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UC San Diego Electronic Theses and Dissertations UC San Diego UC San Diego Electronic Theses and Dissertations Title Cardiac Stretch-Induced Transcriptomic Changes are Axis-Dependent Permalink https://escholarship.org/uc/item/7m04f0b0 Author Buchholz, Kyle Stephen Publication Date 2016 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Cardiac Stretch-Induced Transcriptomic Changes are Axis-Dependent A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Bioengineering by Kyle Stephen Buchholz Committee in Charge: Professor Jeffrey Omens, Chair Professor Andrew McCulloch, Co-Chair Professor Ju Chen Professor Karen Christman Professor Robert Ross Professor Alexander Zambon 2016 Copyright Kyle Stephen Buchholz, 2016 All rights reserved Signature Page The Dissertation of Kyle Stephen Buchholz is approved and it is acceptable in quality and form for publication on microfilm and electronically: Co-Chair Chair University of California, San Diego 2016 iii Dedication To my beautiful wife, Rhia. iv Table of Contents Signature Page ................................................................................................................... iii Dedication .......................................................................................................................... iv Table of Contents ................................................................................................................ v List of Figures .................................................................................................................... ix List of Tables ..................................................................................................................... xi List of Abbreviations ........................................................................................................ xii Acknowledgements .......................................................................................................... xvi Vita ................................................................................................................................ xviii Abstract of the Dissertation ............................................................................................. xix Chapter 1: Introduction ........................................................................................... 1 1.1. Mechanotransduction ...................................................................................... 1 1.1.1. Mechanosensors .................................................................................... 1 1.1.2. Stretch Signaling Pathways................................................................... 6 1.1.3. Stretch-induced Transcription Factors ................................................ 10 1.1.4. Anisotropic Stretch ............................................................................. 14 1.2. RNA Sequencing ........................................................................................... 16 1.2.1. Preparation of RNA-Seq Samples ...................................................... 16 1.2.2. Analysis of RNA-Seq Reads ............................................................... 18 1.2.3. Bioinformatics Analysis...................................................................... 25 1.3. Computational Signaling Network Models ................................................... 28 v 1.3.1. Transcriptional Regulatory Networks ................................................. 29 1.3.2. Underlying Equations in Network Models ......................................... 31 1.3.3. Logic-based Models ............................................................................ 33 1.3.4. Fuzzy Logic ........................................................................................ 35 1.3.5. Normalized-Hill Differential Equation Modeling Approach .............. 36 1.4. Aims of the Dissertation ................................................................................ 38 Chapter 2: RNA-Seq Experiment ......................................................................... 41 2.1. Introduction ................................................................................................... 41 2.2. Methods ......................................................................................................... 42 2.2.1. Micropatterning................................................................................... 42 2.2.2. Isolation and Culture ........................................................................... 43 2.2.3. Stretch and RNA Extraction ............................................................... 43 2.2.4. RNA-Seq and Reverse Transcription Polymerase Chain Reaction .... 44 2.2.5. RNA-Seq Analysis .............................................................................. 45 2.2.6. Gene Ontology and Pathway Analysis ............................................... 47 2.2.7. Immunofluorescence ........................................................................... 47 2.2.8. Sarcomere Length Measurement ........................................................ 49 2.2.9. Western Blotting ................................................................................. 51 2.3. Results ........................................................................................................... 52 2.3.1. DE Genes from RNA-Seq Data .......................................................... 52 2.3.2. Gene Ontology and Pathway Enrichment Analysis ............................ 57 2.3.3. Sarcomere Length Results .................................................................. 63 2.3.4. MAPK Signaling is Induced by Both Stretch Axes ............................ 64 vi 2.4. Discussion...................................................................................................... 66 2.5. Acknowledgements ....................................................................................... 68 Chapter 3: Mechanosignaling Network Model ..................................................... 70 3.1. Introduction ................................................................................................... 70 3.2. Methods ......................................................................................................... 70 3.2.1. Construction of Mechanosignaling Network ...................................... 70 3.2.2. Addition of Target Genes to MSN ...................................................... 71 3.2.3. Network Model Parameters ................................................................ 74 3.2.4. Analysis of Network Topology ........................................................... 76 3.2.5. Validation Literature ........................................................................... 76 3.3. Results ........................................................................................................... 79 3.3.1. Network Topology .............................................................................. 79 3.3.2. Validation of Upstream MSN ............................................................. 80 3.3.3. Activity Change of Nodes in MSN ..................................................... 83 3.4. Discussion...................................................................................................... 87 3.5. Acknowledgements ....................................................................................... 89 Chapter 4: Analysis of RNA-Seq Data with Network Model .............................. 90 4.1. Introduction ................................................................................................... 90 4.2. Methods ......................................................................................................... 90 4.2.1. Sensitivity Analysis ............................................................................ 90 4.2.2. Power Analysis ................................................................................... 91 4.3. Results ........................................................................................................... 91 4.3.1. Comparison of MSN Results with RNA-Seq Data ............................. 91 vii 4.3.2. Distribution of Targets of Each TF ..................................................... 98 4.3.3. Upstream Pathways of Each TF ........................................................ 105 4.4. Discussion.................................................................................................... 108 4.4.1. SRF and MEF2 ................................................................................. 109 4.4.2. NFkB ................................................................................................. 110 4.4.3. CREB ................................................................................................ 111 4.4.4. Potassium Channel Downregulation ................................................. 112 4.4.5. Limitations ........................................................................................ 113 4.4.6. Conclusion ........................................................................................ 113 4.5. Acknowledgements ..................................................................................... 114 Chapter 5: Summary and Conclusion ................................................................. 115 5.1. Summary of Findings .................................................................................
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