Holdings: Identification of atheroprone shear stress responsiv enhancer elements in endothelial cells

Identification of atheroprone shear stress responsiv enhancer elements in endothelial cells:

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Main Author:	Bondareva, Olga 1992- (Author)
Format:	Thesis Book
Language:	English
Published:	Münster 2017
Subjects:	Hochschulschrift
Online Access:	Inhaltsverzeichnis Inhaltsverzeichnis
Physical Description:	126 Blätter Illustrationen 30 cm

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adam_text	TABLE OF CONTENT SUMMARY........................................................................ 4 ACKNOWLEDGMENTS.................................................................................................................................... 6 LIST OF ABBREVIATIONS.................................................................................................................................9 1. INTRODUCTION................................................................................................................................. 11 1.1. SHEAR STRESS AND ENDOTHELIUM...................................................................................................11 1.1.1. ENDOTHELIUM - A DYNAMIC INTERFACE BETWEEN CIRCULATING FLUID AND ORGANS ................ 11 1.1.2. DIFFERENT SHEAR STRESS PROFILES CAUSE MORPHOLOGICAL AND FUNCTIONAL CHANGES IN ENDOTHELIAL CELLS.................................................................................................................................. 11 1.2. EPIGENETIC MECHANISMS OF GENE REGULATION............................................................................ 13 1.2.1. DNA METHYLATION.................................................................................................................14 1.2.2. HISTONE MODIFICATIONS........................................................................................................ 14 1.2.3. ENHANCERS AS CENTRAL REGULATORY ELEMENTS IN GENOME ................................................... 17 1.3. SHEAR STRESS INDUCES EPIGENETIC REPROGRAMMING OF ENDOTHELIAL CELLS ................................. 22 1.3.1. ATHEROSCLEROSIS AS A MAJOR BURDEN IN MODERN SOCIETY.................................................. 22 1.3.2. EPIGENETIC CHANGES FOUND IN ATHEROSCLEROTIC VESSELS ..................................................... 25 1.4. AIM OF STUDY................................................................................................................................. 27 2. MATERIALS AND METHODS............................................................................................................. 28 2.1. MATERIALS AND EQUIPMENT...................................................................................................... 28 2.2. BUFFERS AND SOLUTIONS............................................................................................................. 31 2.3. CELL CULTURE...............................................................................................................................34 2.4. SHEAR STRESS EXPERIMENTS....................................................................................................... 35 2.5. REAL-TIME PCR......................................................................................................................... 36 2.6. WESTERN BLOT........................................................................................................................... 40 2.7. BRDU CELL PROLIFERATION ASSAY..................................................................................................43 2.8. FLUORESCENT MICROSCOPY.........................................................................................................44 2.9. CHROMATIN IMMUNOPRECIPITATION (CHIP).............................................................................. 45 2.10. CHIP AND RNA SEQUENCING (CHIP-SEQ AND RNA-SEQ)............................................................49 2.11. BIOINFORMATICAL ANALYSIS......................................................................................................... 49 2.12. CLONING AND PLASMID PRODUCTION........................................................................................... 51 2.5. LUCIFERASE ASSAY...................................................................................................................... 55 2.6. STATISTICAL ANALYSIS...................................................................................................................56 3. RESULTS.......................................................................................................................................... 57 3.1. RNA-SEQ DATA...............................................................................................................................57 3.1.1. PRE-SEQUENCING RNA QUALITY CONTROL BY RT-PCR.............................................................. 57 3.1.2. QUALITY CONTROLS OF THE RAW DATA AFTER SEQUENCING.........................................................58 3.1.3. DIFFERENTIALLY EXPRESSED GENES IN OSCILLATORY VERSUS STATIC SHEAR STRESS TREATED HUVEC 61 3.1.4. VALIDATION WITH RT-PCR....................................................................................................... 64 3.2. CHIP-SEQ FOR H3K27AC.................................................................................................................66 3.2.1. PRE-SEQUENCING QUALITY CONTROLS OF THE CHIP ................................................................... 