tune
responses to dopamine, whereas other pairs have worked together to help confer
selectivity, with the latter property likely more important for conferring selective
evolutionary advantage.
Contributors: Sung, Yun-Min; Wilkins, Angela D.; Rodriguez, Gustavo J.; Lichtarge, Olivier;
Wensel, Theodore G.
Maria Magdalena Szwarc
Department of Molecular & Cellular Biology
Advisor: Bert O'Malley, M.D.-Department of Molecular & Cellular Biology
Steroid receptor coactivator-2 (SRC-2) is a member of the p160/SRC family of
coregulators, which also includes SRC-1 and SRC-3. Members of this coregulator class exert a
wide-spectrum of physiological processes from mammary morphogenesis, metabolic
homeostasis, to endometrial function. Our group has previously shown that knockout of SRC-2
in the murine endometrium leads to infertility caused by both a defect in embryo attachment and
an inability of the endometrium to undergo decidualization, an essential cellular process that
enables embryo implantation to occur. Importantly, deregulation of SRC expression levels is a
causal factor for many tissue pathologies in both human and mouse. In the case of the
endometrium, clinical studies reveal that SRC-2 and SRC-3 levels are elevated in endometrial
biopsies from patients diagnosed with polycystic ovary syndrome (PCOS). Importantly, the
endometrium of PCOS patients displays severe defects in functionality, including increased
endometrial cancer susceptibility and miscarriage rate. Elevated expression of SRC-2 and SRC3 has also been found in hyperplastic and neoplastic endometrium. Collectively, these
descriptive findings suggest a causal link between elevated expression of one or both of SRCs
and the emergence of these endometrial disorders. To address this proposal further, we
engineered a SRC-2 overexpressor (SRC-2: OE) mouse in which high levels of human SRC-2
expression are targeted to cells that express the progesterone receptor. Although ovulation and
serum hormone levels are normal, breeding studies show that elevated levels of endometrial
SRC-2 result in a severe subfertility defect. Importantly, an artificial decidual response assay
revealed that the SRC-2:OE endometrium exhibits an impaired ability to undergo
decidualization. The incapacity of the SRC-2:OE endometrium to decidualize is also reflected at
the molecular level by significant decrease in the induction of the decidual biomarkers.
Furthermore, estradiol treatment reveals that perturbation of SRC-2 levels markedly potentiates
estradiol-induced uterine epithelial hyperplasia, providing strong support for SRC-2 in the
promotion of unopposed estrogen-action, the main risk factor endometrial cancer development.
These studies are supported by our findings in vitro where we have shown that maintenance of
high SRC-2 levels is required for endometrial cancer cell growth as shown in colony formation
and proliferation assays. Importantly, microarray analysis demonstrated that attenuation of
SRC-2 levels in endometrial cancer cells leads to alteration of cellular signaling pathways
critical for cell proliferation and differentiation. Also, knockdown of SRC-2 leads to a
downregulation of the glycolytic flux and mitochondrial oxidation which is accompanied by a
decrease of metabolic intermediates of the pentose phosphate pathway, which is critical for
cancer cell growth. We conclude that strict control of SRC-2 expression levels is mandatory not
only for normal endometrial functionality but also to prevent unscheduled endometrial
hyperplasia which can lead to cancer.
Contributors: Szwarc, Maria M; Kommagani, Ramakrishna; Jeong, Jae-wook; Wu, San-Pin;
Lanz, Rainer B; Putluri, Nagireddy; Tsai, Sophia Y; Tsai, Ming-Jer; DeMayo, Francesco J;
Lydon, John P; O’Malley, Bert W
Bryan Christopher Tackett
Program in Translational Biology & Molecular Medicine
Advisor: Sundararajah Thevananther, Ph.D.-Department of Pediatrics
Saul Karpen, M.D./Ph.D.-Department of Pediatrics
Background & Aims. Partial hepatectomy (PH) induces hepatocyte proliferation
via step-wise induction of immediate early genes, reorganization of extracellular matrix,
and cytokine and growth factor-mediated signaling early on during liver regeneration.
However, the identity of initial trigger(s) of liver regeneration has remained elusive. ATP
is released into the extracellular milieu within minutes of 70% PH. We hypothesized that
extracellular ATP, via activation of its cognate cell-surface P2Y2 purinergic receptors,
might play a key role in the induction of early events critical for hepatocyte proliferation
in regenerating livers.
Methods. Wild type (WT) and P2Y2 purinergic receptor knockout (P2Y2-/-) mice
were subjected to 70% PH and liver tissues were analyzed for efficiency of hepatocyte
priming and proliferation. Influence of extracellular ATP and P2Y2 puringeric receptor
signaling on hepatocyte proliferation was evaluated in vitro.
Results. Our findings suggest that hepatocyte proliferation in response to 70%
PH was impaired in P2Y2-/- mice. Early activation of p42/44 MAPK (ERK, 5 min), early
growth response-1 (Egr-1) and activator protein-1 (AP-1) DNA-binding activity (30 min)
were attenuated in the remnant livers of P2Y2-/-. Correspondingly, Egr-1 and AP-1
target gene and a key mediator of extracellular matrix remodeling, matrix
metalloprotese-9 (MMP-9) protein induction and HGFα/c-Met signaling were attenuated
in P2Y2-/-. Extracellular ATP alone, via the activation of P2Y2 purinergic receptors, was
sufficient to activate ERK/Egr-1 and proliferation of primary mouse hepatocytes in vitro.
Conclusions. Extracellular ATP-mediated rapid activation of P2Y2 purinergic
receptors plays a key role in the initiation of hepatocyte proliferation in response to PH
in mice.
Contributors: Tackett, Bryan; Sun, Hongdan; Cheruvu, Sayuri; Mei, Yu; Mani, Arunmani;
Hernandez-Garcia, Andres; Vigneswaran, Nadarajah; Karpen, Saul; Thevananther,
Rajeev Babu Tajhya
Department of Molecular Physiology & Biophysics
Advisor: Christine Beeton, Ph.D.-Department of Molecular Physiology & Biophysics
Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular
disorder that primarily causes skeletal muscle wasting. This muscle wasting, due to
reduced myogenesis, is the main cause of disability in patients and there is no cure for
DM1. Errors in alternative splicing due to accumulation of the splice regulators MBNL
and CUGBP at (CUG)n hairpin structures cause prolonged or persistent myoblast
proliferation and delayed myotube fusion. However, the downstream pathways
contributing to reduced myogenesis remain unclear. K+ channels regulate proliferation
of myoblasts and differentiation into myotubes by triggering changes in membrane
potential, cell volume and regulating Ca2+ signaling. Therefore, we investigated the
roles of K+ channels in primary myoblasts obtained from patients with DM1 and healthy
donors. We have identified loss of calcium-activated and voltage-dependent functional
K+ channel KCa1.1 at the plasma membrane of DM1 myoblasts by
immunofluorescence and flow cytometry, and have shown loss of paxilline-sensitive K+
currents by patch-clamp. We hypothesized that the loss of KCa1.1 in DM1 myoblasts
plays role in the delayed myogenesis, and that over-expressing KCa1.1 channels will
rescue the pathological features in vitro. We found that inhibition of KCa1.1 in myoblasts
from healthy donors enhanced proliferation, lowered matrix metalloproteinase-2 (MMP2) production, impaired wound healing, and decreased myotubes formation – all
features of DM1 myoblasts, demonstrating that functional KCa1.1 channels are required
for normal proliferation, migration and myotube fusion. On the contrary, over-expressing
KCa1.1 in myoblasts from patients with DM1 normalized their proliferation to the levels
observed in healthy myoblasts. We also showed that blocking KCa1.1 in healthy
myoblasts increases activated-NFκB which is often associated with muscle wasting. We
conclude that the loss of KCa1.1 in DM1 impairs early stage of myogenesis possibly by
affecting Ca2+-mediated pathways. This suggests that KCa1.1 modulation could be
utilized as a novel strategy to rescue delayed myogenesis in DM1.
Contributors: Tajhya, Rajeev; Hu, Xueyou; Tanner, Mark; Timchenko, Lubov; Beeton, Christine
Kai Li Tan
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M.-Department of Molecular & Human Genetics
Synaptic transmission involves various cellular activities, including protein
synthesis and degradation, vesicular and protein trafficking, and fusion and fission of
membranes. Orchestrating these activities is important to ensure optimal synaptic
function. In a forward genetic screen on the Drosophila X-chromosome to identify
essential genes required for proper synaptic transmission, we identified mutations in ari1, the Drosophila homolog of mammalian HHARI. ari-1 is a highly conserved gene,
which encodes a ring-between-ring E3 ubiquitin ligase. By recording electroretinograms,
we found that mutant photoreceptors display a loss of ON-transients when activated by
light, indicating defects in neurotransmission. To further characterize this synaptic
transmission defect, we turned to the 3rd-instar larval neuromuscular junction (NMJ).
We performed electrophysiological recordings and found that postsynaptic muscle cells
show an increased excitatory junction potential (EJP) upon stimulation when compared
to the control. To identify the cause of the increased evoked response, we performed
transmission electron microscopy and immunostaining for different components in the
synapse, and found that the size of the synaptic vesicles and the amount of postsynaptic receptors remained unchanged. However, the number of active zones
(indicated by Bruchpilot staining), the sites in the synapse where vesicles cluster to
allow neurotransmitter release, is increased in ari-1 mutants. These additional active
zones are potentially functional, since increasing the level of a voltage-gated calcium
channel, Cacophony (Cac) in ari-1 mutant background leads to early animal lethality,
which is likely caused by calcium mediated excito-toxicity. A core component of the
active zone, Bruchpilot (Brp), is homologous to mammalian ELKS, where it functions to
recruit Cac to the active zone. It is likely that Ari-1 directly regulates the level of
Bruchpilot, possibly through its ubiquitination activity. However, over-expressing Brp in
the neuron does not alter the physiological properties and active zone distribution of the
synapse. This indicates that either Ari-1 directly regulates Brp level locally at the
synapse, or Ari-1 indirectly affects Brp level or distribution in the synapse by regulating
other proteins. In order to understand the molecular mechanism of Ari-1 in promoting
optimal synaptic transmission, we will identify the ubiquitinated targets of Ari-1 in the
neuron through biochemical assays.
Contributors: Tan, Kai Li*; Haelterman, Nele*; Nagarkar, Sonal; Lee, Pei-Tseng;
Bellen, Hugo
Mark R. Tanner
Program in Translational Biology & Molecular Medicine
Advisor: Christine Beeton, Ph.D.-Department of Molecular Physiology & Biophysics
David Corry, M.D.-Department of Medicine
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease of largely
unknown etiology that mainly affects diarthrodial joints, leading to joint destruction, pain,
and decreased mobility. The fibroblast-like synoviocyte (FLS) is a resident synovial cell
that has a central role in RA pathogenesis, including the formation of synovial
hyperplasia and increased invasiveness, along with increased cytokine, growth factor,
and protease release. These factors contribute to cartilage and bone degradation within
the joint. Currently, no RA therapy has been developed to specifically target FLS.
We have found that FLS from patients with RA and from rats with the pristaneinduced and collagen-induced arthritis models of RA have increased expression of
KCa1.1 at their plasma membrane when compared with FLS from patients with
osteoarthritis or from healthy rats, respectively. Selectively blocking the function of this
channel reduces many of the pathogenic aspects of RA-FLS, including decreasing
proliferation, invasiveness, and release of cytokines, chemokines, angiogenic factors,
and proteases. Furthermore, decreasing the amount of KCa1.1 expressed in FLS
reduces their invasiveness and opening or increasing membrane expression of KCa1.1
increases invasiveness. This altered invasiveness is likely a result of KCa1.1’s
regulation of the FLS cytoskeleton, as blocking KCa1.1 increases the channel’s
association with actin and causes filamentous actin to have a modified cellular
We have also found that inhibiting KCa1.1 with a selective small-molecule
blocker after onset of clinical signs of arthritis significantly reduced disease severity in
both the collagen-induced and pristane-induced rat models of RA, along with reducing
the ex vivo invasiveness and proliferation of FLS from blocker-treated animals.
These studies indicate the importance of KCa1.1 as a novel target for RA and
emphasize the potential efficacy of directly inhibiting FLS in reducing the severity of this
debilitating disease.
Contributors: Tanner, Mark R.; Hu, Xueyou; Huq, Redwan; Tajhya, Rajeev; Khan, Fatima S.;
Gulko, Percio; Beeton, Christine
Aaron Michael Taylor
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Ching Lau, M.D./Ph.D.-Department of Pediatrics
Background. Ependymomas (EPN) account for ~10% of intracranial tumors in children,
typically presenting in the first five years of life. Several studies to date have described subtypes
of EPN by mRNA expression and DNA copy number that have prognostic significance. Still,
there has been little improvement in survival and the search for effective targeted therapy
continues. We seek to increase our understanding of EPN by adding phenotypes defined by
mRNA regulation in an exclusively pediatric cohort of patients, and in doing so unlock new
routes for therapeutic intervention.
Materials & Methods. We performed expression and miRNA profiling of 65 cases of
pediatric infratentorial EPN collected from a single clinical protocol (TCH n = 16 and Children’s
Oncology Group n = 49), and 16 normal brain controls using the Affymetrix U133 Plus 2.0 and
ABI TaqMan platforms (367 miRNAs), as well as copy number profiling using the Affymetrix
SNP array on a subset of 54 samples. Subjects ranged between 1 day & 18 years of age, with
51% under the age of four. This patient cohort was consistent with the disease epidemiology,
with 38% anaplastic cases (WHO grade III). All but 5 received postoperative radiotherapy, and
75% of initial surgeries were complete resections.
Results. Examination of the two current infratentorial subtypes PFA and PFB, as defined
by a literature-based gene expression signature, revealed no prognostic differences (PFS log
rank p = 0.890). However, supervised mRNA-based profiling based on age at diagnosis
demonstrated further within-phenotype heterogeneity dominated by differential homeobox
(HOX) gene expression. Utilizing this signature we were able to separate all PFB cases with
genomic instability and better outcome from PFB cases with young age, poor prognosis, and
absent expression of the anterior HOX genes, which are involved in embryogenesis and have
been associated with several pediatric brain tumors. Furthermore, miRNA within HOX genomic
loci, which regulate the HOX gene group, also show low expression in this latter subtype,
whereas the homeobox homologue gene EN2 and co-regulated gene CNPY1, markers of early
midbrain-hindbrain development, are strongly overexpressed. The elucidation of this novel
phenotype, designated PFBHOX-, restores prognostic value to the existing subgroup
designations (PFS log rank p = 0.0133).
Conclusions. Examination of existing infratentorial phenotypes in our all-pediatric
samples did not appear prognostic, but reproducible mRNA-based subtypes within the known
phenotypes were discovered. These subtypes, defined by an absence of anterior HOX genes
expression and an overexpression of EN2 and CNPY1, revealed strong differences in clinical
outcome. We intend to validate these markers in an independent set of EPNs while further
exploring their biological implications.
Contributors:Taylor, Aaron; Burstein, Matthew D.; Shen, Jianhe; Chow, Thomas; Su,
Jack; Adesina, Adekunle; Dauser, Robert; Whitehead, William; Jea, Andrew; Curry, Daniel;
Chintagumpala, Murali; Lau, Ching C.
