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Sin, Yuan Yan (Angie) (2012) the Roles of HSP20 in Cardiac Hypertrophy

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Sin, Yuan Yan (Angie) (2012) the Roles of HSP20 in Cardiac Hypertrophy Sin, Yuan Yan (Angie) (2012) The roles of HSP20 in cardiac hypertrophy. PhD thesis http://theses.gla.ac.uk/3581/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] The roles of HSP20 in cardiac hypertrophy A thesis submitted in fulfillment of the requirements for the Degree of Doctor of Philosophy in Molecular Functions in Disease College of Medicine, Veterinary and Life Sciences, University of Glasgow, United Kingdom Yuan Yan (Angie) Sin 2012 THIS THESIS IS DEDICATED TO MY MOST LOVING PARENTS,, HUSBAND AND ONE VERY SPECIAL LITTLE GIRL,, MY DAUGHTER,, EILIDH HONG.. TABLE OF CONTENTS Abstract ………………………………………………………………………………...….i Author’s declaration ……………………………………………………………………..iii Acknowledgements ………………………………………………………………………iv List of figures …………………………………………………………………………..…v List of tables …………………………………………………………………………….vii Abbreviations ………………………………………………...…………………………viii Publications/Conferences ………………………………………………………………..ix Chapter 1: Introduction 1.1. Heat shock protein .................................................................................................. 1 1.1.1. Background ....................................................................................................... 1 1.1.2. Functions ........................................................................................................... 2 1.1.3. Synthesis of heat shock protein ......................................................................... 3 1.1.4. Families of heat shock protein .......................................................................... 3 1.1.5. Structure of heat shock protein ......................................................................... 4 1.2. Heat shock protein 20 ............................................................................................. 5 1.2.1. HSP20 phosphorylation ..................................................................................... 6 1.3. HSP20 in cardiovascular system ............................................................................ 8 1.3.1. Ischaemia/reperfusion injury .............................................................................. 8 1.3.2. Apoptosis ......................................................................................................... 10 1.3.3. Hypertrophy ..................................................................................................... 13 1.4. HSP20 in other diseases ........................................................................................ 15 1.5. Cardiac cell-based model ..................................................................................... 16 1.6. Aims of research .................................................................................................... 17 Chapter 2: Materials and methods 2.1 Materials ................................................................................................................ 18 2.2 Expression and purification of recombinant proteins ...................................... 18 2.2.1 Histidine (His) fusion protein ......................................................................... 18 2.2.2 Glutathione-S-Transferase (GST) fusion proteins ........................................... 19 2.3 Plasmid DNA ........................................................................................................ 19 2.3.1 Transformation of competent cells .................................................................. 19 2.3.2 Isolation of plasmids DNAs ............................................................................. 20 2.3.3 Storage of plasmid DNA .................................................................................. 20 2.3.4 Analysis of plasmid DNA ................................................................................ 21 2.3.5 Quantification of DNA concentration .............................................................. 21 2.4 Mammalian cell culture ....................................................................................... 21 2.4.1 Primary culture of cardiomyocytes ................................................................. 22 2.4.2 HEK293 cells ................................................................................................... 23 2.4.3 Transfection of plasmid DNA .......................................................................... 23 2.4.4 siRNA-mediated gene knockdown in cardiomyocytes .................................... 24 2.5 Preparation of cell lysates .................................................................................... 24 2.5.1 Whole cell lysate ............................................................................................. 24 2.5.2 Subcellular fractionation of cardiomyocytes ........................................... ……25 2.6 Protein analysis .................................................................................................... 26 2.6.1 SDS-PAGE ...................................................................................................... 26 2.6.2 Coomassie staining .......................................................................................... 26 2.6.3 Western immunoblotting .................................................................................. 26 2.7 Protein-protein interactions ................................................................................ 29 2.7.1 ProtoArray ....................................................................................................... 29 2.7.2 In vitro pull-down assay ................................................................................... 31 2.7.3 Co-immunoprecipitation .................................................................................. 31 2.7.4 SPOT synthesis of peptides and overlay experiments ..................................... 32 2.8 In vitro phosphorylation assays ........................................................................... 34 2.8.1 In vitro kinase assay ........................................................................................ 34 2.8.2 In vitro phosphorylation of peptide array......................................................... 34 2.9 Cell-based experiments ........................................................................................ 34 2.9.1 Manual measurements of cell size .................................................................. 34 2.9.2 Real-time xCELLigence measurements ........................................................... 35 2.9.3 Measurement of protein content....................................................................... 36 2.9.4 Disruptor peptide synthesis .............................................................................. 36 2.10 Quantitative real-time PCR analysis of fetal gene expression ......................... 37 2.10.1 RNA extraction ............................................................................................... 37 2.10.2 Reverse transcription of PCR ........................................................................... 37 2.10.3 TaqMan real-time PCR .................................................................................... 38 2.10.4 qPCR data analysis ........................................................................................... 39 2.11 Microscopic analyses ............................................................................................ 40 2.11.1 Differential interference contrast microscopy (DIC) ....................................... 40 2.11.2 Immunostaining and confocal microscopy ...................................................... 41 2.11.3 Phalloidin staining of actin .............................................................................. 41 2.11.4 DuolinkTM proximity ligation assay (PLA) ...................................................... 42 2.12 Statistical analysis ................................................................................................ 42 Chapter 3: Disruption of cAMP PDE4D5-HSP20 complex attenuates β-agonist induced hypertrophic response in cardiomyocytes 3.1 Introduction ........................................................................................................... 44 3.1.1 cAMP signalling and compartmentalisation .................................................... 44 3.1.2 PDE family ....................................................................................................... 47 3.1.3 PDE4 structure and isoforms ........................................................................... 48 3.1.4 cAMP-signalling modulation in the heart ........................................................ 50 3.2 Aims ........................................................................................................................ 54 3.3 Results .................................................................................................................... 55 3.3.1 Characterisation of
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