67 3.2.2. QUALITY CONTROLS OF THE SEQUENCING DATA.......................................................................... 69 3.2.3. PEAK CALLING AND IDENTIFICATION OF DIFFERENTIAL PEAKS IN OSCILLATORY VERSUS STATIC CONDITIONS.............................................................................................................................................72 3.2.4. CHIP-SEQ VALIDATION - HEATMAPS.......................................................................................73 3.2.5. ANNOTATION OF PEAKS TO THE NEARBY GENES........................................................................75 3.2.6. DAVID GO ANNOTATION........................................................................................................ 77 3.2.7. TF BINDING MOTIF ANALYSIS....................................................................................................84 3.3. API TRANSCRIPTION FACTOR ACTIVATION UNDER UNIDIRECTIONAL VERSUS OSCILLATORY SHEAR STRESS .89 3.3.1. API TRANSCRIPTION FACTOR FAMILY MEMBERS EXPRESSION ................................................... 89 3.3.2. JUN ACTIVATION BY PHOSPHORYLATION AND NUCLEAR TRANSLOCATION ..................................... 90 3.3.3. API ACTIVATION BY LUCIFERASE ASSAY....................................................................................92 3.4. YAP/TAZ TRANSCRIPTION FACTOR ACTIVATION IN HUVEC BY OSCILLATORY SHEAR STRESS .................... 94 3.4.1. YAP/TAZ TARGET GENES EXPRESSION.....................................................................................94 3.4.2. PROLIFERATION RATE OF HUVEC SUBJECTED TO 6 H OF SHEAR STRESS ....................................... 95 3.5. LUCIFERASE ASSAY VALIDATION OF ENHANCERS................................................................................ 98 3.5.1. VALIDATION OF THE OSCILLATORY SHEAR STRESS RESPONSIVE ENHANCERS .................................. 98 3.5.2. MUTATION OF TF BINDING SITES IN THE OSCILLATORY SHEAR STRESS RESPONSIVE ENHANCERS .100 4. DISCUSSION...................................................................................................................................103 4.1. TRANSCRIPTOMIC AND EPIGENOMIC ANALYSIS OF ENDOTHELIAL RESPONSE TO OSCILLATORY SHEAR STRESS 104 4.2. API TRANSCRIPTION FACTOR ACTIVATION BY SHEAR STRESS..........................................................108 4.3. YAP/TAZ ACTIVATION BY OSCILLATORY SHEAR STRESS IN HUVEC..................................................109 REFERENCES.............................................................................................................................................114 LEBENSLAUF..............................................................................................................................................125 PUBLICATIONS, CONFERENCE ATTENDANCE.................................................................................................126 8
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spelling	Bondareva, Olga 1992- Verfasser (DE-588)1154451623 aut Identification of atheroprone shear stress responsiv enhancer elements in endothelial cells vorgelegt von Olga Bondareva Münster 2017 126 Blätter Illustrationen 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Westfälische Wilhelms-Universität Münster 2018 Beschränkt für den Austausch (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1154795756/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030607616&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis
spellingShingle	Bondareva, Olga 1992- Identification of atheroprone shear stress responsiv enhancer elements in endothelial cells
subject_GND	(DE-588)4113937-9
title	Identification of atheroprone shear stress responsiv enhancer elements in endothelial cells
title_auth	Identification of atheroprone shear stress responsiv enhancer elements in endothelial cells
title_exact_search	Identification of atheroprone shear stress responsiv enhancer elements in endothelial cells
title_full	Identification of atheroprone shear stress responsiv enhancer elements in endothelial cells vorgelegt von Olga Bondareva
title_fullStr	Identification of atheroprone shear stress responsiv enhancer elements in endothelial cells vorgelegt von Olga Bondareva
title_full_unstemmed	Identification of atheroprone shear stress responsiv enhancer elements in endothelial cells vorgelegt von Olga Bondareva
title_short	Identification of atheroprone shear stress responsiv enhancer elements in endothelial cells
title_sort	identification of atheroprone shear stress responsiv enhancer elements in endothelial cells
topic_facet	Hochschulschrift
url	http://d-nb.info/1154795756/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030607616&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA
work_keys_str_mv	AT bondarevaolga identificationofatheroproneshearstressresponsivenhancerelementsinendothelialcells

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