Burak Tepe
Program in Developmental Biology
Advisor: Benjamin Arenkiel, Ph.D.-Department of Molecular & Human Genetics
The mammalian brain has a remarkable capacity for continued neurogenesis
throughout life. Every day, thousands of adult-born neurons are generated in the
subventricular zone and migrate anteriorly along the rostral migratory stream where
they eventually populate the granule cell layer of the olfactory bulb (OB). During this
process, however, only fifty percent of adult-born neurons will survive long-term to
populate the OB circuitry. This dramatic reduction suggests that certain selection
events act on these neurons to determine which ones will be integrated. To elucidate
how survival dynamics modulate adult-born neuron maturation, plasticity, and
integration in the brain, we hypothesize that the balance between the death rate of
resident granule cells (GC), and survival rate of adult-born neurons, determines the
integration efficiency of adult born neurons.
To determine if integration of adult-born neurons depends on the loss of resident
neurons, we propose to increase neuronal cell death in the OB and determine the
effects on long-term survivability and circuit integration of incoming adult-born neurons.
To do so, we genetically targeted resident OB interneurons for toxin-mediated cellspecific ablation. Upon death of resident OB neurons, we injected animals with the
proliferation marker EdU to follow a population of adult-born neurons and investigate
their integration probability and spatial distribution. Preliminary data suggest that the
loss of resident GCs increases the integration probability of newly integrating neurons.
To examine the increased survival of resident and adult born GCs, we generated a
novel mouse strain which we have targeted the ROSA26 locus for insertion of a Cre
recombinase-dependent expression construct that contains viral apoptosis inhibitor p35.
By injecting these mice with Cre-expressing virus at different time points of adult-born
neuron development, we propose to increase the survival of these neurons and observe
their integration efficiency. Similarly, we will target resident OB neurons for inhibition of
apoptosis by targeting them with a Cre-expressing virus while assessing effects on
adult-born neuron integration.
Contributors: Tepe, Burak; Arenkiel, Benjamin R.
María Elisa Terrón-Díaz
Integrative Program in Molecular and Biomedical Sciences
Advisor: Olivier Lichtarge, M.D./Ph.D.-Department of Molecular & Human Genetics
One-third of all available medications target G-Protein Coupled Receptors
(GPCR). Therefore, specificity towards ligands and downstream effectors is important
for understanding protein function and the development of selective therapeutic drugs
with fewer side effects. The serotonin (5-HT2AR) and D2 dopamine (D2R) receptors are
both part of the bioamine subfamily of GPCRs and have similar sequence identity,
however, they discriminate stringently in their cellular responses between endogenous
agonists. Using Difference Evolutionary Trace (ET) analysis, functional determinants
were previously identified and rationally targeted by swapping residues from 5-HT2AR
into D2R. This swap altered D2R ligand potency, affinity, and specificity. Beyond the
ligand bias, we aim to utilize these sixteen D2R mutants to determine the effector bias
in respect to the β-arrestin2 pathway, involved in receptor desensitization and
resensitization, using a live cell-based assay known as Bioluminescent Resonance
Energy Transfer2 (BRET2). The BRET2 assay is being used to detect the proximity
between D2R-RlucII and beta-arrestin2-GFP10 at less than a distance of 100Å. Five
plasmids for the BRET assay have been generated: GFP10 fused to RLucII (positive
control), GFP10 (negative control), RLucII (negative control), D2R-RLucII (donor), and
beta-arretin2-GFP10 (acceptor). Plasmid titrations are currently being tested and sitedirected mutagenesis is being performed on D2R-RLucII to generate the 16 pointmutations. We hypothesize that one or more of the 16 D2R mutants that where
previously shown to have ligand bias will also have beta-arrestin2 bias. The betaarrestin2 bias will be detected in the BRET assay as an increased GFP10/RLucII signal
in the mutant D2R after agonist treatment as compared to wild type. This project will
give light as to how allosteric regulation of D2R can influence effector bias aiding in the
understanding of pathway selectivity.
Austen Lee Terwilliger
Integrative Program in Molecular and Biomedical Sciences
Advisor: Anthony Maresso, Ph.D.-Department of Molecular Virology & Microbiology
Bacterial infections are sustained by acquiring essential nutrients (e.g. carbon,
nitrogen, phosphorous, sulfur, and metals) from the host to support cellular metabolism
and replication. Necessary for bacterial survival, numerous studies focus on iron and its
acquisition systems within blood borne pathogens, many of which target the oxygen
carrier protein hemoglobin as an iron reservoir. However amino acid acquisition
systems, supplying carbon, nitrogen and sulfur, remain uncharacterized and
understudied as therapeutic targets. Blood, the likely source of nutrients during
bacteremia, contains free amino acids as well as a multitude of plasma proteins that
could fulfill a bacterium’s nutritional needs. Employing a novel defined medium to mimic
human blood serum and Bacillus anthracis as a model pathogen, we determined that
hemoglobin and other serum proteins can serve as an amino acid source during nutrient
depravation. This effect is independent of the iron-bound heme in hemoglobin, and
genetic analysis reveals the immune inhibitor metalloprotease, InhA1, as the main
proteolytic contributor. These studies highlight the emergence of amino acid acquisition
as a novel point for therapeutic intervention and challenge a previous model, which
views hemoglobin solely as an iron source for bacterial pathogens during infection.
Contributors: Austen Terwilliger, Anthony Maresso
Gengwen Tian
Department of Pathology & Immunology
Advisor: Leonid Metelitsa, M.D./Ph.D.-Department of Pediatrics
CD1d-restricted type-I Natural Killer T cells (NKTs) have been shown to mediate
antitumor responses in mouse models and are associated with improved outcome in
several types of cancer. However, the therapeutic application of NKTs has been limited
by low numbers and functional defects of these cells in patients with cancer. To provide
a means for safe and effective ex-vivo NKT-cell expansion for cell and gene therapy
applications we explored native and engineered properties of K562 cells to function as
artificial antigen-presenting cells (aAPC). Although clinical grade K562-based aAPC
products have been shown to be effective for T- and NK-cell expansion for therapeutic
use in patients, K562 cells express HLA-Cw3, an NK-cell activating ligand which
stimulates expansion of NK and potentially alloreactive T cells that causes competition
with NKTs in culture and poses a clinical hazard. Because HLA-C is the only HLA allele
in K562, we rendered K562 cells HLAnull by eliminating HLA-C gene from K562 cell
genome using a HLA-C-specific zinc finger nuclease. We then transduced parental and
HLAnull K562 cells with CD1d cDNA followed by single cell sorting and clonal
expansion. The clones were pulsed with αGalactosylceramide and tested as aAPC for
NKTs using CFSE proliferation assay. We found that in contrast to K562/CD1d,
HLAnullK562/CD1d clones selectively expanded NKTs when added to primary PBMC,
and clones with an intermediate level of CD1d expression induced the highest rate of
NKT-cell proliferation. Next, a selected HLAnullCD1dmed clone was further modified to
express CD86 alone or in combination with 4-1BBL and/or OX40L followed by single
cell sorting and clonal expansion. The comparison of the APC function allowed us to
select a clone with the phenotype HLAnullCD1dmedCD86highOX40Lmed4-1BBmed (B8-2) which consistently induced the highest rate of NKT-cell expansion. B-8-2 cells were
able to efficiently expand both primary NKTs as well as NKTs modified to express a
chimeric antigen receptor (CAR) specific to CD19 antigen, a clinically validated
therapeutic target for B-cell malignancies. CAR expression in NKTs rendered them
highly cytotoxic against CD19+ Raji leukemia cells while preserving their CD1drestricted specificity. Importantly, CAR-NKTs expanded with B-8-2 cells exhibited a Th1skewed cytokine profile and had potent therapeutic activity in Raji leukemia model.
Therefor, the engineered K562 B-8-2 can be used as highly efficient aAPC for ex vivo
propagation of primary human as well as gene modified NKTs amenale for adoptive cell
therapy application for human patients. Contributors: Gengwen Tian1, Bipulendu Jena2, Daofeng
Liu1, Andras Heczey1, Hiroki Torikai2, Dean Lee2, Laurence Cooper2, and Leonid Metelitsa11Texas
Children's Cancer Center, Center for Cell and Gene Therapy, Departments of Pediatrics, Pathology and
Immunology, Baylor College of Medicine, Houston, TX, 77030 2Cell Therapy, Children's Cancer Hospital,
Department of Immunology, MD Anderson Cancer Center, Houston, TX, 77030
Lin Tian
Department of Biochemistry & Molecular Biology
Advisor: Xiang Zhang, Ph.D.-Department of Molecular & Cellular Biology
Tumor-associated vasculatures are usually considered to be abnormal and lack
the support of pericytes. High percentage of pericyte coverage is considered to be the
good prognosis marker for some cancers such as colorectal cancer and breast cancer.
However, the field lacks the information on whether the adaptive immune system is
involved in tumor-associated vascular normalization. We utilized a p53-null murine
mammary tumor model to determine the role of adaptive immune cells in vascular
normalization. The p53-null tumor pieces were inoculated into the mammary gland of
immunocompetent (Balb/c) mice and immunodeficient (nude) mice. We found that
pericyte coverage in tumors of Balb/c mice was significantly higher than that of nude
mice. To directly test the function of T lymphocytes in vascular normalization, we
reconstituted T lymphocytes in tumor bearing nude mice, and found that pericyte
coverage of tumor-associated vasculatures increased significantly in nude mice with T
cell reconstitution. This study indicates that adaptive immune cells function to normalize
tumor-associated vasculature by increasing pericyte coverage, which might prevent
cancer cells entering into circulation and further decrease metastasis frequency.
Contributors: None
Baouyen Tran
Department of Neuroscience
Advisor: Edward Cooper, M.D./Ph.D.-Department of Neurology
KCNQ2/3 voltage-gated potassium channels underlie the M-current (IM)
regulating neuronal excitability. About 150 mutations in KCNQ2 have been published,
about half leading to mild neonatal-onset epilepsy (Benign Familial Neonatal Seizures)
and half leading to severe epileptic encephalopathy. High concentrations of functional
KCNQ2/3 channels are found at the axon initial segment (AIS) where they exert control
over action potential generation. Experimental determination of the pathophysiological
mechanisms in severe KCNQ2 encephalopathy is important for guiding therapy in this
disorder. Candidate mechanisms include alterations in voltage-dependent gating,
trafficking, or stability. We used immunofluorescence and confocal imaging mice to
determine localization of KCNQ2/3 in both the cortex and hippocampus of transgenic
hKCNQ2-G279S, and in transfected cultured rat hippocampal neurons. We used
surface biotinylation assays in CHO cells transfected with mutant KCNQ2 in order to
determine whether or not localization of the KCNQ2/3 channels are altered or
abnormally degraded. Immunofluorescence microscopy performed on tissue sections
from transgenic mice over-expressing the dominant negative mutant G279S revealed a
strikingly aberrant pattern: KCNQ2 was completely absent from the AIS and was
retained at numerous intracellular puncta in the soma and dendrites. KCNQ3 weakly
labeled the AIS, and was partially redistributed to these puncta. Voltage-gated sodium
channel concentration at the AIS was normal. In addition, cultured hippocampal neurons
electroporated with mutant KCNQ2 at the pore show absence or abnormal pattern of
KCNQ2 labeling at the AIS at DIV7. However, surface biotinylation assays in CHO cells
show no changes in mutant KCNQ2’s ability to be detected at the surface. Some
KCNQ2 mutations may act by preventing trafficking to the AIS, leading to an imbalance
in sodium and potassium channel activity, excessive action potential initiation, and
cellular hyperexcitability. KCNQ2 pore mutations appear to fall into this category. Since
such effects that may not be easily revealed through heterologous expression, further
development of neuronal expression and in vivo models is warranted.
Contributors: Tran, Baouyen; Li, Li; Xu, Mingxuan; Cooper, Edward
Linda Lien Tran
Integrative Program in Molecular and Biomedical Sciences
Advisor: David Rowley, Ph.D.-Department of Molecular & Cellular Biology
In prostate cancer, reactive stroma is observed to co-evolve with disease and be
indicative of rate of progression, rate to biochemical recurrence after prostatectomy, and
mortality. Previous xenograft studies in our lab involving the recombination of
carcinoma and stromal cells have shown that reactive stroma is tumor promoting.
Myofibroblasts are a key component of this microenvironment, yet little is known about
the mechanisms guiding their expansion differentiation.
p53 is a transcription factor that has been widely studied for its role in the
regulation of DNA damage response. Modifications to this protein can determine
whether a cell is fated to apoptosis or cell cycle arrest. The Cooney lab and
collaborators have implicated p53 in the differentiation potential of mouse embryo
fibroblasts (MEFs). p53-deficient MEFs spontaneously differentiated into several
different cell types.
RUNX1 is a transcription factor critical in normal hematopoiesis. Recent work
performed in the Rowley lab has identified RUNX1 as a key transcription factor in TGFeta1-mediated activation of human prostate mesenchymal stem cells (hpMSCs) to
myofibroblasts. These data indicate a role for RUNX1 in stem cell biology and
While p53 status appears to be important for differentiation commitment and
resistance to plasticity, RUNX1 seems to be essential for transit amplification of MSCs
and maintenance of their plasticity. RUNX1 is known to complex with p53 and affect
transcriptional activity of downstream target genes. Therefore RUNX1 and p53 may be
coordinating MSC expansion and myofibroblast differentiation. We hypothesize that as
RUNX1 levels decrease with myodifferentiation, p53 levels/activity will increase,
signaling the commitment of these cells to a specific lineage and removing them from
the multipotent pool.
To determine the correlation of p53 levels with myodifferentiation, hpMSCs were
treated with TGF-beta1 and RNA isolated at 6, 12, 24, and 48 hours. To investigate
whether RUNX1 regulates p53 expression, RUNX1 was knocked down in hpMSCs
using siRNA and treated with TGF-beta1. RNA was isolated at 6, 12, 24, and 48 hours
and analyzed by qRT-PCR. Preliminary studies show that RUNX1 knockdown appears
to raise p53 threshold expression levels and illicit a more pronounced response to TGFbeta1 treatment. Interestingly, there was a decrease in p53 expression at 12 and 24
hours; timepoints at which reactive stroma markers TNC and ACTA2 were at their peak
expression. This data is to be corroborated with protein expression via Western blots
and IHC.
Contributors: Tran, Linda; Cooney, Austin; Rowley, David
Chang-Ru Tsai
Program in Developmental Biology
Advisor: Michael Galko, Ph.D.-Biochemistry and Molecular Biology
Efficient wound closure is critical for survival and is conserved across species.
Epidermal cell migration into the wound gap requires both soluble chemoattractants and
mechanical cues. However, it remains mysterious how these different types of
directional information are integrated by the migrating cell. Previously our lab identified
the Drosophila Vascular Endothelial Growth Factor (VEGF) ligand and receptor
(VEGFR) as wound closure genes. Surprisingly, VEGFR appears to function as both a
chemoreceptor and a mechanoreceptor during wound healing, suggesting integration of
chemical and mechanical cues may occur at the level of the same receptor. Both the
VEGF ligand and mechanical stimuli/wounding act through the downstream transcription
factor Yorkie (Yki). Yki is also required for wound closure but is typically thought to
function in the context of Hippo signaling, a growth control signaling pathway. Upon
wounding, Yki translocates from the cytoplasm into the nucleus nearly instantaneously
and even at large distances from the wound.
