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The Role of Citrullination and the Type III Intermediate Filaments in Retinal Gliosis John Wizeman University of Connecticut, [email protected]

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The Role of Citrullination and the Type III Intermediate Filaments in Retinal Gliosis John Wizeman University of Connecticut, Wizeman@Uchc.Edu University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 9-1-2016 The Role of Citrullination and the Type III Intermediate Filaments in Retinal Gliosis John Wizeman University of Connecticut, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Wizeman, John, "The Role of Citrullination and the Type III Intermediate Filaments in Retinal Gliosis" (2016). Doctoral Dissertations. 1295. https://opencommons.uconn.edu/dissertations/1295 The Role of Citrullination and the Type III Intermediate Filaments in Retinal Gliosis John William Wizeman, Ph.D. University of Connecticut, [2016] Abstract: Visual disorders are a massive economic and personal burden. Two leading causes of blindness in the United States, Glaucoma and Age-Related Macular Degeneration (AMD), currently have no cure [1, 2]. A major secondary issue in these diseases, often untreated, is the development and ongoing presence of scars in the eye [2, 3, 4]. While scarring is a byproduct of the wound healing process [5], this scarring in the eye can lead to blindness [6, 7]. In the retina the scarring process is a result of ongoing retinal gliosis, mediated by the structural Müller glial cells [8, 9, 10]. Two well-characterized markers of this process are the cytoskeletal Intermediate Filament (IF) proteins vimentin and the glial fibrillary acidic protein (GFAP), which become overexpressed as a stress response [11, 12]. Importantly, interfering with the soluble, non- cytoskeletal forms of these two proteins with the small molecule withaferin A (WFA) has anti-proliferative [13] and anti-migratory effects [14, 15], two cellular processes integral to scarring [16, 17, 18]. As with many other proteins, GFAP and vimentin can be regulated by a number of different post-translational modifications (PTM). One PTM which the IF proteins are subjected to is citrullination [19]. Here, I have identified citrullination as an injury-induced response in the retina. Increases in citrullination are seen as early as 1 day after injury. This citrullination occurs along GFAP and vimentin filaments. I have identified two sites on GFAP that are citrullinated after retinal injury. The enzyme responsible for this injury-induced citrullination in the retina was PAD4. The citrullinated form of GFAP was targeted initially in a retinal explant system by the pan-PAD inhibitor Cl-amidine using an intravitreal injection model. The treatment decreased the levels of GFAP protein indicating a reduction of the gliotic phenotype. These findings mimicked what was seen in a mouse model of glaucoma, the DBA/2J mouse line. In this system, changes in citrullination within the glaucomatous eyes preceded changes in GFAP protein levels. These findings identify citrullination of GFAP as a major response to i injury in the retina, mimicking the disease pathology of glaucoma. The ability to target these proteins within the retina, initially, and in the whole eye with an intravitreal injection to reduce a major gliotic marker has implications for the future development of therapeutics for treating the often undiagnosed gliosis associated with retinal disorders. ii The Role of Citrullination and the Type III Intermediate Filaments in Retinal Gliosis John William Wizeman, Ph.D. B.S., Saint Michael’s College, [2008] A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut [2016] iii Copyright by John William Wizeman, Ph.D. [2016] iv APPROVAL PAGE Doctor of Philosophy Dissertation The Role of Citrullination and the Type III Intermediate Filaments in Retinal Gliosis Presented by John William Wizeman, Ph.D. Major Advisor ___________________________________________________________________ Royce Mohan, Ph.D. Associate Advisor ___________________________________________________________________ Elisa Barbarese, Ph.D. Associate Advisor ___________________________________________________________________ Stephen J. Crocker, Ph.D. Associate Advisor ___________________________________________________________________ Betty A. Eipper, Ph.D. Associate Advisor ___________________________________________________________________ Zhao-Wen Wang, Ph.D. University of Connecticut [2016] v Acknowledgements I would first and foremost like to thank my thesis advisor, Dr. Royce Mohan, for his unending support throughout my time in his lab. I have learned an incredible amount from him. In five years, we have had discussions and debates about every aspect of life, from science and training to politics and culture and beyond. His guidance has helped me in not only honing my scientific skills but in time management and helping me to learn how to focus properly in more ways than I can recount on paper. From our first conversation at recruitment weekend to