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UC San Diego UC San Diego Electronic Theses and Dissertations UC San Diego UC San Diego Electronic Theses and Dissertations Title Serial Post-Transcriptional Splicing Establishes Negative Feedback Regulation in HIV-1 Expression / Permalink https://escholarship.org/uc/item/1rv5h1dt Author Wen, Yi Publication Date 2013 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Serial Post-Transcriptional Splicing Establishes Negative Feedback Regulation in HIV-1 Expression A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Bioinformatics and Systems Biology by Yi Wen Committee in charge: Professor Leor Weinberger, Chair Professor Douglas Richman, Co-Chair Professor John Guatelli Professor Alexander Hoffmann Professor Gurol Suel 2013 Copyright Yi Wen, 2013 All rights reserved. The Dissertation of Yi Wen is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Co-Chair Chair University of California, San Diego 2013 iii DEDICATION This dissertation is dedicated to my beloved family, my husband, and my future son. To my grandmother A-pen Yeh-Lee, who brought me up till I was 7 years old, for giving me an optimistic personality. To my father Chan-hwa Wen and mother His-ju Chen, for educating me and supporting me with their love and understanding. To my husband Panos, for always there for me when I felt discouraged with my research, and for supporting me with his love, patience, and cheerful “humor”. I also want to delicate this dissertation to my future son, who is still in my belly at the moment, for making me feel strong and invincible to all the challenges in the last couple of months of my Ph.D. life. I would like to delicate this dissertation to two other very special people in my life. First, to my junior high school teacher Chin-ling Lin, for her mentorship that continues to deeply influence my character for the rest of my life, even though I am not under her guidance any more. Last, I want to dedicate my dissertation to my deceased friend, Joanne Chen, who was once my best and closest friend. Without her, I would have had a very different life, probably a less happy one, and would not have come to UCSD for my Ph.D. iv EPIGRAPH Stop wasting your time being sorry, just do it. -----Daniele Micciancio v TABLE OF CONTENTS SIGNATURE PAGE ..................................................................................................... iii DEDICATION .............................................................................................................. iv! EPIGRAPH .................................................................................................................... v! TABLE OF CONTENTS .............................................................................................. vi! LIST OF ABBREVIATIONS ........................................................................................ x! LIST OF FIGURES ...................................................................................................... xii! LIST OF TABLES ....................................................................................................... xv! ACKNOWLEDGEMENTS ........................................................................................ xvi! VITA ......................................................................................................................... xviii! ABSTRACT OF THE DISSERTATION ................................................................... xix! CHAPTER 1: Introduction ............................................................................................. 1! CHAPTER 2: Post-Transcriptional Splicing Cascade Is Required to Generate the Rev Negative Feedback ....................................................................................................... 10! Abstract .................................................................................................................... 10! Materials and Methods ............................................................................................. 11! Cloning ......................................................................................................................... 11! Cell Culture and Cell Lines .......................................................................................... 11! Infection and Imaging .................................................................................................. 12! vi Mathematical Modeling and Computational Analysis ................................................ 13! Results and Discussion ............................................................................................. 14! HIV-1 Gene Expression Exhibits Rev Negative-Feedback Regulation in Diverse Cell Types ............................................................................................................................ 14! Negative Feedback Is Generated by Rev-Dependent Nuclear Export of Unspliced RNA ............................................................................................................................. 16! Acknowledgements .................................................................................................. 19! Appendix 2-1 ............................................................................................................ 34! Appendix 2-2 ............................................................................................................ 35! Construction of the HIV-1 Full-Length Gene Regulatory Models .............................. 35! CHAPTER 3: HIV-1 RNA is Post-transcriptionally Spliced ...................................... 40! Abstract .................................................................................................................... 40! Materials and Methods ............................................................................................. 41! Results and Discussion ............................................................................................. 43! Acknowledgements .................................................................................................. 45! Appendix 3 ............................................................................................................... 51! Probe Designs of Single Molecule mRNA Fluorescence in situ Hybridization (smFISH) ...................................................................................................................... 51! Image Processing and Data Analysis of smFISH ........................................................ 57! CHAPTER 4: Developement of SATURN (Splicing After Transfection of Unspliced pre-mRNA into Nucleus) Assay .................................................................................. 61! Abstract .................................................................................................................... 61! Materials and Methods ............................................................................................. 62! vii Cloning ......................................................................................................................... 62! In vitro Transcription ................................................................................................... 63! Transfection of RNA into the Nucleus ........................................................................ 63! BlaM Assay and Flow Cytometry ................................................................................ 63! Results and Discussion ............................................................................................. 65! Acknowledgements .................................................................................................. 67! Appendix 4 ............................................................................................................... 70! CHAPTER 5: Prediction and Characterization of Rev Negative Feedback ................ 71! Abstract .................................................................................................................... 71! Materials and Methods ............................................................................................. 72! Ordinary-Differential Equation Models and Stochastic Simulations .......................... 72! Rev Overexpression ..................................................................................................... 72! Results and Discussion ............................................................................................. 73! Acknowledgements .................................................................................................. 75! Appendix 5 ............................................................................................................... 80! Fitting and Analysis of the ODE models ..................................................................... 80! Characterization of HIV-1 Gene Regulatory Circuitry ................................................ 81! Using Stochastic Models to Estimate the Cooperativity of Rev-Dependent Nuclear Export ........................................................................................................................... 81! Prediction of HIV-1 Gene Regulatory Circuits with Rev Over-Expression ................ 84! CHAPTER 6: Discussion ............................................................................................. 92! Significance and Implications
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