A major goal of my project is to figure out exactly how this spectacular
response occurs. With the power of fly genetics, I will manipulate the expression
pattern and function of both VEGF and VEGFR in the epidermis and monitor Yki
translocation upon mechanical stimulation and/or wounding. This approach will test the
roles of VEGF and VEGFR during both signal initiation and propagation. Second, to
obtain more detailed insights into the unique properties of the VEGF/VEGFR/Yki
signaling axis, an in vivo structure/function study of these factors will be performed.
Different genomic deletion constructs of VEGFR and VEGF will be engineered into
VEGFR and VEGF mutant backgrounds, respectively, and their abilities to rescue
chemoattraction and mechanotransduction will be assessed. This project is likely to
lead to fundamental insights into how cells respond to damage cues and perceive their
mechanical environment. Given that both the VEGFR and Hippo/Yki signaling
pathways are important for cancer signaling, these insights will likely be relevant not just
to wound healing but also to cancer metastasis and tissue growth regulation.
Contributors: Tsai, Chang-Ru; Michael Galko
Wei-Chih Tsai
Department of Molecular Virology & Microbiology
Advisor: Richard Lloyd, Ph.D.-Department of Molecular Virology & Microbiology
Stress granule (SG) is formed when eukaryotic cells cope with different external stresses,
including virus infection. These stressors are sensed by different eIF2a kinases, and eventually shutdown
global translation initiation. In the meanwhile, RasGAP-SH3-binding protein 1 (G3BP1) will be
dephosphorylated, targeting to stalled mRNP/mRNA complexes to nucleate SG assembly.
In contrast, viruses have evolved to manipulate SG formation via digesting or sequestering
G3BP1 and other essential SG components. Our previous data has demonstrated the localization and
activity of PKR in G3BP1 induced SGs. In addition, expression of G3BP1 dampens enteroviruses titer
and stimulates NFκB reporter activity. Given the important role for SGs in innate immune response;
however, it is still unclear how G3PB1 be regulated in this process. Type I protein arginine
methyltransferases (PRMT) are the family of enzymes in eukaryotic cells that are responsible for arginine
methylation in the RGG domains of many RNA-binding proteins, including G3BP1. Herein, we
demonstrated a dramatic reduction of SG assembly in stressed PRMT3 (40%) and PRMT4 (80%)
knockout mouse embryonic fibroblast (MEFs). These data implies the importance of arginine methylation
in SG formation. Sequence analysis of G3BP1 reveals a C-terminus RGG domain where contains four
potential arginine methylation sites. Deletion of the RGG domain abolished SG assembly in stressed
G3PB1 KO MEFs and repressed NFκB reporter activity. In addition, there was an average of 40%
reduction of SGs with the 435, 443, or 447 methyl mimetic (R to F) mutants. Taken together, these data
illustrate that arginine methylations are new SG regulation signals in the RGG domain of G3PB1. That
can also modulate innate immunity by controlling NFκB activation.
Stress granules (SGs) are dynamic cytoplasmic structures, which contain aggregates of
translationally stalled messenger ribonucleoprotein (mRNP) complexes. SGs are formed in response to
environmental stressors such as heat shock, oxidative stress, amino acid deprivation, and viral infection.
Protein arginine methyltransferases (PRMTs) are the enzymes responsible for arginine methylation,
which provides a means to modulate the charge and localization of target proteins. A central component
of SGs is Ras-GTPase activating protein SH3 domain-binding protein 1 (G3BP1), which contains an
arginine-rich RGG domain in the C-terminus. It has been shown that PRMT1 can also bind in this region;
however, the role of methylation in SGs dynamics is yet to be determined.
The first aim of this study is to investigate whether G3BP1 methylation is required for SG
formation. We found three potential methylation sites (R435, R443, R447) in the RGG region of G3BP1.
In order to characterize the functional significance of these sites, I will generate methylation mimics (R to
F) and deficient mutants (R to K). These mutants will be transfected into U2OS cells and microscopy will
be used to study SG behavior under arsenate and thapsigargin stresses. It is reasonable to infer that
G3BP1 will switch its binding partners after changing its charge. The second aspect of this aim is to verify
the components of G3BP1 complex to depict the possible biological function of G3BP1 via pull down
assay and Ingenuity pathway analysis software.
The second aim of this study will be to investigate the role of methylation in SG dynamics. For
this aim, methylation deficient mouse embryonic fibroblasts will be used to ablate specific methylation
pathways. Stress granules will be induced in these cells and stress granule assembly and disassembly
kinetics will be monitored by immunofluorescence microscopy. Additionally, in vitro and in vivo
methyltransferase assays using PRMTs found to be important in the above SG dynamics studies will be
employed with G3BP1 as a substrate. These studies will provide insight into whether R435, R443 and
R447 are modified by PRMTs.
The last part of this study will focus on the relationship between innate immunity and methylation
modification on G3BP1. First, I will overexpress those mutants in either G3BP knockout or PRMT
knockout cells. Then infect cells with enterovirus to investigate viral replication rates. If methylation
effects viral replication, I will investigate cytokine expression to determine whether SG modulation of
various cytokines is partly responsible for the observed changes in virus replication.
Contributors: Wei-Chih Tsai, Lucas C. Reineke, Jon Dougherty, Richard E. Lloyd
Yen-Kuei Tu
Integrative Program in Molecular and Biomedical Sciences
Advisor: Kimberley Tolias, Ph.D.-Department of Neuroscience
Synapses are specialized sites that mediate communication between neurons.
Most excitatory synapses in the brain reside on actin-rich structures called dendritic
spines. The formation, regulation and maintenance of excitatory synapses are crucial
for normal cognitive function.
Recently, we have identified the adhesion G protein-coupled receptor (GPCR)
brain-specific angiogenesis inhibitor 1 (BAI1) as a key regulator of synapse
development. We show that BAI1 is highly localized to spines. Furthermore, knockdown
of BAI1 results in decreased spine and synapse density in both cultured hippocampal
neurons and cortical neurons from intact mouse brains. Synaptic loss caused by BAI1
knockdown can be rescued by full-length BAI1, but not by a BAI1 truncation mutant,
which fails to interact with the Par3/Tiam1 polarity complex. Tiam1 is a Rac1-guanine
nucleotide exchange factor (GEF) that promotes spine and synapse development by
inducing Rac1-dependent actin remodeling. Tiam1 is restricted to spines by the polarity
protein Par3, enabling for spatial control of Rac1 activation. We show that BAI1
regulates spine and synapse development by recruiting the Par3/Tiam1 complex to
spines, resulting in localized Rac activation and actin polymerization. Although these
findings elucidate how BAI1 signals inward to promote post-synaptic development, it is
unclear whether BAI1 also signals across the synapse to induce pre-synaptic
differentiation. Utilizing a COS7 cell-neuron co-culture system, we show that BAI1
increases pre-synaptic termini formation on the axons of cultured hippocampal neurons
that contact BAI1-expressing COS7 cells. These results indicate that BAI1 can induce
pre-synaptic as well as post-synaptic development. Our future investigations are twofold: (1) We have also shown that BAI1 interacts with important ligands that regulate
synaptic development such as Neuroligin-1 and the complement factor C1ql3.
Investigating the importance of N-terminal domains that interact with these ligands will
further elucidate mechanisms for BAI1's regulation of synaptogenesis. (2) Given that the
two other BAI family members are also highly expressed in the brain, affect neuron
morphology, and have been linked to neurological disease, we are investigating their
role as regulators of spine and synapse development. Results from our study should
help to elucidate the mechanisms that regulate excitatory synapse development, and
provide potential therapeutic targets for the treatment of neurological disease.
Contributors: Tu, Yen-Kuei; Duman, Joseph; Tolias, Kimberley
Hui-Ying Tung
Department of Pathology & Immunology
Advisor: David Corry, M.D.-Department of Medicine
The immune basis of allergic versus non-allergic diseases differ vastly, however,
both utilize the immune pattern like recognition receptor- Toll like receptor (TLR) 4.
While the canonical TLR4 ligand, lipopolysaccharide or LPS, induces innate and
adaptive responses characterized by T helper (Th) 1 type of immune response; our
laboratory has recently shown that during allergic airway infections, LPS-free fibrinogen
cleavage product (FCP) triggers a Th2 type immune response in a TLR4-dependent
manner. We hypothesize that TLR4 exhibit biased agonism in response to different
ligands, such as LPS and FCP, and aim to determine the fibrinogen-derived TLR4
ligand that drives allergic responses and the distinct signaling mechanisms activated by
FCP through TLR4.
Column fractionation of fibrinogen hydrolysates, in addition with coomassie blue
staining analysis and NFkB seap reporter assay, yielded two large size fractions that
are potentially the TLR4 ligand; one of which had the molecular size similar to that of Ddimer, a known FCP that has been implicated in many diseases. Reverse phase protein
array (RPPA) of LPS vs FCP treated RAW 264.7 cells demonstrated a distinctive
signaling pathway induced by FCP in comparison to that triggered by LPS. Western blot
analyses further showed distinctive kinetic differences between LPS and FCP in
activating various NFkB subunits and phosphorylation of Mitogen-activated protein
kinases (MAPKs) such as ERK 1/2 and p38. Moreover, as FCP can induce both mucin
gene expression and airway eosinophilia, both of which are induced by IL-13 through
STAT6, our western blot results based on RAW 264.7 cells and bone marrow derived
macrophages (BMDMs) showed that STAT6 was clearly induced by FCP, but not LPS.
These results indicate that LPS and FCP, both ligands for TLR4, activate NF-kB
and MAPKs through distinct signaling pathways and that activation of STAT6 by FCP
but not by LPS further support our hypothesis. Collectively, TLR4 appears to exhibit
biased agonism for controlling immune responses elicit by different ligands.
Contributors: Tung, Hui-Ying; Landers, Cameron; Yuping, Qian; Luz, Roberts; Corry, David.
Berrak Ugur
Program in Developmental Biology
Advisor: Hugo Bellen, Ph.D./D.V.M.-Department of Molecular & Human Genetics
Mitochondria are highly dynamic organelles that are responsible for energy
production, steroid biosynthesis, Ca+2 homeostasis, cell differentiation and cell cycle
progression. Defects encompassing any of the mitochondrial metabolic pathways cause
mitochondrial diseases and a breath of different symptoms in humans including
myopathies and gastrointestinal dysfunction. The mitochondrial proteome contains
approximately 1200 nuclear encoded genes and the vast majority of these genes
remain uncharacterized. Hence, studying mitochondrial function in model organisms will
provide important insights into the complex etiology and phenotypic heterogeneity of
different disorders. We performed an unbiased, forward genetic screen for essential
genes on the Drosophila X-chromosome that cause late developmental defects and
neurodegeneration (X-screen). We isolated 165 genes, 100 of which were previously
uncharacterized. Interestingly, 32/165 genes function in mitochondria and are highly
conserved in humans. Mutations in 45% of these mitochondrial genes have already
been reported to be causal in human disease. The remaining 55% are likely to be novel
disease or lethality causing genes. We decided to perform a detailed phenotypic
analysis of these 32 mutants to assess mitochondrial morphology, reactive oxygen
species (ROS) levels, mitochondrial potential, lipid droplets (LD) in larval muscles, and
morphology and function of homozygous mutant clones in the retina. All the Xmitochondrial mutants, except those that are cell lethal, cause neuronal defects in
photoreceptors. We therefore rationalized that a detailed phenotypic analysis of the
mutants will allow us to define specific pathways by which mitochondria cause neuronal
demise. We isolated multiple phenotypic groups including two novel genes that encode
proteins that may localize to mitochondria but have not yet been associated with them.
For example, we observed that the loss of the fly homolog of human RFT1 (CG3149)
causes excessive LD accumulation and increased ROS levels in the larval muscles. In
parallel, 3 other X-mitochondrial mutants display increased ROS and muscle LD
accumulation and hence define a phenotypic group. We are finishing the phenotypic
analysis and grouping of X-mitochondrial mutants and we aim to investigate the
molecular mechanisms that underlie these phenotypes.
Contributors: Jaiswal, Manish; Sandoval, Hector; Haelterman, Nele A.; Yamamoto, Shinya;
Bellen, Hugo J.
Kevin Ung
Program in Developmental Biology
Advisor: Benjamin Arenkiel, Ph.D.-Department of Molecular & Human Genetics
Adult neurogenesis in the mammalian brain represents an extraordinary example
of continued cellular and structural neuronal plasticity. Although much has been learned
about the general phenomenology associated with adult-born neuron development, the
molecular and genetic mechanisms that guide adult-born neuron synapse formation,
synapse maintenance, and circuit integration are not well understood. Previous studies
show that multiple cellular interactions and diverse forms of presynaptic input affect the
proliferation, survival, and synapse formation of adult-born neurons. However, the
nature of how these presynaptic inputs and/or cellular interactions translate to the adultborn neuron circuit development remains unknown.
We have recently combined Rabies Virus (RV)-mediated transsynaptic circuit
tracing and mouse genetics to elucidate cell types that provide presynaptic inputs to
adult-born neurons. Interestingly, in addition to diverse neuron-neuron interactions, we
revealed numerous direct contacts between adult-born neurons and local resident
astrocytes. We have also found that targeted activity manipulations of astrocytes
directly influence proper adult-born neuron development and function.
Astrocytes have been implicated in many aspects of neuronal development,
ranging from trophic support to neuromodulation and plasticity. Moreover, defective
astrocytes have been associated with several conditions, such as Alzheimer’s disease,
schizophrenia, epilepsy, and tumorigenesis. Taking into account the known functional
roles of astrocytes along with our preliminary data, we are currently testing the
hypothesis that: specific local astrocyte populations play essential roles in adult-born
neuron synapse formation and circuit integration in the olfactory bulb.
Elucidating the molecular and cellular interactions between neurons and
astrocytes that influence postnatal synaptogenesis and circuit integration will enhance
our understanding of circuit formation in the developing, aging, and diseased brain.
Uncovering novel functional roles for astrocytes toward adult-born neurons may not only
better inform us of normal neural development and brain function, but also help explain
how defects in astrocytes may contribute to neurodegenerative diseases.
Yasmin1 Margarita Vasquez
Department of Molecular & Cellular Biology
Advisor: Francesco Demayo, Ph.D.-Department of Molecular & Cellular Biology
The nuclear receptor subfamily 5, group A, member 1 (Nr5a1), is a transcription
factor expressed at high levels in the gonads where it promotes the expression of key
steroidogenic enzymes. In the endometrium, the promoter of Nr5a1, is heavily
methylated and thus the gene is silenced. However, in women with endometriosis, a
chronic gynecological disease characterized by the growth of endometrial tissue outside
the uterine cavity, this epigenetic regulation is lost and the gene is aberrantly
expressed. The presence of endometriosis causes inflammation and severe pain, and it
is associated with infertility lacking any other identified causes. We hypothesize that
high expression of Nr5a1 in endometrial tissues results in the local production of
estrogen and the disruption of hormone signaling. To test our hypothesis in vivo, we
developed a mouse model in which Nr5a1 was conditionally expressed in the uterus.
Nr5a1 expression resulted in the rapid development of cystic endometrial glands,
observable by 6 weeks of age. Following a 6-month breeding trial, mice presented
depleted stromal and myometrial compartments, a worsening of the cystic gland
morphology, and were infertile. Endometriosis was induced by auto-transplantation of a
2 mm punch biopsy of uterine tissue sutured to the mesenteric membrane of cycling
mice at 6 weeks of age. Ectopic lesions were excised four weeks later, revealing that
those from Nr5a1 expressing mice were on average 4-fold larger than control lesions.
