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Understanding the Alternative Splicing-Regulated Transcriptomic Changes University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 10-6-2015 Understanding the Alternative Splicing-regulated Transcriptomic Changes During Mouse Retinal Development and Disease Through Global and Gene-centric Approaches Devi Krishna Priya Karunakaran University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Karunakaran, Devi Krishna Priya, "Understanding the Alternative Splicing-regulated Transcriptomic Changes During Mouse Retinal Development and Disease Through Global and Gene-centric Approaches" (2015). Doctoral Dissertations. 930. https://opencommons.uconn.edu/dissertations/930 ABSTRACT Understanding the Alternative Splicing-regulated Transcriptomic Changes During Mouse Retinal Development and Disease Through Global and Gene-centric Approaches Devi Krishna Priya Karunakaran, Ph.D University of Connecticut, 2015 Alternative splicing (AS) is an important layer of gene regulation and has been shown to control various cellular processes including splicing, mRNA export, translation and cell cycle. Mis-regulation in AS has been implicated in many diseases. But, the role of AS in the retinal development and diseases remains unexplored. To this end, I employed a two- pronged approach, (i.e), gene-centric approach and an en-mass transcriptome analysis approach to address this question. For the first approach, I studied the role of an alternative splicing factor, Sfrs10 and a kinase, Citron Kinase (CitK) and its spliced isoforms in murine retinal development and diseases. Expression analysis of Sfrs10 in mouse and human retinae showed that unlike mouse, it was not expressed in normal human retina but was observed only in AMD retina, suggesting a specific role in response to oxidative stress. In parallel, I showed that the loss of CitK affected the cell division of a subset of retinal progenitor cells which in turn affected the late neurogenesis, specifically that of the Islet1+ bipolar neurons. In the second approach, global analyses were performed by employing RNA deep sequencing on cytoplasmic and nuclear fractions of developing retinal tissue. We investigated if the nuclear transcriptome would be ahead of that of the cytoplasm where it simultaneously executes the current molecular program whilst preparing for the next program i.e., de novo transcription. Also, I employed a custom bioinformatics pipeline to reverse-engineer the order in which the molecular programs are set up as the retinal tissue develops. Further, I extended the study to Nrl i gene knockout to identify the perturbation of molecular pathways in the absence of the gene. Here, our bioinformatics strategy could predict the perturbed molecular programs well before its histological manifestation. We also compared our methodology with the existing methods of data analysis and show that our pipeline could give information on transcription kinetics of genes segregated into each bin. Thus, this pipeline was employed in the temporal comparison of a triple microRNA cluster knockout and its wild type counterpart across different stages of development. ii Understanding the Alternative Splicing-regulated Transcriptomic Changes During Mouse Retinal Development and Disease Through Global and Gene-centric Approaches Devi Krishna Priya Karunakaran B.S., Madras University, 2006 M.S., Madurai Kamaraj University, 2008 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2015 iii Copyright 2015 by Devi Krishna Priya Karunakaran iv APPROVAL PAGE Doctor of Philosophy Dissertation Understanding the Alternative Splicing-regulated Transcriptomic Changes During Mouse Retinal Development and Disease Through Global and Gene-centric Approaches Presented by Devi Krishna Priya Karunakaran, B.S., M.S. Major Advisor: ______________________________________ Rahul Kanadia, Ph.D. Associate Advisor: ___________________________________ Akiko Nishiyama, Ph.D. Associate Advisor: ___________________________________ Daniel Mulkey, Ph.D. Associate Advisor: ___________________________________ Barbara Mellone, Ph.D. Associate Advisor: ___________________________________ Marie Cantino, Ph.D. University of Connecticut 2015 v Dedicated to my Grandmother! vi ACKNOWLEDGEMENTS I would like to thank God for His blessings that he has showered on me throughout these years and for helping me realize my dream. First and foremost, I would like to sincerely thank Dr. Rahul Kanadia for being a great mentor that he is to me. He not only taught me to do science but also to think like a scientist. I thank him for giving me the opportunity to work in his lab. He has always been a strong support and a great motivator. He had the trust in my capabilities when I was not sure whether I could do certain things. A constant push given by him has made me achieve what I wanted. I am very grateful to have had a mentor, who was more like a father figure. I would like to sincerely thank him for his belief and trust in me as well as for the encouragement with everlasting patience. Special