our last in the lab, it was always good to have someone whose interest in science could drive interesting and lively discussion about new ideas, and helped remind me why I wanted to undertake this process. I also want to thank Dr. Paola Bargagna- Mohan, with whom I have been fortunate to share a lab space for the past five years. Paola has always been willing to discuss scientific techniques and tips, as well as many other topics during my time in the lab. Paola and Royce have been my lab family for a long time, and their ever-present guidance and support will be missed as I move on in my career. I hope to make them proud as a scientist. I would also like to thank the members of my thesis committee who helped me in shaping a more focused project from a more broad initial interest early in graduate school. Dr. Elisa Barbarese, Dr. Stephen Crocker, Dr. Betty Eipper, and Dr. Zhao-Wen Wang were instrumental in keeping the efforts for this project on task and clear, and for that I am extremely grateful. I was also fortunate to do a rotation with Dr. Barbarese, and would also like to thank her for guidance during that time. I would also like to thank the Neuroscience Department at UConn Health, including Dr. Mains, for support throughout my time here. The office, including Jody Gridley, Anna Sagan, Diana Mikulak and Mary Nordgren have been extremely helpful in answering what I’m sure at times felt like an endless onslaught of questions. I also want to thank the Biomedical Science Ph.D. program at UConn, specifically Dr. Barbara Kream and Dr. Lynn Puddington, who were helpful during my time on the GSO. I also want to thank Stephanie Rauch and Dorothy Linhoff for their help during this time. Of course, I would like to thank my wife, Kate Ely Wizeman, for her support. For five years she has gracefully helped me through many different trials. Through sleepless nights, anxiety and fear, vi frustration, and a number of hours where I could only stare at the computer screen, she has helped keep me tethered to the world beyond graduate school. I have known her for 12 years, and during this time I have yet to meet someone more selfless, more caring, more loving, more devoted than her. She has always had more patience than anybody, and that was indescribably important to me as I finished graduate school. We have been fortunate to be married for four of the five years in this time, bought a house, got a dog, and will soon welcome our first child into the world. I cannot think of anyone I could possibly hold in higher esteem, or love more, than Kate. I am genuinely lucky to have met and married such a wonderful person. I want to thank my immediate family for their support during this time, including their understanding when I was short or irritated after long and frustrating days of experiments in the lab. My mother has been supportive through my entire life, and I think that sometimes she does not understand how important she has been to me. I can only hope the work in this thesis will have made her proud. I am also lucky to have two sisters who have been supportive over the past five years (and more). Whether through making meals, or through simple conversations, they have been invaluable throughout graduate school. I also have three nephews, Tate, Cooper, and Gordon, who I was thrilled to help welcome into the world. I met the oldest the day before I started graduate school, and celebrated his fifth birthday the day after I defended this thesis. They have been a joyful part of my life in that time. Lastly, I want to thank my father, who unfortunately passed a few short months before this thesis was completed from multiple glioblastoma. Although he was not able to see its completion, his passing reminds me daily that although sometimes our bench research may not seem to be making headline grabbing breakthroughs, everything we do is to benefit the sick, the dying, those losing hope. Every small victory at the bench is a major step in relieving that pain, frustration and fear that those facing untreatable illnesses face daily. I want to also thank my in-laws, Dr. Matthew Ely and Susan Ely, for their tremendous support and help throughout graduate school. Kate and I could not have possibly gotten through the past five years without their help, including their generosity in opening their home to us as we moved back to vii Connecticut from Boston. From watching the dog, to helping with meals, to everything else they have given us, I am extremely grateful for their positive influence on my life. I would lastly like to thank the friends who I have been fortunate to share both triumphs and troubles with during my time in graduate school. This includes many students in the graduate program, as well as the students of the neuroscience graduate program, who often share a sense of camaraderie. Specifically, Kyle Denton, Maegan Gross, and Bhavita Walia have been very good friends for the past 5 years, and I look forward to even better times beyond graduate school as we all move on in our careers.
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