Thus, Nr5a1 expression conferred distinct advantages to ectopic endometrial lesions.
To determine which genes are misregulated in the endometrium in the presence of
Nr5a1, microarray analysis was conducted, demonstrating 1992 genes to be
differentially regulated. Pathway analysis identified these genes to be involved in
development (Dcc, Klf9, Notch1 and Msx1), cellular movement and morphology (Shh,
Cdkn1c, Gli2 and Wnt4) and vascular development and function (Hoxa10, Vegfa and
Vcam1). Most notably, we identified a significant increase in the expression of genes
involved in the activation of the immune response including the migration of
lymphocytes and leukocytes, indicating that aberrant activation of Nr5a1 expression not
only disrupt the hormone signaling pathways required for proper uterine morphology,
function and fertility, but it also promotes endometriotic pathogenicity by inducing
pathways responsible for a robust immune response. The present results indicate the
Nr5a1 pathology in endometriosis may not be merely due to the production of estrogen
as previously postulated, but also have a direct role in endometrial gland function and
activation of inflammatory pathways. This research was supported by R01 HD042311
and U54 HD007495 to FJD.
Contributors: Wu, San-Pin; Anderson, Matthew L.; Hawkins, Shannon M.; Creighton, Chad J.;
Tsai, Sophia Y.; Tsai, Ming-Jer; Lydon, John P. and DeMayo, Francesco J.
Hugo Villanueva
Department of Molecular & Cellular Biology
Advisor: Michael Lewis, Ph.D.-Department of Molecular & Cellular Biology
Background: Smoothened (SMO), the primary Hedgehog signaling effector, is
overexpressed in breast cancers, and constitutive activation in mouse mammary glands
leads to paracrine stimulation of proliferation. SMO can function canonically via GLI
transcription factor activation, or non-canonically as a heterotrimeric G-protein coupled
receptor (GPCR). Whether SMO functions non-canonically in mammals is not known.
Our goal is to determine whether SMO signals as a GPCR in the mammary gland.
Experimental Design and Methods: Differential gene expression analysis was
performed using RNAseq, with validation by qRT-PCR in FACS-enriched SMOoverexpressing or non-expressing cells vs. wild type cells. G i function was assessed
pharmacologically by in vivo treatment with pertussis toxin (PTX), and genetically using
G i null alleles, in the presence or absence of activated SMO. GLI transcription factor
function was assessed using a small molecule antagonist.
Results: Gene expression analysis indicated that G i1 was the only G subunit
upregulated in SMO-overexpressing mammary epithelial cells. Consistent with this
observation, in vivo PTX treatment of SMO-induced mammary gland proliferation
suggests G i function. Genetic deletion of G i2, and not G i1 or G i3, in the
mammary gland in the presence of activated SMO suggests G i2 mediates SMOinduced proliferation. Gli expression was upregulated in SMO overexpressing cells, and
treatment with a GLI antagonist is ongoing.
Conclusion: Our data support a model of SMO function as a GPCR coupling via
the PTX sensitive G i2 G-protein.
Contributors: Villanueva, Hugo; Yu, Peng; Wu, Jessie; Plummer, Nicholas; Birnbaumer, Lutz;
Hilsenbeck, Sue; Shaw, Chad; Lewis, Michael
Megan Brittany Vogt
Integrative Program in Molecular and Biomedical Sciences
Advisor: Rebecca Rico, M.P.H.-Department of Molecular Virology & Microbiology
Dengue virus (DENV) is the most prevalent cause of viral hemorrhagic fever
worldwide, with 500,000 cases of dengue hemorrhagic fever (DHF) and 50,000
DENV-related deaths occurring each year. Mechanisms of DENV pathogenesis are not
well understood, primarily because this virus only causes disease in humans; however,
DHF is often correlated with high blood DENV titers and infection with specific DENV
strains. All DENV strains possess a single stranded positive sense RNA genome with a
single 3’ untranslated region (3’UTR). In order to replicate the genome during viral
replication, the host cell translation machinery must be utilized to translate the viral
polymerase off of the viral genome. The genomes of DENV strains commonly
associated with severe disease have distinctive putative RNA folding patterns, notably
pseudoknots, in the 3’UTR that are thought to recruit the translation machinery and the
viral polymerase to the viral RNA. Thus, we hypothesize that RNA folding patterns in
the 3’UTR improve replication of DENV, ultimately increasing disease severity. To
determine the impact of 3’UTR folding patterns on DENV genome replication, chimeric
viruses will be created in which the 3’UTR of a more severe strain replaces the 3’UTR of
a less severe strain and vice versa. Genome replication and production of infectious
virus in vitro will be assessed upon infection of these chimeras into human dendritic
cells, the only cell culture system that mimics natural infection. The impact of 3’UTR
folding patterns on DENV genome translation will be assessed in vitro via luciferase
assay with reporter constructs engineered with the 3’UTRs of specific DENV strains. If
the 3’UTR structure does impact DENV genome replication and translation, then
chimeras and reporter constructs containing the 3’UTR of more severe DENV strains
should produce more infectious virus and have higher luciferase activity than those
containing the 3’UTR of less severe DENV strains. The effects of these 3’UTRs on
DENV pathogenesis will be further assessed in vivo using a humanized mouse model
that develops dengue disease and mimics natural DENV infection in humans. Selected
chimeras will then be evaluated for production of dengue disease in these humanized
mice (NOD/SCID/IL-2r gamma null mice engrafted with human stem cells) by evaluating
signs of disease, such as fever, viremia (virus titers in blood), thrombocytopenia
(platelet loss), and rash. This study could improve our knowledge of DENV replication
and help measure viral determinants of pathogenesis.
Contributors: Vogt, Megan; Rico-Hesse, Rebecca
Zer Vue
Program in Developmental Biology
Advisor: Richard Behringer, -Molecular Genetics - M.D. Anderson
Endometrial glands secrete substances (or histotrophs) that are essential for
endometrial receptivity to the embryo, implantation, conceptus survival, development
and growth. During pregnancy, in response to increasing demands of the developing
conceptus for histotrophs, endometrial glands undergo extensive hyperplasia and
hypertrophy. In models where uterine glands have been phenotypically knocked out
either through the use of genetics (mouse) or hormones (sheep), the females are
infertile due to defects in implantation and early pregnancy loss, which suggests that
uterine glands are essential for fertility. Uterine adenogenesis is the formation of glands
within the stroma of the uterus of mammalian females. Like other tissues, uterine
glands develop from fundamental processes such as tubulogenesis and branching
morphogenesis. Uterine gland development is a unique postnatal process that occurs
between Postnatal Day [P] 5 and P12 in the mouse. During P5, the luminal epithelium
(LE) of the uterus will invaginate into and form epithelial buds. By P12, we have found
that these initial buds extend into the adjacent stroma as single, essentially unbranched
tubes. We are currently using various methods to examine later stages of
adenogenesis. Uterine glands are considered to be highly coiled and branched, except
for rodents. However, many of these conclusions were based on two-dimensional
histological analysis of the uterus and limited data exists on the three-dimensional
shape of a uterine gland.
Contributors: Vue, Zer; Stewart, C. Allison; Gonzalez, Gabriel; Behringer, Richard
Edgar Yasuhiro Walker
Department of Neuroscience/M.D.-Ph.D. Program
Advisor: Andreas Tolias, Ph.D.-Department of Neuroscience
How the visual system resolves ambiguity is a fundamental problem in vision
science. A classic example is the aperture problem, in which a moving grating is viewed
through an aperture. Although the stimulus is consistent with many motion directions, it
typically produces a reliable motion direction percept. However, since very few studies
have explored the effect of the shape of the aperture and of its orientation relative to the
grating on perceived motion direction, the brain’s strategy for resolving ambiguity in this
problem is not fully understood. We conducted an experiment in which subjects
reported the perceived motion direction of a grating moving behind an elliptical or
rectangular aperture with a variable aspect ratio and variable relative orientation. We
found strong effects of relative orientation, aspect ratio, and shape on the perceived
motion direction. These effects could not be captured by previous models – one based
on a prior favoring low speeds, and one based on line terminators. Instead, we reframed
the observer’s decision process as Bayesian inference on the motion direction of an
infinitely long patterned strip with fixed but unknown width viewed through the aperture.
In the model, the observer a) computes for each candidate motion direction the speed
and minimum strip width consistent with the scene, b) assigns posterior probability
using both the low-speed prior and a prior we propose here, which favors narrower
strips, and c) reports the posterior mean. The resulting model not only outperformed the
other two models, but also captured the observed dependencies with high accuracy.
One potential interpretation of the narrow-strip prior is as a “little-unseen stuff” prior,
favoring scenes that require the fewest assumptions about unobserved regions of the
scene. Perhaps our brain resolves ambiguity by performing a process analogous to
model selection, where simpler models are favored over complex ones.
Contributors: Walker, Edgar; Ma, Wei Ji
Chih-Chuan Wang
Integrative Program in Molecular and Biomedical Sciences
Advisor: Matthew Rasband, Ph.D.-Department of Neuroscience
Myelination and the formation of nodes of Ranvier have been proposed to be
an evolutionarily critical advance allowing saltatory and rapid propagation of action
potentials in axons. The saltatory conduction of action potential depends on the nodal
clustering of Na+ and K+ channels. It has been proposed that AnkyrinG is of critical
importance in the clustering of Na+ and K+ channels. AnkyrinG interacts with NF-186,
IV-spectrin, Na+ channels and K+ channels. NF-186 is the axonal receptor of glialderived extracellular matrix (ECM) and mediates ECM-based clustering of ion channels.
IV-spectrin is the scaffold protein bridging nodal protein complex, including Na+ and
K+ channels, to the actin cytoskeleton. However, we found that both AnkyrinG and IVspectrin are dispensable for nodal clustering of Na+ channels. In the AnkyrinG
conditional knockout mice and IV-spectrin mutant mice, the nodal clustering of Na+
channels is still intact. We also found that the nodal clustering of Na+ channels is
compensated by AnkyrinR and I-spectrin. Although it has been shown that Na+ and
K+ channels share conserved domains that interact with Ankyrin, our results indicated
that AnkyrinG and IV-spectrin are required for nodal clustering K+ channels. In
contrast to Na+ channels, the nodal clustering of K+ channels cannot be compensated
by AnkyrinR and I-spectrin in the AnkyrinG conditional knockout mice and IVspectrin mutant mice.
In summary, we found that AnkyrinG and IV-spectrin are dispensable for
nodal clustering of Na+ channels. Their function in nodal clustering of Na+ channels can
be compensated by AnkyrinR and I-spectrin. However, the nodal clustering of K+
channels cannot be compensated by AnkyrinR and I-spectrin in the AnkyrinG
conditional knockout mice and IV-spectrin mutant mice. There are different affinities of
the AnkyrinR/ I-spectrin complex for Na+ and K+ channels.
Contributors: Wang, Chih-Chuan; Ho, Tammy Szu-Yu; Rasband, Matthew N.
Feng Wang
Department of Molecular & Human Genetics
Advisor: Rui Chen, Ph.D.-Department of Molecular & Human Genetics
Retinitis pigmentosa (RP) is a genetically heterogeneous disease with over 60
causative genes known to date. Nevertheless, approximately 40% of RP cases remain
genetically unsolved, suggesting many novel disease-causing genes are yet to be
identified. Here, we identified a glucose metabolism-related kinase gene as a novel
causative gene for autosomal dominant RP (adRP). This is the first report that
associates the glucose metabolic pathway with human retinal degenerative disease,
suggesting a potential new disease mechanism. A large adRP family with negative
result from known-retinal-disease-gene screening was recruited. Linkage analysis
identified a minimal disease region of 8 Mb with a peak parametric LOD score of 3.500.
Further whole-exome sequencing identified a heterozygous missense mutation that
segregated with the disease phenotype in the family. Screening over 200 unsolved RP
patients identified two unrelated simplex RP cases carrying an identical mutation which
is only 11 amino acids away from the initial mutation identified in the adRP family,
suggesting that both alleles may impact the same functional domain. Biochemical
assays showed that the mutation does not affect kinase enzymatic activity or the protein
stability, suggesting that the mutation may impact other uncharacterized function or
result in a gain-of-function.
Contributors: Feng Wang1,2*, Yandong Wang3*, Bin Zhang1,2*, Huajin Li4, Li Zhao1,5, Keqing
Wang1,2, Mingchu Xu1,2, Yumei Li1,2, Frances Wu6, Cindy Wen6, Paul S. Bernstein7, Hui
Wang1,2, Ruifang Sui4#, Kang Zhang6,8#, Rui Chen1,2,5,9,10#
Gao T. Wang
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Suzanne Leal, Ph.D.-Department of Molecular & Human Genetics
Traditionally, linkage analysis was used to map Mendelian diseases and genes
within the linked regions were sequenced to identify the causal variants. Recent
advances in next generation sequencing (NGS) make it possible to directly sequence
genomes and exomes of individuals with Mendelian diseases and identify causal
mutations by filtering variants in an affected individual(s) family member(s), removing
those variants with higher allele frequency, e.g. >0.1% in variant databases. Linkage
analysis of SNP data are sometimes used in conjunction with NGS to increase the
success of identifying the causal variant. With the reduction in cost of NGS, DNA
samples from entire families can be sequenced and linkage analysis can be performed
directly using NGS data. Inspired by “burden” tests which are used for complex trait rare
variant association studies, we developed the collapsed haplotype pattern (CHP)
method to generate markers from sequence data for linkage analysis. To demonstrate
the power of the CHP method compared to analyzing individual variants, we analyzed
and performed empirical power calculations using the allelic architecture for several
known non-syndromic hearing loss genes, i.e. GJB2, SLC26A4, MYO7A & MYH6.
Power analysis demonstrated that the CHP method is substantially more powerful than
analyzing individual SNVs in the presence of inter-allelic familial heterogeneity, i.e.
families have different pathological variants within a gene or intra-familial heterogeneity
e.g. compound heterozygotes. Specifically for an autosomal recessive model with allelic
heterogeneity and locus heterogeneity of 50%, it requires 12 families for the CHP
method to achieve a power of 90% for the SLC26A4 gene, while analyzing individual
SNVs requires >50 families to achieve the same power at a genome-wide significance
level of α=0.05. Unlike the commonly practiced filtering approaches used for NGS data,
the CHP method provides statistical evidence of the involvement of a gene in Mendelian
disease etiology. Additionally because it incorporates inheritance information and
penetrance models it is less likely than filtering to exclude causal variants in the
presents of phenocopies and/or reduced penetrance. We recommend the use of the
CHP method in parallel to filtering methods to take full advantage of the power of NGS
in families. The CHP method is incorporated in the SEQLinkage software which is freely
Contributors: Gao Wang, Di Zhang, Biao Li, Hang Dai, Suzanne M. Leal
Li Wang
Department of Molecular & Human Genetics
Advisor: Huda Zoghbi, M.D.-Department of Pediatrics
SHANK3 haploinsufficiency causes Phelan-McDermid Syndrome whereas
SHANK3 duplications lead to manic-like behavior and epilepsy, suggesting a proper
dosage of SHANK3 is critical for normal brain function. Currently, however, treatment
approaches for normalizing SHANK3 abundance are lacking. Post-translational
modifications like phosphorylation are common mechanisms for controlling protein level.