thanks to those speeches that woke me up to reality as well as for those times you have made me laugh! I would like to thank my colloborators, Dr. Ion Mandoiu and Dr. Sahar Al Seesi of Computer Science and engineering department at the University of Connecticut for always being there to help me out with the bioinformatics troubleshooting and Sahar for patiently teaching me how to understand the scripts and to be independently working with bioinformatics. I would like to thank my committee members, Dr. Akiko Nishiyama, Dr. Daniel Mulkey and Dr. Barbara Mellone for their constant support and valuable suggestions during the meetings. They were instrumental in keeping me to stay focused as well as give constructive criticism in my thesis work. I would like to thank the people who made lab a fun place to be in. First, I would like to thank my past lab member, Dr. Abdul Rouf Banday, who was very helpful in teaching me bio informatics tools and giving me valuable suggestions in my project. I would also like to thank my undergraduate mentees, Christopher Lemoine, who is currently pursuing his PhD in the Kanadia lab, Nisarg Chhaya and Katery Hyatt, who were great to work with and were ready to help me anytime I needed help. I would like to thank former Research for Undergraduates (REU) students, Marybeth Baumgartner, who is currently pursuing her Ph.D in the Kanadia lab, Katherine Delaney and Anouk Olthof for helping me in my project during their time as summer research fellows. I would like to thank the undergraduates Amye Black, Whitney Washburn for briefly helping me in my project. I would like to thank Dr. Marie Cantino for teaching me Electron microscopy and helping me with my Sfrs10 project. I would also like to thank Steve Daniels, David Serwanski of Nishiyama lab for helping me with the same. I would like to thank Dr. Joanne Conover and Dr. Joseph Loturco for giving me valuable insights during our tri-lab meets. I would like to thank Dr. Xinnian Chen, Kristen Kimball, Penny Dobbins, Ed Lechowicz and Ann Hamlin for making PNB 2274 teaching a fun. I would like to thank Kathy Keheller, Linda Armstrong for helping me with the administrative side of the course, whenever I needed them. vii I would like to thank other faculty members, staff and other students of PNB for encouraging me during my journal club talks. I would like to thank the animal facility staff, especially Kevin and Teresa and other members of the facility for taking good care of my mice and helping me whenever I needed one. I would like to thank Bevan, who has been a constant support and help whenever I needed one. I would like to thank him for all his help like a brother in a country outside of my home country. I would like to thank Ashley, his fiancé and my past lab mate, who is a great fun to be with. I would like to extend my heartfelt thanks to Seethe, who was, is and will always be a great friend, sister and a great person that shared living space with me and made me feel at home away from home as well as took care of me during my times of frustration and ill-health. Thanks a ton ma! I would like to thank my friends, Raji, Priyanka, Anitha, Hems, Shankar, Shanmu and Prathiba who are in constant touch and have always been supportive and encouraging me of my work. Last but not the least, I would like to extend my heartfelt thanks to my family, without whom I would not have made it to this point in my career. I would like to thank my Paati, who is no more with me, my Appa and Amma and Matty for letting me achieve my dream of pursuing my doctorate studies in neuroscience miles away from them. My sisters and brothers-in-law are my strong pillars of support when I felt like I missed home. I would like to thank Dekka, Lavan, Ratty, Arjun, BIL and Jiju for truly making my PhD an enjoyable journey. I would also like to thank my nieces and nephews, who were my primary stress-busters. I am truly blessed to have them! My acknowledgements would be incomplete without thanking my husband, Vishnu, who put up with me and patiently waited for me to finish my studies to start our life together. He has been a great source of encouragement and support, without which I could not have managed to finish my PhD. I would also like to thank my in-laws for supporting me in this journey. Thank you! viii TABLE OF CONTENTS Acknowledgements …………………………………………………………………………..v List of Figures………………………………………………………………………………...xiii List of Tables………………………………………………………………………………….xv Chapter 1: Retinal Development .................................................................................. 1 1.1 Retina as a model system ...................................................................................... 2 1.2 Structure of the vertebrate retina ............................................................................ 3 1.3 Development of the mouse retina ........................................................................... 4 1.4 Gene regulation in the developing retina ...............................................................
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