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University of Florida Thesis Or Dissertation Formatting TRANSCRIPTIONAL AND EPIGENETIC PROFILING OF THE HIPPOCAMPUS AND THE MEDIAL PREFRONTAL CORTEX: RELATIONSHIP TO AGING AND COGNITIVE DECLINE By LARA IANOV A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017 © 2017 Lara Ianov To my spouse, Okie Ianov, and my dear friends Lana McMillan and Guido Pardi ACKNOWLEDGMENTS First, I would like to thank my mentor, Dr. Thomas C. Foster, for providing me the opportunity to work on an exciting area of research, along with his trust in my skills and his time and patience to mentor me. I would also like to acknowledge my committee members, Dr. Leonid Moroz, Dr. Jorg Bungert and Dr. Martha Campbell-Thompson for their guidance and suggestions in all committee meetings. In addition, Dr. Alberto Riva, deserves special recognition for his devoted support to me with a section of the data analysis shown in this dissertation. I would also like to thank Dr. Jennifer Bizon and Dr. B. Sofia Beas for their help with the set shifting task. Furthermore, I would also like to thank the past and present Genetics and Genomics program coordinators including Dr. Wilfred Vermerris who admitted me into the program, Dr. Jorg Bungert and Dr. Connie Mulligan, as past coordinators, and the current coordinators, Dr. Maurice Swanson and Dr. Doug Soltis. I would also like to thank Dr. Patrick Concannon, for his support to the program and the Genetics Institute. Finally, I would like to thank Hope Parmeter for signing me up for all classes each semester along with all student-related requirements. Additionally, the scientific and moral support from the Foster lab family was very helpful and I am grateful to each one of you, both past and present: Dr. Ashok Kumar, Asha Rani, Dr. Michael Guidi, Dr. Linda Bean, Dr. Brittney Yegla, Costa Kyritsopoulos, Jolie Barter, Olga Tchigrinova and Semir Karic. Finally, I would like to thank Okie Ianov, Lana McMillan and Guido Pardi for their constant encouragement in this chapter of my life. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 13 CHAPTER 1 INTRODUCTION .................................................................................................... 15 Goals of Current Research ..................................................................................... 15 Aging and Age-Related Cognitive Decline .............................................................. 16 Estrogen Receptors and the Hippocampus ............................................................ 17 The Prefrontal Cortex and Cognitive Flexibility ....................................................... 21 Transcriptomic Studies in Aging and Cognition ...................................................... 23 DNA Methylation in Aging and the Prefrontal Cortex .............................................. 25 2 EPIGENETIC REGULATION OF ESTROGEN RECEPTOR ALPHA CONTRIBUTES TO AGE-RELATED DIFFERENCES IN TRANSCRIPTION ACROSS THE HIPPOCAMPAL REGIONS CA1 AND CA3 ................................... 30 Background ............................................................................................................. 30 Materials and Methods............................................................................................ 31 Animals............................................................................................................. 31 Surgery and Tissue Collection .......................................................................... 31 RNA Isolation and Reverse Transcription Quantitative Polymerase Chain Reaction ........................................................................................................ 32 Sodium Bisulfite Sequencing ............................................................................ 33 Statistical Analysis ............................................................................................ 34 Results .................................................................................................................... 35 Region and Aging Effects on ERα mRNA Expression ...................................... 35 Methylation of the ERα Promoter ..................................................................... 36 Discussion .............................................................................................................. 38 Acknowledgement of Financial Support to Chapter 2 ............................................. 43 3 TRANSCRIPTIONAL PROFILE OF AGING AND COGNITION-RELATED GENES IN THE MEDIAL PREFRONTAL CORTEX .............................................. 49 Background ............................................................................................................. 49 Materials and Methods............................................................................................ 51 5 Animals............................................................................................................. 51 Behavioral Studies ........................................................................................... 51 Set shifting operant task ............................................................................ 51 Morris water maze ..................................................................................... 55 Tissue Collection .............................................................................................. 57 RNA and Library Preparation ........................................................................... 57 Reverse Transcription Quantitative Polymerase Chain Reaction ..................... 58 Sequencing, Bioinformatics, and Statistical analysis ........................................ 58 Results .................................................................................................................... 61 Behavior ........................................................................................................... 61 Gene Expression .............................................................................................. 62 Gene expression related to aging .............................................................. 62 mPFC gene expression related to behavior ............................................... 65 Discussion .............................................................................................................. 68 Acknowledgement of Financial Support to Chapter 3 ............................................. 72 4 DNA METHYLOME OF AGING AND EXECUTIVE FUNCTION IN THE MEDIAL PREFRONTAL CORTEX ........................................................................................ 94 Background ............................................................................................................. 94 Materials and Methods............................................................................................ 95 Animals............................................................................................................. 95 Behavior and Gene Expression ........................................................................ 95 Genomic DNA Isolation, Sodium Bisulfite Conversion and Library Preparation .................................................................................................... 96 Sequencing, Bioinformatics and Statistical Analysis ........................................ 97 Results .................................................................................................................. 100 DNA Methylome Profiling at CpGs of the Aging mPFC .................................. 100 DNA Methylome Profiling at CpGs of Cognitive Flexibility in the mPFC ......... 104 Non-CG Methylation in the mPFC .................................................................. 107 Non-CG methylation and aging ................................................................ 107 Non-CG methylation and cognitive flexibility ............................................ 109 Discussion ............................................................................................................ 111 Acknowledgements to Chapter 4 .......................................................................... 115 5 CONCLUSIONS AND FUTURE DIRECTIONS .................................................... 145 General Conclusions............................................................................................. 145 Future Directions .................................................................................................. 149 APPENDIX A SUPPLEMENTARY FIGURES TO RNA-SEQ PROFILING .................................. 153 B SUPPLEMENTARY FIGURE AND TABLES TO GENOME-WIDE PROFILING OF DNA METHYLATION ...................................................................................... 155 6 C SEQUENCING DEPTH CONSIDERATIONS FOR RNA-SEQ AND WHOLE GENOME BISULFITE SEQUENCING .................................................................
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