We explored Shank3 phosphorylation pattern and identified 16 phosphorylated
residues, suggesting a phospho-dependent regulation. We hypothesize that kinase
pathways regulate the stability and abundance of Shank3, and that targeting these
pathways could restore SHANK3 level and ameliorate corresponding Shankopathies.
To identify such kinase pathways, I made a cell line to stably express a DsRedIRES-EGFP-Shank3 construct. The ratio of EGFP to DsRed serves as a read-out for
Shank3 levels controlling for transgene transcription efficiency. Using this cell line, I
performed a kinome-wide RNA interference (RNAi) screen with each of the 638 kinases
targeted by 3 siRNAs independently. The RNAi hits with the greatest change in
EGFP/DsRed ratio are considered positive. The screen revealed that multiple
components of ERK1/2 pathway are involved in SHANK3 regulation. To validate this
pathway, I treated mouse primary cortical neurons with two highly selective MEK1/2
inhibitors and detect a significant increase in Shank3 level. By q-RT-PCR, I found that
this increase is not dependent on higher transcription of Shank3, suggesting
translational or post-translational modifications. Considering that kinases in ERK1/2
pathway may directly phosphorylate SHANK3 and determine its stability, I performed a
bimolecular fluorescence complementation (BiFC) assay to detect transient interactions
between Shank3 and kinases in ERK1/2 pathway as well as other hits coming out of my
screen. MEK1, Erk1 and Erk2 are all found to interact with Shank3. In parallel, immunoprecipitation followed by mass-spectrometry analysis identified ERK2 as one of the top
interactors of Shank3. Currently I am performing an in vitro kinase assay to explore
kinase-substrate interaction between SHANK3 and ERK. At the same time, I am
generating shRNA lenti-viruses to knock down Erk1/2 pathway for further validation. In
the future, I will validate other kinase hits for potential combination therapy to reduce
side effects of strong inhibition of a single kinase.
Contributors: Wang, Li; Han Kihoon; Holder Jimmy; Zoghbi, Huda
Mengyu Wang
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Ido Golding, Ph.D.-Department of Biochemistry & Molecular Biology
Gene regulation consists of a series of stochastic, single-molecule events,
resulting in substantial randomness in mRNA production between individual cells, and
even between the individual copies of the same gene within a single cell. To
characterize the stochastic kinetics of transcription in E. coli, we combine fluorescently
labelled DNA-binding proteins and single-molecule fluorescence in situ hybridization
(smFISH), to simultaneously detect a gene of interest and measure its transcriptional
activity, in individual bacteria. Our preliminary results, using the lactose promoter (Plac),
indicate that we can reliably detect the site of active transcription. They also indicate
that active transcription involves a change in the gene’s spatial position in the cell.
Under conditions of strong repression, we find that transcription is more likely to occur
close to the time of gene replication. Next, we will use mathematical modeling to map
the observed nascent and total mRNA copy-number statistics to the underlying
stochastic kinetics of transcription at a single gene locus. We will examine how this
stochastic kinetics is modulated by transcription factors that regulate gene expression,
and whether the activity of multiple copies of the same gene is correlated within the
single cell.
Contributors: Wang, Mengyu; Zhang, Jing; Sepulveda, Leonardo; Golding, Ido.
Rui Wang
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Wah Chiu, Ph.D.-Department of Biochemistry & Molecular Biology
In the United States, heart attack and stroke are two major killers, which are the
products of arterial thrombosis and thromboembolism.
Platelets, blood cellular
fragments that are key components in hemostasis, play a critical role in the thrombosis
pathophysiology. Platelets adhere to the exposed subendothelium of ruptured
atherosclerotic plaques, aggregate and thereby forming a life-threatening occlusive
thrombus that blocks blood flow and kills the surrounding cardiac or neural tissue.
Platelet activation, which is obligatory to both hemostasis and thrombosis,
catalyzes their granular contents, thereby propagating aggregation. Within this context,
it is clear that a better understanding of the structural mechanisms underlying platelet
activation will aid in the development of the appropriate interventions for thrombosis and
bleeding disorders. However currently, the structural changes associated with platelet
activation are poorly understood.
Although conventional electron microscopy reveals structural details of platelet, it
fails to uncover platelet structure in its native solution state. Cryo-electron tomography
(CET) is a cellular imaging technique that offers some great advantages over other
methods, particularly in the study of platelet structure. CET allows direct visualization of
cellular structures at molecular resolution. Importantly, CET could deconvolute platelet
structure in its native solution state without chemical embedding and fixation. CET and
time-dependent rapid vitrification methods also permits kinetic analysis of activation
according to changes in morphology. Unlike conventional methods that require
chemical pretreatment that distort structure, CET reveals platelet morphology in a native
physiological state.
Contributors: Wang, Rui; Chiu, Wah; Michael Schmid; Dong, Jing-Fei; Wensheng Sun; Khant,
Yue Wang
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Gad Shaulsky, Ph.D.-Department of Molecular & Human Genetics
Dictyostelium, commonly known as social amoeba, live as single cells with
nutrition. After nutrition is consumed, thousand of cells aggregate to form multicellular
structures and develop into fruiting bodies. In the final fruiting body, there are two types
of cells: 80% of them are spores in the sorus, and the remaining 20% become
vacuolized and make up the stalk. Only spores can germinate into cells again. TgrC1 is
an adhesion molecule that has been demonstrated essential in both cell type
differentiation and kin recognition. Knockout of tgrC1 results in a mutant defective in
both differentiation and kin recognition. Protein Kinase A (PKA) regulates every stage of
the development and its over-expression has been shown to overcome many
developmental gene defects. However, the over-expression of PKA fails to rescue
tgrC1-, which indicates that tgrC1 may represent a parallel pathway that is equally
important as the PKA pathway for Dictyostelium development. How tgrC1 regulates
differentiation and kin recognition therefore has been of great interests for scientists
studying Dictyostelium and for those studying differentiation and kin recognition. We
started to investigate the TgrC1 pathway by identifying the downstream players of
TgrC1. Through an unsaturated suppressor screen we have discovered three genes
that are likely to act downstream of TgrC1. They are stcA, stcB and stcC, stc short for
suppressor of tgrC. All mutations partially rescue the tgrC1- developmental defects. We
then investigated TgrC1’s role in differentiation and kin recognition using stcAins as a
probe. Our study revealed TgrC1 has distinctive downstream pathways for cell type
differentiation and for kin recognition. Furthermore, TgrC1 regulates stalk differentiation
in a cell-autonomous way, whereas it regulates spore differentiation in a non-cell
autonomous way. We propose that TgrC1 may function as a central regulator that
integrates and responds to different signal inputs from cell adhesion, differentiation and
kin recognition through the development of Dictyostelium.
Contributors: Wang, Yue; Shaulsky, Gad
Yumeng Wang
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Patrick Barth, Ph.D.-Department of Pharmacology
How specific protein associations regulate the function of membrane receptors
remains poorly understood. Conformational flexibility currently hinders the structure
determination of several classes of membrane receptors and associated oligomers. In
absence of monomeric protein structures and experimental information on their binding
sites, the prediction of protein-protein interactions and associated structures remains
also very challenging. To address this problem, we have developed a general method to
predict transmembrane (TM) helical protein homooligomeric structures from sequence
guided by co-evolutionary constraints. We show that a combination of sequence-based
prediction of TMH binding surfaces and stringent convergence criteria in folding
simulations allow to considerably enrich sequence covariation signals in inter-monomer
residue pairwise contacts. When applied to all 13 structurally characterized right and left
handed homodimers and 3 higher-order oligomers, the method predicts near-native
binding interface structures (Cα-RMSD ≤ 2.5 Å) and reach near-atomic accuracy (CαRMSD ≤ 1.5 Å) for 15 and 12 of these targets, respectively, using less than 3 contacts
on average. The method also successfully predicts the structure of alternative
oligomeric receptor conformational states and of combined TM and juxtamembrane
(JM) receptor domains. Blind predictions of structurally-uncharacterized FGFR and KIT
receptors provide testable hypothesis on the molecular mechanisms of diseaseassociated point TM and JM mutations and potential binding surfaces for the rational
design of novel selective inhibitors. The method sets the stage for uncovering novel
sequence/structure determinants of molecular recognition and mechanisms of signaling
in single-spanning membrane receptor homo-oligomers.
Contributors: Wang, Yumeng; Barth, Patrick
Xing Wei
Integrative Program in Molecular and Biomedical Sciences
Advisor: Li Xin, Ph.D.-Department of Molecular & Cellular Biology
Prostate cancer (PCa) is one of the most common malignancies in the United
States. Although most of the local tumors can be cured through surgery, chemo- and
radio-therapy, a high percent of cases will eventually relapse as metastatic androgenrefractory PCa which is still lethal so far. The cell-of-origin of PCa has been indicated to
be related with disease progression and potential resistance to androgen-deprivation
therapy. As the tumorigenic mutation hits in cells with different differentiated statuses
may cause diverse outcomes, the study on lineage hierarchy of prostate epithelia can
shed light upon distinct properties of these cell subpopulations and specific therapeutic
targets on the transformed cells. The long term goal of my project is to investigate the
function of CD133 as a putative molecular marker for normal adult stem cells and/or
cancer stem cells. Although CD133 together with some other cell surface molecules has
been used to identify a subpopulation of prostate basal epithelia containing the capacity
of self-renewal and differentiation, the function of this protein is still elusive in the
prostate. In addition, the indeterminacy of CD133 antibody generates a lot of
controversies in the previous stem cell marker studies. In my project, I will utilize better
approaches to investigate the role of CD133 as a stem cell marker and to elucidate the
function of CD133 in the prostate. My study shows that most of the CD133-expressing
cells in murine prostate are luminal cells, but they can also be detected in the basal and
stromal compartments. By performing lineage tracing with a CD133-CreER/R26-LSLYFP mouse model, I find that the percentage of CD133-expressing cells doesn't change
after the induction of prostate epithelia turnover. And after castration-induced prostate
involution, there is not an enrichment of CD133-expressing cells. These results indicate
that prostate stem cells are not enriched in CD133-expressing cells. By using BrdU
staining and transplantation-regeneration assay, I will determine the proliferation ability
and in vivo stem cell capacity of CD133-expressing cells. As for the function study,
CD133 knockout mice have been examined, and they have normal prostate pathology
and in vitro sphere-forming ability. By overexpressing CD133 in human prostate cancer
cell line PC-3, I find that the colony-forming and migration abilities are not affected,
indicating that the function of CD133 is not essential. In the end, my thesis work will
generate a definitive conclusion about the role of CD133 in prostate stem cell biology
and prostate cancer cell biology, and will provide a solid basis for determining whether
CD133 can be employed as a potential therapeutic target for PCa.
Contributors: Wei, Xing; Xin, Li
Margeaux Wetendorf
Integrative Program in Molecular and Biomedical Sciences
Advisor: Francesco Demayo, Ph.D.-Department of Molecular & Cellular Biology
In the nation today, millions of women suffer from the inability to reproduce. The
female uterus is a hormone-responsive organ dependent on the presence of the ovarian
steroid hormones, estrogen and progesterone, functioning via their receptors, the
estrogen receptor and progesterone receptor (Pgr). Progesterone signaling is
necessary for the initiation and continuance of a healthy pregnancy. Although
comprised of two isoforms, the Pgr-A isoform is the most abundant within the murine
uterus. During early murine pregnancy, the Pgr is expressed in the uterine epithelium on
days 2-3 and decreases in expression by day 4, the time of embryo implantation. This
decrease in epithelial Pgr expression is thought to initiate the beginning of what is
known as the “window of receptivity” or the limited time frame in which the uterus is
prepared to receive the embryo. We hypothesize that this unique Pgr expression pattern
is critical for embryo implantation and if the Pgr is expressed in the epithelium through
the window, implantation will fail to occur. To test this hypothesis, a conditional mouse
model continually expressing the Pgr-A isoform was generated utilizing a Wnt7acre
driven lox-STOP-lox system. Through continuous expression of Pgr-A within the uterine
epithelium, the mice proved to be sterile due to defects in implantation and
decidualization. The implantation defect was attributed to the decreased expression of
leukemia inhibitory factor (LIF), a cytokine absolutely necessary for embryo
implantation. Although not a direct target of the Pgr, Lif is thought to be regulated by a
variety of transcription factors as the proximal promoter consists of Nfkb, Ets, and AP-1
binding sites. Furthermore, in vitro experiments using human endometrial
adenocarcinoma cells expressing high levels of the Pgr-A isoform demonstrate that Lif
is downregulated upon treatment with progestin. Our data suggest an indirect role of
Pgr in the inhibition of Lif at the time of implantation. This mouse model and utilization of
human endometrial cells will further our understanding of Pgr function in pregnancy and
disease. This work was supported by NIH Grants: R01HD042311, 5U54HD007495 (to
FJD) and R01CA77530 (to JPL) and NURSA grant: U19DK62434 (to MJT, SYT &
Contributors: Wu, San-Pin; Lydon, John; Tsai, Sophia; Tsai, Ming-Jer; DeMayo, Francesco
Joshua James White
Department of Neuroscience
Advisor: Roy Sillitoe, Ph.D.-Department of Pathology & Immunology
Dystonia is a severe neurological disorder that can cause painful muscle
contractions. Although recent work has identified several brain regions that are altered
in dystonia, a major obstacle to understanding why and how the brain sends erroneous
signals to the muscles has been the lack of an accessible animal model. While
genetically engineered mouse models have been useful for defining the molecular
mechanisms of dystonia, none of these mice show dystonic motor behaviors. And
although spontaneous mutant models do show dystonia, they also have unrelated
pathological defects. Additionally, despite the severe dystonia that can be induced by
certain chemicals, the methods of delivery often damage brain circuits and the dystonia
that’s induced is usually transient and variable. To overcome these limitations, we
developed a novel dystonia model by using a conditional genetic approach to remove
vesicular glutamate transporter 2 (Vglut2) from olivo-cerebellar synapses, a connection
that may be central to the defects in humans and animal models with dystonia. Here, we
show using high-resolution anatomy and in vivo, awake electrophysiology that
constitutive loss of olivo-cerebellar synaptic transmission alters the patterning of
Purkinje cell dendrites, induces abnormal burst firing within the cerebellar circuit, and
causes severe muscle co-contractions that produce dystonic movements. We also used
a surgical approach that transiently inhibits the abnormal cerebellar output to overcome
the dystonic phenotype. Our new model offers a unique opportunity to determine the
circuit defects that trigger dystonia and an ideal approach for testing drug efficacy in a
model that displays obvious dystonic movements and severe brain dysfunction.
Contributors: White, Joshua J; Sillitoe, Roy V
LaTerrica Chemise Williams
Program in Translational Biology & Molecular Medicine
Advisor: Stephen Gottschalk, M.D.-Department of Pediatrics
Xiao-Tong Song, Ph.D.-Department of Pathology & Immunology
Background: T-cell immunotherapy with genetically modified T cells expressing
chimeric antigen receptors (CARs) has shown promise in preclinical models as well as
early clinical studies. However, patients with solid tumors often do not respond as well
as patients with hematological malignancies. This lack of efficacy for solid tumors is
most likely due to several factors including a) emergence of immune escape mutants,
and b) inability of tumor-specific T cells to recognize and destroy the vascular bed of
solid tumors, which is critical for their malignant growth. The aim of this project is to
generate CARs specific for tumor endothelial marker (TEM)8, and evaluate their antivasculature and anti-tumor activity in preclinical tumor models.
