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HOOVER-THESIS-2016.Pdf (6.708Mb) Characterization of non-coding RNAs in regulating thymic epithelial cell responses to pathophysiological stress APPROVED BY SUPERVISORY COMMITTEE Nicolai S.C. van Oers, PhD. Lora V. Hooper, PhD. Ondine B. Cleaver, PhD. Joshua T. Mendell, MD. PhD. Acknowledgments First, I would like to thank my mentor Nicolai van Oers. You have helped me grow so much as a student and as a scientist throughout my graduate career. No matter the situation you were there to support, guide, and allow me to make my own conclusions/decisions. Your mentorship and guidance has helped me become a more successful and independent scientist, and for that I am truly grateful. Dr. Ondine Cleaver and the members of your laboratory. I would have never been able to do any of the embryo analysis, in situ hybridization, or immunohistochemistry without the help and guidance from you and your lab. Your help and supervision allowed me explore a new realm of science that I had never thought about before. Thank you for being like a second mentor to me Ondine. I would also like to thank my committee members, Dr. Lora Hooper, Dr. Joshua Mendell, Dr. Ondine Cleaver, and Dr, Felix Yarovinsky for suggestions and advice throughout my thesis. To past and present van Oers’ lab members. Thank you for all of the help and laughs that come along with working together over the years. Dr. Igor Dozmorov and Dr. Maite de la Morena, you both have been there through the majority of my graduate career. Thank you for all that you have done for me. Lastly, I would like to thank my family. You all have been an endless pillar of support and helped me push forward through the ups and downs of graduate school. To my husband Daniel, thank you for your continuous support and encouragement. The road to a PhD. would have been much more difficult without you. Characterization of non-coding RNAs in regulating thymic epithelial cell responses to pathophysiological stress by ASHLEY RENAE HOOVER DISSERTATION / THESIS Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas, Texas August, 2016 Copyright by Ashley Renae Hoover, 2016 All Rights Reserved Characterization of non-coding RNAs in regulating thymic epithelial cell responses to pathophysiological stress Publication No. Ashley Renae Hoover, PhD. The University of Texas Southwestern Medical Center at Dallas, 2016 Supervising Professor: Nicolai S.C. van Oers The thymus is uniquely sensitive to several forms of stress. Stress initiates a transient involution that can reduce overall thymic volume up to 90%. The thymus is predominantly composed of developing thymocytes and specialized epithelial cells. The type of stress predicates whether the thymocytes or epithelial cells initiate the involutionary response. MicroRNAs (miRs) are small non-coding RNAs ~18-22 nucleotides in length that maintain cellular homeostasis and regulate stress responses. Previous work in the laboratory identified several microRNAs involved in regulating thymocyte responses to stress. Thymocytes have been the main v population studied in response to stress. However, it has become increasingly clear that the epithelial cells play a critical role in thymus involution and the subsequent recovery of thymopoiesis. The work presented in this thesis characterizes an epithelial specific miR, miR-205, and its surrounding long noncoding RNA, MIR205.001 in regulating TEC functions. A deficiency of miR-205 specifically in TECs renders these cells more susceptible to stress mediated thymic involution. This is revealed by a significant loss in developing thymocytes, altered migration, delayed recovery of single positive thymocyte selection, and proliferative defects in cortical TECs. Gene expression comparisons revealed miR-205 deficient TECs had reduced levels of the TEC master transcriptional regulator, Foxn1, as well as the expression of multiple chemokines. MiR-205 mimics introduced into miR-205 deficient fetal thymic organ cultures were able to restore the levels of Foxn1 and selected chemokines. This work demonstrates that miR-205 positively regulates Foxn1 and chemokine expression following stress. MiR-205 resides within a putative long noncoding RNA (lncRNA), MIR205.001. TECs deficient in this lncRNA are also sensitive to stress, but do not experience a delay in thymopoiesis nor cortical TEC proliferation defects. This suggests these noncoding RNAs have non-overlapping functions within the TECs. Mice deficient in MIR205.001 also have distinct phenotypes, displaying reduced mendelian ratios indicative of a lethality. The surviving animals display reduced body vi size, weight, and fat mass. Current experiments are addressing whether this is a metabolic defect or due to changes in feeding behavior. vii TABLE OF CONTENTS ABSTRACT ............................................................................................................... v LIST OF PUBLICATIONS ........................................................................................ xii LIST OF FIGURES ................................................................................................. xiv LIST OF TABLES .................................................................................................. xviii LIST OF ABBREVIATIONS .................................................................................... xx CHAPTER 1: INTRODUCTION ............................................................................... 1 The functional role of the thymus ........................................................................ 1 The thymus is an extremely stress responsive organ ......................................... 6 Pathophysiological induced thymus involution .............................................. 8 Physiological induction of thymus involution ............................................... 11 Hypotheses associated with transient and age associated thymic involution ... 14 Hypotheses associated with acute thymus involution ................................. 14 Theories and hypotheses of age associated thymic involution .................... 17 MicroRNAs and Functions ................................................................................ 22 MicroRNAs regulating TEC responses to stress and homeostasis ............. 23 Conclusions ...................................................................................................... 27 CHAPTER 2: MATERIALS AND METHODS ........................................................ 38 Mice .................................................................................................................. 38 Conditional knockout of MIR205.001 ................................................................ 39 viii Thymic epithelial cell isolation and purification ................................................. 41 RNA isolation and analysis ............................................................................... 42 Fetal thymic organ culture ................................................................................ 43 Immunofluorescene and immunohistochemistry ............................................... 43 Induction of stress responses ........................................................................... 44 Microarray analysis ........................................................................................... 44 Patient samples ................................................................................................ 45 Single cell suspension of lymphocytes and flow cytometry .............................. 46 Castration and 5-DHT treatment of male mice ................................................. 46 Statistical analysis ............................................................................................ 46 Whole mount in situ hybridizations ................................................................... 47 Section in situ hybridizations ............................................................................ 48 Riboprobe synthesis ......................................................................................... 50 EchoMRI ........................................................................................................... 50 Serum Chemistries ........................................................................................... 51 CHAPTER 3: MICRORNA-205 MAINTAINS T CELL DEVELOPMENT FOLLOWING STRESS BYREGULATING FOXN1 AND SELECTED CHEMOKINES....................................................................................................... 58 INTRODUCTION .............................................................................................. 58 RESULTS ......................................................................................................... 63 MiR-205 positively regulates thymic cellularity following stress ................... 63 ix MiR-205 maintains Foxn1 levels in TECs.................................................... 68 MiR-205 regulates several pathways involved in cell-cell communication and TEC maintenance ................................................................................ 70 Male mice lacking miR-205 in TECs develop a thymic hypoplasia .............. 73 DISCUSSION ................................................................................................... 74 CHAPTER 4: A LONG NONCODING RNA,
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