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University of California, San Diego UNIVERSITY OF CALIFORNIA, SAN DIEGO The post-terminal differentiation fate of RNAs revealed by next-generation sequencing A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by Gloria Kuo Lefkowitz Committee in Charge: Professor Benjamin D. Yu, Chair Professor Richard Gallo Professor Bruce A. Hamilton Professor Miles F. Wilkinson Professor Eugene Yeo 2012 Copyright Gloria Kuo Lefkowitz, 2012 All rights reserved. The Dissertation of Gloria Kuo Lefkowitz is approved, and it is acceptable in quality and form for publication on microfilm and electronically: __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ __________________________________________________________________ Chair University of California, San Diego 2012 iii DEDICATION Ma and Ba, for your early indulgence and support. Matt and James, for choosing more practical callings. Roy, my love, for patiently sharing the ups and downs of this journey. iv EPIGRAPH It is foolish to tear one's hair in grief, as though sorrow would be made less by baldness. ~Cicero v TABLE OF CONTENTS Signature Page .............................................................................................................. iii Dedication ..................................................................................................................... iv Epigraph ........................................................................................................................ v Table of Contents ......................................................................................................... vi List of Abbreviations .................................................................................................... ix List of Figures ............................................................................................................... xi List of Tables ............................................................................................................... xii Acknowledgements .................................................................................................... xiii Vita ............................................................................................................................. xiv Abstract of the Dissertation ......................................................................................... xv Chapter 1: The study of variation and genetics 1.1 Introduction ................................................................................................. 1 1.2 Characterizing genomic variation ................................................................ 1 1.3 Linking genomic variation with expression variation ................................. 8 Chapter 2: Hair as a source of variation 2.1 The external hair ........................................................................................ 11 2.2 Evolutionary context of hair ...................................................................... 12 2.3 Hair follicle development and cycling ...................................................... 14 2.4 Structures of the hair follicle ..................................................................... 15 2.5 Genetic variations affecting hair ............................................................... 16 vi 2.6 Gene Expression in hair shafts as a phenotype ......................................... 21 Chapter 3: Hair shaft library 3.1 Introduction ............................................................................................... 25 3.2 Mouse hair shaft RNA ............................................................................... 25 3.3 Generation of a human hair RNA-Seq library ........................................... 27 3.4 RNAs of the human hair shaft ................................................................... 30 3.5 Methods ..................................................................................................... 31 Chapter 4: RNA preservation 4.1: Introduction .............................................................................................. 52 4.2: Cornification ............................................................................................. 52 4.3: DNA fragmentation occurs during cornification ...................................... 55 4.4: RNA degradation during cell death and life ............................................. 55 4.5: RNA coverage of viable cells and external hair ....................................... 57 4.6: Keratin associated proteins in the hair shaft library ................................. 58 4.7: Methods .................................................................................................... 62 Chapter 5: Persistence of RNA 5.1: Introduction .............................................................................................. 70 5.2: Scalp hair .................................................................................................. 70 5.3: Testing RNA retention of other tissues .................................................... 72 Chapter 6: Conclusions, future directions, and implications 6.1: Summary of project .................................................................................. 84 6.2: Feasibility of application in population studies ........................................ 84 vii 6.3: Personalized medical screening ................................................................ 85 6.4: Retrospective screening ............................................................................ 85 6.5: Discovery of novel transcripts or novel functions of transcripts .............. 87 6.6: Localization and developmental cues ....................................................... 88 6.7: Clues to tensile strength and other physical properties of hair ................ 89 6.8: Biological significance of RNA retention ................................................ 91 6.9: Summary of potential future work ........................................................... 92 viii LIST OF ABBREVIATIONS CNV ............................................................................................. copy number variation DMSO ................................................................................................ dimethyl sulfoxide DNA ............................................................................................ deoxyribonucleic acid EDA ....................................................................................................... ectodysplasin-A EDAR ...................................................................................... ectodysplasin-A receptor eQTL ........................................................................... expression quantitiative trait loci FGF ............................................................................................ fibroblast growth factor FGFR ........................................................................... fibroblast growth factor receptor GWAS ......................................................................... genome wide association studies HF ................................................................................................................. hair follicle HS ..................................................................................................................... hair shaft INPP5B ........................................... Type II inositol-1,4,5-trisphosphate 5-phosphatase MALAT-1 ................................ metastasis associated lung adenocarcinoma transcript 1 NEAT1 ............................................ noncoding nuclear-enriched abundant transcript 1 NHEK ................................................................. normal human epidermal keratinocyte Krt ......................................................................................................................... keratin KRTAP ................................................................................... keratin associated protein MapK ........................................................................... mitogen activated protein kinase miRNA .......................................................................................................... microRNA OCRL ............................................................... oculocerebrorenal syndrome of Lowe 1 qRT-PCR ............................................ quantitative real time polymerase chain reaction ix QTL ............................................................................................ quantitiative trait locus Ras ................................................................................................................ rat sarcoma RNA ....................................................................................................... ribonucleic acid SNP .............................................................................. single nucleotide polymorphism TCHH ........................................................................................................... trichohyalin x LIST OF FIGURES Figure 2.1: Hair follicle ............................................................................................... 23 Figure 2.2: Signaling pathways affecting hair morphology .......................................
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