Methods/Results: We generated a retroviral encoding a TEM8-specific CAR
consisting of the TEM8-specific single chain variable fragment AF344, a
hinge/transmembrane domain, and a CD28.41BB.z endodomain. CD3/CD28-actiavted
T cells were transduced with RD114-pseudotyped retroviral particles to generate TEM8specific T cells and CAR expression was confirmed by FACS analysis. To evaluate the
functionality of TEM8-specific T cells we used TEM8-negative cell lines (U373, A549,
LM7, 293T) and 293T cells that were genetically modified to either express human
TEM8 (293T.hTEM8) or murine TEM8 (293T.mTEM8). TEM8-specific T cells
recognized target cells in an antigen-dependent fashion as judged by their ability to
secrete pro-inflammatory cytokines (IFN-g and IL-2) in coculture assays, and kill TEM8positive target cells. Importantly, TEM8-specific T cells readily recognized mTEM8positive target cells, which will allow us to evaluate the safety and efficacy of TEM8specific T cells in xenograft and immune competent murine tumor models.
Conclusion: We have constructed a TEM8-specific CAR and have shown that T
cells expressing this CAR recognize and kill hTEM8- or mTEM8-positive target cells.
Animal studies are in progress to determine their safety and efficacy. Targeting the
tumor vasculature with TEM8-specific T cells may improve current T-cell
immunotherapies for solid tumors.
Contributors: Williams, LaTerrica; Krebs, Simone; Karla, Mamta; Kakarla, Sunitha; Phung, Thuy;
Rowley, David; Gottschalk, Stephen
Chun-Ting Wu
Department of Neuroscience
Advisor: Daoyun Ji, Ph.D.-Department of Molecular & Cellular Biology
Previously people have shown that rats that have fear memory shows increased
freezing while observed conspecifics being shocked. My hypothesis is that during
socially induced freezing, previous fear memory is recalled, and the hippocampal
representations associated with the place of fear experience is reactivated. To test this,
we set up a task in which the rats can observe a conspecific being shocked at the place
where they had been shocked before.
In the set-up, there are two boxes separated by a transparent glass. One rat
(Observer) in non-shock box (NS) can observe the other rat (Demonstrator) in the shock
box (S) being shock. These two boxes have distinct cues so that rats can differentiate
two contexts. There were two groups of animals - Shocked Observer and Naïve
Observer. Shocked Observer was shocked in S on Day1 but Naïve Observer was not.
Both groups were in NS to observe Demonstrator being shocked in S on Day2, and the
freezing levels of two groups were measured. To make sure that the freezing in
Shocked Observer is not induced by the context of NS box, in another set of
experiments (Control), rats are shocked in S on Day1 and then freezing level was tested
on Day2 while rats are in NS but without Demonstrator in S. The preliminary result
showed that Naïve Observer didn’t have increased freezing during observation. The
freezing level of Shock Observer is low before the Demonstrator was shocked, but
increased significantly after Demonstrator was shocked, indicating that freezing in
Shock Observer is induced by the shock event of Demonstrator. For the Control rats,
the freezing level is also low. In summary, I have demonstrated that rats had increased
freezing while conspecifics were shocked in the environment where they had been
shocked before, and this response is dependent on previous fear memory. To see the
hippocampal representations during socially induced retrieval of fear memory, one rat
with 9 place cells have been recorded simultaneously. In these cells, 5 cells have place
field in S and 4 cells have place field in NS. While the rat was in NS, before
Demonstrator was shocked, place cells encoding NS were activated. However, after
shock to Demonstrator, place cells encoding S occasionally fire together in a short time
period (~200 ms). This activity co-occurred with freezing behavior. The finding supports
our hypothesis. I will confirm this by recording larger population of hippocampal neurons
and finishing the quantitative analysis.
Contributors: Wu, Chun-Ting; Ji, Daoyun
Jun Xia
Integrative Program in Molecular and Biomedical Sciences
Advisor: Susan Rosenberg, Ph.D.-Department of Molecular & Human Genetics
Two broad functional classes of cancer genes are the “gatekeepers”, and the
“genomic caretakers”. The caretakers are DNA-repair and -damage-response genes,
mutations in which increase mutation rate, which promotes cancer. The DNA damage
that caretaker pathways ameliorate results mostly from endogenous cellular sources.
We reasoned that the genes that affect endogenous DNA-damage levels are potentially
cancer genes if either mutations in, or overexpression of these genes increase
endogenous DNA damage to levels above repair capacity. This would create cells that
act like caretaker (repair-defective) mutants, without having a caretaker mutation. We
developed a high-throughput screen using E. coli that fluoresce upon DNA damage and
screened an ordered E. coli overexpression library to model cancer-causing gene
amplifications. We report a DNA-damage-control network of 214 damage-up and 24
damage-down genes that cause more or less DNA damage, respectively, when
overexpressed. We validated all positives from our screen by sensitive flow-cytometric
assay. We find that the vast majority are not traditional caretakers, indicating a new
class of genes important to DNA damage. We identified 282 human homologues of the
E.coli damage-control genes and found that these genes are significantly overrepresented in cancer-genome databases, mostly as amplifications, supporting the
expectation that their overproduction promotes cancer. Only 5% of these human
homologs are traditional caretaker genes. These data indicate that the human
homologs are new class of cancer genes. We are characterizing the reasons for
increased DNA damage in the E. coli model: (1) many of the DNA damage-control
genes, when overexpressed, show sensitivity to DNA damaging agents, implying that
excessive DNA damage overwhelms DNA repair pathways. (2) Fifteen of 15 selected
high damage-up overexpressers show increased mutation rate, validating the proposal
that excessive DNA damage leads to increased mutagenesis in E.coli. (3) We
developed high-throughput microscopy to scan for overexpressers that have increased
DNA double-strand breaks (DSBs) visualized as foci of an engineered fluorescent DSBbinding protein developed by our lab (eLife 2013). (4) We also showed increased
stalled replication forks visualized as fluorescent foci of a new protein we have
developed. (5) We also developed flow-cytometric assays to screen for overexpressers
with chromosome-segregation defects and (6) increased oxygen radicals. Our data
show the existence and functions of a new class of genes controlling genome stability:
the DNA damage control genes, and show that in human, these are a new class of
cancer gene.
Contributors: Xia, Jun; Nehring, Ralf; Bravo, Maria; Guzman, Meztili; Mei, Qian; Frisch, Ryan;
Gibson, Janet; Herman, Christophe; Miller, Kyle; Rosenberg, Susan;
Wei Xie
Integrative Program in Molecular and Biomedical Sciences
Advisor: Huda Zoghbi, M.D.-Department of Pediatrics
Proper development of the cerebellum is highly dependent on the basic helix loop
helix (bHLH) transcription factor Atoh1. Previous studies on Atoh1 have demonstrated
its requirement for both proliferation and differentiation of cerebellar granule cell
precursors (CGPs), which give rise to all granule cells of the mature cerebellum. Our
lab’s previous studies have also implicated Atoh1 in the formation of Sonic hedgehogdriven medulloblastoma, the most common solid pediatric brain tumor. How Atoh1
promotes both cell proliferation and differentiation is not known. A better understanding
of this mechanism not only expands our basic understanding of how a transcription
factor can regulate seemingly opposing functions, but will also shed light on the
mechanism of medulloblastoma formation.
We hypothesized that phosphorylation and/or different interactors of Atoh1 can
regulate its function, ultimately affecting CGP proliferation and differentiation status.
Based on evolutionary conservation, serine 193 (S193) emerged as a potential
phosphorylation site for regulation of Atoh1 function. In vitro studies in our lab
demonstrated that altering S193 phosphorylation affects Atoh1 transcriptional activity.
Further studies showed that the DNA binding properties of the S193A phosphomutants
were altered. In sum, our results pointed to a pivotal role of S193 phosphorylation on
Atoh1 function. Therefore, we generated a phosphomutant knock in mouse in order to
investigate the role of S193 phosphorylation in vivo.
Concurrently, we performed an in vivo immunoprecipitation and mass
spectrometry screen to identify novel interactors of Atoh1. Several proteins from the
screen have been further validated with two assays: bimolecular fluorescence and in
vitro immunoprecipitation. We are currently pursuing knockdown studies to identify
interactors that affect Atoh1 function.
Future studies will focus on characterizing the Atoh1 S193 phosphomutant knock
in mice and investigating the functional interaction between Atoh1 and its interactors.
Ultimately, our studies will shed light on the underlying mechanisms in which a single
transcription factor can drive seemingly opposing functions in a specific cell population.
Contributors: Xie, Wei; Klisch, Tiemo; Zoghbi, Huda
Mingchu Xu
Department of Molecular & Human Genetics
Advisor: Rui Chen, Ph.D.-Department of Molecular & Human Genetics
Familial exudative vitreoretinopathy (FEVR) is a rare genetic disease that cause
visual impairment and retinal detachment due to abnormal retinal vascularization To
date, five disease-causing genes, including NDP, FZD4, LRP5, TSPAN12 and ZNF408,
have been associated with FEVR and together account for ~50% of FEVR cases. We
collected a cohort of about 100 FEVR patients and a two-step strategy was used to
unravel the molecular etiology. First, targeted panel NGS sequencing was performed to
screen mutations in all known FEVR-causing and other retinal disease genes. Patients
who are negative during the first step are subject to whole exome sequencing (WES) as
they are likely caused by mutations in novel disease genes. By capture panel
sequencing, we have solved ~40% cases. We also identified many novel variants in
known FEVR-causing genes, especially in LRP5 (15 missense variants). For the
unsolved cases, we have enriched some candidate genes both in large families and in
sporadic cases. These genes have functional implications in tissue vascularization
based on mouse phenotypes. Their mutations also recurred in multiple cases, which
make them good candidates. They are now undergoing genetic validation and probably
some of them will be tested functionally.
Contributors: Xu, Mingchu; Salvo, Jason; Wang, Hui; Wang, Keqing; Li, Yumei; Nguyen, Duy;
Luo, Hongrong; Zhang, Kang; Chen, Rui
Xiaowei Xu
Department of Biochemistry & Molecular Biology
Advisor: Rachel Schiff, Ph.D.-Department of Medicine
Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination,
is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12week neoadjuvant trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall
pathologic complete response (pCR) rate was 27%. To investigate resistance mechanisms our
lab developed 10 HER2+ BC cell lines resistant (R) to these drugs (LR/TR/LTR). To discover
potential predictive markers/therapeutic targets to circumvent resistance, we completed
genomic profiling of the cell line panel and a subset of pre-treatment specimens from
TBCRC006. Methods: Parental (P) lines and LR/TR/LTR derivatives of 10 cell line models were
profiled with whole exome/RNA sequencing. Mutations detected in R lines but not in P lines of
the same model were identified. Single cells of the BT474AZ-LR line were cloned and
sequenced. Mutation-specific Q-PCR was designed to sensitively quantify mutations. Whole
exome sequencing (>100X) and Ion AmpliSeq of 17 TBCRC006 baseline tumor/normal pairs
were performed. Results: We found and validated the HER2 L755S mutation in the
BT474ATCC-LTR/BT474AZ-LR lines (~30% of DNA/RNA/cDNA in BT474AZ-LR), in which the
HER pathway was reactivated for resistance. Overexpression of this mutation was previously
shown to induce LR in HER2-negative BC cell lines, suggesting a role as an acquired L/LT
resistance driver in HER2+ BC. Sequencing of BT474AZ-LR single cell clones found the HER2
L755S mutation in ~30% of HER2 copies in every clone. Using mutation-specific Q-PCR, we
found statistically higher HER2 L755S levels in BT474ATCC-P/ BT474AZ-P compared to other
HER2+ BC parentals. These data suggest that this mutation exists subclonally within BT474
parentals and was selected as the more dominant population in the two resistant lines. The
HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of
BT474ATCC-LTR/AZ-LR cells (IC50: Afa 0.02µM vs. L 3 µM). Western blots confirmed HER
and downstream Akt and MAPK signaling inhibition in the LR cells by Afa. Whole exome
sequencing/Ion Ampliseq of TBCRC006 baseline found the HER2 L755S mutation in 1/17
subjects. This patient did not achieve pCR. The variant was present in 2% of the reads,
indicating a subclonal event. Conclusion: Acquired resistance in BT474 LR/LTR lines is due to
selection of HER2 L755S subclones present in parental cells. The higher HER2 L755S levels in
BT474 parentals compared with other parentals, and detection of its subclonal presence in a
pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be
needed to identify patients with potentially actionable HER family mutations in primary tumor.
Treating this patient group with an irreversible TKI like Afa may prevent resistance and improve
clinical outcome of this subset of HER2+ BC.
Contributors: Xu, Xiaowei; Nardone, Agostina; Qin, Lanfang; Hu, Huizhong; Nanda, Sarmistha;
Heiser, Laura; Wang, Nicholas; Covington, Kyle; Chen, Edward; Renwick, Alexander; Wang,
Tao; De Angelis, Carmine, Contreras, Alejandro; Gutierrez, Carolina; Fuqua, Suzanne;
Chamness, Gary; Shaw, Chad; Wheeler, David; Gray, Joe; Hilsenbeck, Susan; Rimawi,
Mothaffar; Osborne, C. Kent; Schiff, Rachel.
Yang Xu
Department of Pathology & Immunology
Advisor: Gianpietro Dotti, M.D.-Department of Medicine
The clinical efficacy of chimeric antigen receptor (CAR)-redirected T cells
remains marginal in solid tumors compared to leukemia. Several immunosuppressive
mechanisms may be responsible for this lack of efficacy including the low oxygen
tension or hypoxia that is a common feature of the solid tumor microenvironment. In
particular, hypoxia is known to severely impair proliferation and viability of human
peripheral blood T cells (PBT cells). However, it remains unknown if hypoxia uniformly
and equally suppresses all T-cell subsets, and specifically whether it affects CAR-T cells
generated for clinical use. We thus generated T-cell lines by expanding ex vivo T
lymphocytes from PBT after activation with CD3/CD28 cross-linking antibodies in the
presence of IL-2 (TEXP cells), and tested them in 1% O2 tension. In sharp contrast with
freshly isolated PBT cells, TEXP cells featured enhanced cell divisions in hypoxia vs
normoxia (proliferation index: 1.8±0.2 vs 1.3±0.1, respectively; p<0.01). In addition,
viability of activated TEXP cells was significantly higher in hypoxia (71±1%) than
normoxia (48±6%)(p<0.01). Cytotoxic function of TEXP cells redirected with a
GD2-specific 2nd generation CAR was also enhanced in hypoxia as residual
neuroblastoma cells were reduced in hypoxia (9±5%) than in normoxia (227%) (p<0.05)
in co-culture assays. Using in vivo hypoxia labeling in NSG mice, we found higher
percentages of CAR-redirected TEXP cells actively cycling within hypoxic tumor areas
(KI67+ T cells: 68±21%), than in normoxic tumor areas (KI67+ T cells: 35±11%),
suggesting that CAR- redirected TEXP cells are functionally superior in hypoxic
environment. As the great majority of TEXP cells consist of effector-memory (EM) cells,
we dissected whether their enhanced function in hypoxia reflected an endogenous
property of EM cells or was rather acquired through ex vivo cultures. We found that EM
cells directly isolated from PBT showed comparable properties to TEXP cells in hypoxia
(proliferation index: 0.36±0.13 in normoxia vs 0.94±0.05 in hypoxia; p<0.05; viability
42±6% in normoxia vs 61±7% in hypoxia; p<0.05), while freshly isolated naive and
central-memory cells were highly suppressed. Resistance to hypoxia of TEXP cells was
associated with an increased expression of hypoxia ¬adaptation genes and glycolytic
activities. HIF¬1α and downstream glycolytic genes are highly expressed in EM and
TEXP cells. Consequently, glucose uptake and lactate production were also significantly
elevated in these cells compared to PBT ±. Our findings thus suggest a
“pre¬-adaptation status” of EM and TEXP cells to hypoxia that can be further exploited
to enhance the functionality of ex-vivo generated tumor-specific T cells.
Contributors: Xu, Yang; Savoldo, Barbara; Metelitsa, Leonid; Dotti, Gianpietro
Zenghui Xue
Department of Biochemistry & Molecular Biology
Advisor: Anna Sokac, Ph.D.-Department of Biochemistry & Molecular Biology
While Myosin-2 motor activity is required for actomyosin ring contraction during
cytokinesis in some cell types, recent findings suggest that motor activity is dispensable
in other cell types, including mammalian cultured cells and budding yeast. One simple
possibility is that different cell types use different mechanisms of actomyosin contraction
during cytokinesis. Another possibility is that Myosin-2 dependent and independent
mechanisms act in the same cell type, but make different contributions to contraction. In
this work, we address the contribution of Myosin-2 motor activity during the cytokinetic
event of cellularization in early Drosophila embryos.
During cellularization, actomyosin rings contract to build the bottom of newly
forming epithelial cells. By quantitative live cell imaging, we find that ring contraction
proceeds in two morphologically and kinetically distinct phases. In Phase 1, hexagonal
rings become circular, and the contraction rate is slow (0.17 ± 0.04 μm/min, mean ±
S.E.M.). In Phase 2, rings get smaller, and ring contraction is fast (0.66 ± 0.03 μm/min,
mean ± S.E.M.). F-actin and Myosin-2 levels increase or stay constant, respectively,
during Phase 1, but both decrease during Phase 2, suggesting that the Phases are
mechanistically distinct. Indeed, using genetic mutants and drug strategies to
manipulate Myosin-2 motor activity and F-actin polymerization dynamics, we find that
contraction during Phase 1 depends on Myosin-2 motor activity, while Phase 2 does
not. Instead, Phase 2 contraction depends on F-actin depolymerization.
Taken together, our work shows that two back-to-back mechanisms drive distinct
phases of actomyosin ring contraction during cellularization. Our data supports a model
whereby Myosin-2 dependent and independent mechanisms conspire to drive ring
contraction, within the same cell type, and even during the same cytokinetic event.
Cellularization now provides a unique opportunity to compare distinct contraction
mechanisms in the same cell type, and to understand what molecular components
control the switch between Myosin-2 dependent and independent mechanisms.
Contributors: Xue, Zenghui; Sokac, Anna
Eric Yota Yang
Clinical Scientist Training Program
Advisor: Christie Ballantyne, M.D.-Department of Medicine
BACKGROUND: Increased systemic arterial stiffness has been demonstrated in
and is thought to contribute to heart failure with preserved ejection fraction (HFpEF).
Pulmonary arterial hypertension [PAH] results from vascular dysfunction through
multiple pathways, some of which may be shared with the systemic circulation. In
concert with this a similar female preponderance has been observed in patients with
HFpEF and in those with PAH. We hypothesized that a shared perturbation in both
pulmonary and systemic arterial stiffness as evaluated by carotid-femoral pulse wave
velocity [PWV] may be present in PAH patients.
METHODS: PWV and derived central pressures were measured with applanation
tonometry in sequential consenting hemodynamically confirmed PAH patients (N=25).
Exclusion criteria included hypertension, diabetes, scleroderma, liver disease, renal
disease, congenital heart disease.
RESULTS: No patient was treatment naïve though 2 were on only calcium
channel blocker therapy. 15 patients had idiopathic PAH; 4, heritable PAH; 7 PAH,
associated with connective tissue disease; and 1, anorexiant associated PAH. WHO
functional classes 2 and 3 were present in 17 and 10 patients respectively. The group
was middle-aged (median 43 [interquartile range (IQR) 34, 54] years) and mostly female
(89%) with brachial systolic and diastolic blood pressures (BP) of 105 (IQR 100, 115)
and 65 (IQR 60, 70) mm Hg, respectively. These BP were correlated (p<0.01) with
central systolic (95 [IQR 91, 102] mm Hg, tau-b = 0.60) and diastolic BP (67 [IQR 61,
72] mm Hg, tau-b = 0.93). The median PWV was 6.7 m/s (IQR 5.9, 7.7). No difference
(p=0.75) in PWV was noted between PAH patients and controls reported in literature.
CONCLUSIONS: Systemic arterial stiffness appears to be normal in patients with
pulmonary arterial hypertension although the impact of PAH specific therapy cannot be
excluded. Further evaluations of systemic arterial stiffness should be considered in
treatment naïve PAH patients.
Contributors: Yang, Eric; Nambi, Vijay; Frost, Adaani
Liubin Yang
Department of Molecular & Human Genetics/M.D.-Ph.D. Program
Advisor: Margaret Goodell, Ph.D.-Department of Pediatrics
Epigenetic modifiers including the de novo DNA methyltransferase (DNMT) 3A
are amongst the most frequently occurring recurrent mutations in hematologic
malignancies, yet the mechanism by which DNMT3Amut contributes to leukemic
transformation remains unknown. Interestingly, 41% of these mutations coexist with the
FLT3-ITD oncogene. Here, we report that Dnmt3a haploinsufficiency cooperates with
FLT3-ITD to generate acute myeloid leukemia and Dnmt3a loss generates FLT3-ITD
early immature T-cell-like leukemia, arising from transformed HSCs, by conditional
knockout murine models. We found that Dnmt3a loss results in upregulation of stem-cell
and myeloid gene signatures likely causing hypomethylation of critical distal regulatory
regions in hematopoietic stem and progenitor cells. Our results demonstrate that
Dnmt3a loss of function releases otherwise suppressed stem-cell and myeloid genes to
participate in leukemogenesis. More broadly, our findings support a role for Dnmt3a as
a guardian of distal regulatory regions important in the epigenetic control of leukemic
Contributors: Yang, Liubin; Rodriguez, Benjamin; Mayle, Allison; Luo, Min; Jeong, Mira, Curry,
Choladda; Ruau, David; Zhang, Xiaotian; Challen, Grant; Rau, Rachel; Gottgens, Berthold; Li,
Wei; Goodell, Margaret
Dimitri Yatsenko
Department of Neuroscience
Advisor: Andreas Tolias, Ph.D.-Department of Neuroscience
Ambitious projects aim to record the activity of ever larger and denser neuronal
populations in vivo. Correlations in neural activity measured in such recordings can
reveal important aspects of neural circuit organization. However, estimating and
interpreting large correlation matrices is statistically challenging. Estimation can be
improved by regularization, \ie by imposing a structure on the estimate. The amount of
improvement depends on how closely the assumed structure represents dependencies
in the data. Therefore, the selection of the most efficient correlation matrix estimator for
a given neural circuit must be determined empirically. Importantly, the identity and
structure of the most efficient estimator informs about the types of dominant
dependencies governing the system. We sought statistically efficient estimators of
neural correlation matrices in recordings from large, dense groups of cortical neurons.
Using fast 3D random-access laser scanning microscopy of calcium signals, we
recorded the activity of nearly every neuron in volumes 200 µm wide and 100 µm deep
(150–350 cells) in mouse visual cortex. We hypothesized that in these densely
sampled recordings, the correlation matrix should be best modeled as the combination
of a sparse graph of pairwise partial correlations representing interactions between the
observed neuronal pairs and a low-rank component representing common fluctuations
and external inputs. Indeed, in cross-validation tests, the covariance matrix estimator
with this structure consistently outperformed other regularized estimators. The sparse
component of the estimate defined a graph of interactions. These interactions reflected
the physical distances and orientation tuning properties of cells: The density of positive
‘excitatory’ interactions decreased rapidly with geometric distances and with differences
in orientation preference whereas negative ‘inhibitory’ interactions were less selective.
Because of its superior performance, this ‘sparse+latent’ estimator likely provides a
more physiologically relevant representation of the functional connectivity in densely
sampled recordings than the sample correlation matrix.
Contributors: Yatsenko, Dimitri; Josic, Kresimir; Ecker, Alexander; Froudarakis, Emmanouil;
Cotton, Ronald; Tolias, Andreas.
Szu-Ying Yeh
Program in Developmental Biology
Advisor: Huda Zoghbi, M.D.-Department of Pediatrics
Cellular and circuit excitability of the nervous system is essential for important
rhythmic behaviors, including breathing and heartbeats. Respiration and cardiac cycle
continue to maintain physiological homeostasis throughout life, and cardiorespiratory
coupling ensures precise and efficient gas exchange in organisms through highly
overlapping respiratory and cardiac networks within the brainstem. Prematurity and
several brain disorders are associated with deterioration of breathing and cardiac
rhythms such as apneas of prematurity (AoP), Rett syndrome, sudden infant death
syndrome (SIDS), and sudden unexpected death in epilepsy (SUDEP). Dissecting the
mechanism underlying breathing control is of central importance to understand the
basis of cardiorespiratory neural circuits and life-threatening cardiorespiratory disorders.
I focus on a recently identified ion channel NALCN, which is highly expressed in
respiratory rhythm generators, the preBötzinger complex and the retrotrapezoid
nucleus/parafacial respiratory group. NALCN is critical for maintaining respiratory
activity and sustaining life. Also, NALCN current contributes to about 72% of the basal
Na+ leak current, which is the characteristic of all pacemaker cells that generate
autonomous neuronal activity, and has been purposed to underlie neuromodulation of
neuronal bursting rhythm. Using genetic mouse models, we will delete Nalcn gene in
cells within the hindbrain to determine the NALCN-dependent neuronal population that
is essential for neonatal survival. On the other hand, we purpose that the NALCN
current govern the respiratory rhythm generation and/or modulation. Combining genetic
mouse models and electrophysiological approaches, we will assess the biophysical
properties of rhythm generators within the respiratory center. We expect that loss of
NALCN current in respiratory rhythm generators would lead to perturbations, or even
cessations in spontaneous oscillation and responses to neuromodulators, and ultimately
lethality that will illuminate the functional roles of NALCN. We will also exploit this model
to study whether the rhythm generators themselves are sufficient to drive motor
behavior and whether the circuit excitability dictates respiratory activity. Revealing the
functions of NALCN in our study will advance our understanding of the mechanism in
respiratory rhythm and pattern generation.
Contributors: Yeh, Szu-Ying; Huang, Wei-Hsiang; Zoghbi, Huda
Shuo-Ting Yen
Program in Developmental Biology
Advisor: Richard Behringer, -Molecular Genetics - M.D. Anderson
Tubulogenesis is a fundamental process in development. Several mechanisms
are already well-studied in many tubular systems, such as neural tube, kidney, as the
trachea system in fly. However, the tube formation of reproductive tract development
still remains unclear. The female reproductive tract primordium, Müllerian duct, forms in
an intriguing manner. Its formation is now defined as three phase: initiation,
invagination, and elongation. Several models of Müllerian duct formation have been
proposed but none of them agree with each other. New Methodologies should be
applied to elucidate the cellular mechanism of Müllerian duct formation. By time-lapse
imaging of Wnt7a-Cre; R26R-YFP mouse embryo, we have observed digit like cell
process and changes in diameter of the tube. These findings may provide new clues to
explain Müllerian duct formation. Furthermore, Müllerian duct formation is unique in its
dependence on Wolffian duct. Most tube formation does not depend on another existing
tube. It is known that some molecules, such as WNT9B produced by the Wolffian duct,
play a tropic role in Müllerian duct formation (Carroll et al., 2005; Grünwald, 1937).
However, the detailed mechanisms of this Wolffian-duct dependent guidance are still
needed to be elucidated.
Contributors: Yen, Shuo-Ting; Huang, Cheng-Chiu; Behringer, Richard
Michael Joseph Yetman
Department of Neuroscience
Advisor: Joanna Jankowsky, Ph.D.-Department of Neuroscience
The entorhinal cortex (EC) plays a central role in episodic learning and memory
formation, and is among the earliest sites of neuronal loss and neurofibrillary tangle
formation in Alzheimer’s disease. The EC has therefore been an attractive target for
genetic manipulation to selectively modify gene expression or neuronal function in
various models of neurological disease. Many such conditional models utilize the
neuropsin (Nop) promoter to limit spatial distribution of the tetracycline transactivator
(TTA). When crossed with a second tet-responive transgenic line, the resulting bigenic
mice will express the transgene of interest in neurons where TTA is active. The NopTTA mouse line was reported to restrict tet-responsive transgenes to the superficial
layers of medial EC and parts of the pre- and parasubiculum (Yasuda and Mayford,
Neuron, 2006), and Nop-TTA mice have been used in several experimental studies
examining functional properties of the entorhinal-hippocampal circuitry. The utility of this
transgenic driver line is contingent on the specificity of the spatially restricted gene
expression, yet detailed neuroanatomical mapping of its expression has not yet been
done. We therefore crossed the Nop-TTA driver line with a reporter strain expressing βgalactosidase and green fluorescent protein, and established an online histological atlas
of Nop-TTA regulated gene expression. This atlas resource (available through the
Rodent Brain Workbench, was used to perform a detailed brainwide
analysis of β-galactosidase labeling in bigenic (Nop-TTA-LacZ) mice. Our findings
highlight strong expression in regions beyond the EC and suggest caution in interpreting
experiments that depend on precise localization of gene products controlled by the NopTTA driver.
Contributors: Yetman, Michael; Lillehaug, Sveinung, Bjaalie, Jan, Leergaard, Trygve,
Jankowsky, Joanna
Jiani Yin
Department of Molecular & Human Genetics
Advisor: Huda Zoghbi, M.D.-Department of Pediatrics
15q13.3 microdeletion syndrome is a rare genetic disorder caused by a deletion
of a segment of chromosome 15. The deletion is commonly 1.5Mb in length and
encompasses 6 genes, but patients with small deletions, which only encompass the
CHRNA7 gene, have also been reported. The clinical phenotypes associated with this
syndrome are variable, but commonly include developmental delay/intellectual disability
and impaired social interaction. Other clinically important features include epilepsy,
impulsive behavior, aggression, and schizophrenia. A mouse model deficient of
CHRNA7 was generated and reported to be grossly normal in growth, anxiety-like
behaviors, learning and memory, as well as sensorimotor gating. However, deficits in
social interaction and repetitive behavior have not been assessed in this mouse model.
We tested heterozygous and homozygous CHRNA7 mutant mice and their wildtype
littermates for repetitive behaviors in self-grooming, holeboard exploration, and marble
burying test, and for social interaction behaviors in the three-chamber paradigm,
partition test, and social interaction video scoring. A detailed assessment of the
aforementioned behaviors will be presented and be discussed in the context of human
15q13.3 microdeletion phenotypes.
Contributors: Yin, Jiani; Schaaf, Christian
Lin-Kin Yong
Program in Translational Biology & Molecular Medicine
Advisor: Qizhi Yao, M.D./Ph.D.-Department of Surgery
William Fisher, M.D.-Department of Surgery
Pancreatic cancer (PC) is the 4th leading cause of cancer death in the US with a 5-year
survival rate of less than 6%. One characteristic feature of PC is that it has dense stroma which
makes PC cells especially difficult to respond to treatment and often develop resistance to
chemotherapeutic drugs. Hence, there is an urgent need for better understanding of PC stroma.
Two recent large-scale genomic analyses of human PC have uncovered increased copy
numbers of an axon-guidance gene SEMA3E that codes for the glycoprotein Semaphorin-3E
(Sema3E). Several recent reports have implicated Sema3E in metastasis of breast and colon
cancers. Sema3E has also been reported to have functions in inflammation and immune cell
migration. Hence, we hypothesize that Sema3E contributes to PC progression by promoting
metastasis of PC, as well as modulating immunosuppression in PC stroma.
Using qRT-PCR of matched patient tumor and adjacent normal tissue, we found that
Sema3E was significantly upregulated (>2-fold) in 46% of samples, while the receptor of
Sema3E, PlexinD1, was significantly upregulated in 33% of samples. We also performed
immuno-histochemical (IHC) staining for Sema3E and PlexinD1 expression in patient and
mouse PC (KrasG12D/-;P53R172H/-;PDX-1-Cre PC mouse model) tissue samples, and found
that, for both human and mouse tissues, Sema3E and PlexinD1 were expressed at much higher
levels in the epithelial tumor cells compared to normal acinar cells, highlighting the significance
of the roles Sema3E and PlexinD1 play in PC. We are currently in the process of generating
and characterizing Sema3E-overexpression and –knockdown PC cell lines to further understand
the precise effects Sema3E has on PC.
Given reports of Sema3E’s role in mediating inflammation in atherosclerosis, we
hypothesize that Sema3E can modulate immunosuppression in PC stroma by skewing
macrophage polarization towards the protumoral M2 subtype. Mouse bone marrow-derived
macrophages (BMDM) were first polarized to an M1 or M2 subtype via addition of LPS or IL4+IL-13, respectively, followed by addition of recombinant Sema3E to the cells, after which the
cells were harvested for analysis. qRT-PCR and WB analysis showed that the levels of iNOS, a
M1 macrophage marker, were suppressed upon adding Sema3E to M1-polarized cells. Flow
cytometry analysis revealed that addition of Sema3E to BMDM enhanced the production of a
M2 macrophage-related cytokine, IL-10. These preliminary results suggest that Sema3E can
skew polarization of macrophages towards the M2 subtype, which may point towards an
immunosuppressive role of Sema3E in the tumor stroma.
Altogether, we have shown that Sema3E is overexpressed in majority of pancreatic
cancer, and plays a role in modulating immunosuppressive stroma by skewing macrophage
polarization towards the protumoral subtype. These findings indicate that Sema3E could be an
attractive novel therapeutic target by acting on both PC tumor cells and tumor stroma.
Contributors: Yong, Lin-Kin; Li, Dali; Liang, Zhengdong; Fisher, William; Chen, Changyi Johnny;
Yao, Qizhi Cathy
Nejla Yosef
Department of Molecular Physiology & Biophysics
Advisor: Mary Dickinson, Ph.D.-Department of Molecular Physiology & Biophysics
In mouse development, the first macrophages appear in the yolk sac at
embryonic day 7.5 (E7.5), and between E8.0 and E9.5 they can be found in both extraembryonic and embryonic tissues [1]. Several studies suggested that embryonic
macrophages (EMs) play various roles during the development and remodeling of the
vascular system [2], but the mechanisms behind this diversity are not fully understood.
Relatively little is known about the phenotypic and functional capabilities of embryonic
macrophages (EMs) at the molecular level. Here, we report the establishment of a
highly efficient protocol to isolate EMs using the Csf1r-EGFP+/tg mouse reporter line
expressed within EMs. E9.5 Csf1r-EGFP+/tg transgenic embryos were enzymatically
digested to generate a single cell suspension and EMs were sorted via fluorescence
activated cell sorting (FACS) using GFP signal. Further marker analysis showed that it
is possible to sort brightly >98% purified EMs. Such a pure EM population is suitable for
a wide range of molecular and functional studies to define specialized properties of EMs
that are different from other macrophage classes
Ran You
Department of Pathology & Immunology
Advisor: Farrah Kheradmand, M.D.-Department of Medicine
Environmental pollutants and cigarette smoke are major causative factors for
lung diseases, such as COPD (chronic obstructive pulmonary diseases) and lung
cancer, which are respectively the 3rd leading cause of death and the 1st cancerrelated death, however the shared pathogenic substance by all risk factors for these
diseases is still unknown. In our study, carbon black (CB), as the component of coal
mine dust, cigarette smoke and airborne particles, is found to deposit in the lung
antigen-presenting cells (APCs) of emphysema/COPD patients. Therefore, we
hypothesized that CB-induced activation of APCs and lung inflammation promote
progression of emphysema and lung cancer.
By using mouse model with the intranasal challenge of CB, we found that CBchallenged mice developed similar immunopathological changes in emphysema
patients and mice exposed to chronic smoke, including enlarged lung volume, infiltration
of immune cells into the lungs, upregulated disease-related gene and strong Th17
responses. Moreover, CB directly activate APCs by upregulation of pro-Th17 cytokines,
IL-6 and IL-1β. Furthermore, by comparing the soluble nanoparticle polyethylene glycol
(PEG)-CB (hydrophilic) and elemental CB (hydrophobic), we found that hydrophobic CB
induced more severe inflammation and emphysema, indicating that surface features of
CB contribute to its pathogenesis.
More interestingly, CB-induced inflammation also decreases lung tumor latency.
In airway specific Pten/Smad4 deficient mice, CB promotes early lung cancer
progression concomitant with early stomach metastasis. Further on, CB directly
increases Pten-deficient bronchoepithelial cell invasion.
To figure out the underlying mechanism of how hydrophobic CB induces
emphysema and promotes cancer progression, we did RPPA analysis of CB-treated
cells and found that CB directly induced DNA double strand break and Erk signaling,
which can stimulate genomic instability, inflammation and cell invasion. APCs treated
with the inhibitors to either protein kinases sensing DNA damage or MAPK kinase were
less sensitive to CB treatment indicated by less IL-6 production.
In all, CB, as the shared pathogenic substance in environmental pollutants and
cigarette smoke, induces severe inflammation and promotes emphysema and early lung
cancer progression by direct induction of DNA damage. Potential mechanisms of CBinduced APC activation revealed in our study provide new insights into the
immunopathogenesis of lung diseases caused by environmental risk factors.
Contributors: You, Ran; Shan, Ming; Lu, Wen; Cho, Sungnam; Seryshev, Alexander; Marcano,
Daniela, Song, Lizhen; Yuan, Xiaoyi; Demayo, Francesco; Tour, James; Corry, David;
Kheradmand, Farrah
Melvin Chen Young
Department of Biochemistry & Molecular Biology
Advisor: Patrick Barth, Ph.D.-Department of Pharmacology
G protein-coupled receptors (GPCRs) commonly exhibit an inherently high level
of promiscuity in ligand and effector binding and activation that hinders accurate
understanding of their regulation and specific role in diseases. Consequently, many cell
and gene therapy strategies are limited by the inability to reprogram receptor signaling
properties without affecting endogenous cellular signaling pathways. We hypothesize
that orthogonal, highly specific, GPCR-mediated signaling complexes will allow for the
properties of specific receptor and downstream effector systems to be finely tuned
without perturbing the function of related receptors and alternative pathways.
A combination of Rosetta’s multi-state design (MSD) and docking protocols have
been used to evolve and select in-silico novel and highly specific binding interfaces
unrelated to native GPCR/G protein complexes. The MSD protocol does not allow for a
flexible backbone, limiting the available sequence space. To compensate for this
limitation, we use the docking protocol to produce a larger diversity of GPCR/G protein
binding interfaces that allows access to a substantially larger sequence space than
MSD alone.
The most promising computationally designed orthogonal GPCR/G protein
complex has been experimentally cross validated by monitoring agonist-induced specific
activation of design and WT complexes using cell-based assays. Our activation assay
shows that the change in membrane potential with the designed GPCR and designed G
protein pair compares favorably with the WT/WT pair, while both design/WT pairs have
drastically lower activation as intended by the design process. This suggests that our
methodology should greatly aid in the creation of highly specific GPCR-mediated
signaling complexes.
Contributors: Young, Melvin Chen; Chen, Kuang-Yui Michael; Barth, Patrick
Bo Yuan
Integrative Program in Molecular and Biomedical Sciences
Advisor: James Lupski, M.D./Ph.D.-Department of Molecular & Human Genetics
Cornelia de Lange syndrome (CdLS) is a genetically heterogeneous disorder
manifesting extensive phenotypic variability. To date, mutations in NIPBL, SMC1A,
SMC3, RAD21 and HDAC8, which encode subunits or regulators of the cohesin
complex, are found in about 65% of patients. Wiedemann-Steiner syndrome (WDSTS),
caused by mutations in KMT2A, shares phenotypic features with CdLS. We utilized
genomic approaches to determine additional molecular etiologies for CdLS-like
phenotypes and investigate molecular underpinnings of shared clinical features. Whole
exome sequencing (WES) of two male siblings clinically diagnosed with WDSTS
revealed a hemizygous, predicted-deleterious, missense mutation in SMC1A. Extensive
clinical evaluation and WES of a Turkish cohort of 30 patients clinically diagnosed with
CdLS revealed a de novo heterozygous nonsense KMT2A mutation in one patient
without characteristic WDSTS features. Moreover, a de novo heterozygous frameshift
mutation in SMC3 was identified in a patient with combined CdLS and WDSTS features.
Furthermore, in families from two separate world populations segregating an autosomal
recessive disorder with CdLS-like features, we identified homozygous mutations in
TAF6, which encodes a core component in a transcriptional regulation pathway. Our
findings suggest CdLS and related phenotypes may result from a “transcriptomopathy”
rather than a cohesinopathy: a conclusion supported by recent transcriptomic studies.
Contributors: Yuan, Bo; Pehlivan, Davut; Karaca, Ender; Patel, Nisha; Gambin, Tomasz;
Gonzaga-Jauregui, Claudia; Sutton, V. Reid; Yesil, Gozde; Bozdogan, Sevcan; Tos, Tulay;
Beck, Christine R.; Gu, Shen; Aslan, Huseyin; Yuregir, Ozge Ozalp; Rubeaan, Khalid; Nakeeb,
Dhekra; Alshammari, Muneera; Bayram, Yavuz; Atik, Mehmed M.; Aydin, Hatip; Geckinli, Bilge;
Seven, Mehmet; Ulucan, Hakan; Fenercioglu, Elif; Jhangiani, Shalini; Muzny, Donna M.;
Boerwinkle, Eric; Baylor-Hopkins Center for Mendelian Genomics; Tuysuz, Beyhan; Alkuraya,
Fowzan S; Gibbs, Richard A.; Lupski, James R.
Xiaoyi Yuan
Department of Pathology & Immunology
Advisor: Farrah Kheradmand, M.D.-Department of Medicine
Smoking-induced chronic obstructive pulmonary disease (COPD), which
encompasses chronic bronchitis and emphysema, is a progressive inflammatory lung
disease with no known effective treatment. Sterile inflammation induced by cigarette
smoke can activate lung antigen presenting cells (APCs) expressing CD11b/CD11c
markers that can differentiate T helper type 1 (Th1) and Th17 cells. Maturing APCs are
associated with expression of complement components because a large number of their
proteins and receptors have been detected in mice and humans. In particular whereas
most other complement factors are synthesized in the liver, the first component of the
classical complement pathway, C1q, is primarily produced by the APCs and
macrophages. Interestingly deficiency of C1q in humans and mice is associated with a
lack of immunoregulatory response that results in inflammation and autoimmunity
characteristic of systemic lupus erythematosus (SLE). Thus we hypothesized that C1q
serves a regulatory role in the inflammation and autoimmunity observed in emphysema.
We show here that C1q mRNA level is reduced in lung APCs from emphysema
patients and mice exposed to cigarette smoke, and smokers with emphysema showed
lower plasma C1q concentration, which correlates with decreased lung function.
Moreover, C1q augments the differentiation of T regulatory cells in vitro and in vivo, with
the increase of IL-10 production. Furthermore, APCs treated with C1q also exhibit
inhibitory phenotype marked by elevated CD103 expression and the ability to induce
more robust T regulatory cells differentiation in co-culture assays. Finally, relative to
vehicle control, complement C1q intranasally treated mice showed attenuated smokeinduced emphysema with reduced lung pathology measured by Micro-CT, less
inflammatory cells infiltration to the airway and antigen presenting cell (APCs) infiltration
to the lung, as well as reduction in inflammatory cytokine production. These findings
suggest a critical role for C1q in the inhibition of smoke-induced lung inflammation, and
should be further explored to develop specific new therapeutic targets for the treatment
of emphysema.
Contributors: Yuan, Xiaoyi; Kheradmand, Farrah
Yuan Yuan
Program in Structural and Computational Biology and Molecular Biophysics
Advisor: Han Liang, Ph.D.-Bio informatics and Computational Biology
Although individual pseudogenes have been implicated in tumor biology, the
biomedical significance and clinical relevance of pseudogene expression have not been
assessed in a systematic way. Here we generate pseudogene expression profiles in
2,808 patient samples of seven cancer types from The Cancer Genome Atlas RNA-seq
data using a newly developed computational pipeline. Supervised analysis reveals a
significant number of pseudogenes differentially expressed among established tumor
subtypes; and pseudogene expression alone can accurately classify the major
histological subtypes of endometrial cancer. Across cancer types, the tumor subtypes
revealed by pseudogene expression show extensive and strong concordance with the
subtypes defined by other molecular data. Strikingly, in kidney cancer, the pseudogeneexpression subtypes not only significantly correlate with patient survival, but also help
stratify patients in combination with clinical variables. Our study highlights the potential
of pseudogene expression analysis as a new paradigm for investigating cancer
mechanisms and discovering prognostic biomarkers.
Contributors: Han, Leng; Yuan, Yuan; Zheng, Siyuan; Yang, Yang; Li, Jun; Edgerton, Mary;
Diao, Lixia; Xu, Yanxun; Verhaak, Roeland; Liang, Han
Zachary A Zalewski
Department of Molecular & Human Genetics
Advisor: David Nelson, Ph.D.-Department of Molecular & Human Genetics
FXTAS is hypothesized to arise by an RNA-mediated toxic gain-of-function
mechanism, by which the rCGG sequesters RNA binding proteins (RBPs), inhibiting
their normal function. Molecular hallmarks of FXTAS include ubiquitin-positive
intranuclear inclusion bodies throughout the brain and marked dropout of cerebellar
Purkinje neurons. We have previously developed transgenic mouse models to drive
premutation-length rCGGs specifically in Purkinje neurons. Purkinje neuron-specific
promoter L7/Pcp-2 drives the expression of 90 CGG repeats fused to the cDNA of Fmr1
or EGFP. These mice have been shown to successfully model human phenotypes
including inclusion formation (in Purkinje neurons), Purkinje neuron loss, and impaired
motor coordination. CUG-Binding Protein Elav-Like Family Member 1 (CELF1, aka
CUGBP1) is an RBP that has previously been demonstrated in a Drosophila model to
be a potent modifier of the rCGG premutation. We predict that, in the mouse, CELF1
protein expression is inversely proportional to phenotypic severity – the less CELF1 is
being expressed, the more severe the phenotype and vice versa.
We have developed a novel CELF1 knockout mou