Regulation of cardiac hypertrophy through histone deacetylases: from molecular characterization to novel therapeutic strategies
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in engl. und dt. Sprache |
| Beschreibung: | 124 Bl. Ill., graph. Darst. |
Internformat
MARC
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| 003 | DE-604 | ||
| 005 | 20120816 | ||
| 007 | t | ||
| 008 | 120815s2012 ad|| m||| 00||| eng d | ||
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| 100 | 1 | |a Chen, Lan |e Verfasser |0 (DE-588)1024916790 |4 aut | |
| 245 | 1 | 0 | |a Regulation of cardiac hypertrophy through histone deacetylases |b from molecular characterization to novel therapeutic strategies |c [presented by Lan Chen] |
| 264 | 1 | |c 2012 | |
| 300 | |a 124 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a Zsfassung in engl. und dt. Sprache | ||
| 502 | |a Heidelberg, Univ., Diss., 2012 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025220071&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 999 | |a oai:aleph.bib-bvb.de:BVB01-025220071 | ||
Datensatz im Suchindex
| _version_ | 1804149411079520256 |
|---|---|
| adam_text | IMAGE 1
INDEX OF CONTENTS
INDEX OF CONTENTS 5
OVERVIEW OF FIGURES AND TABLES 9
ABSTRACT 11
ZUSAMENFASUNG 13
1. INTRODUCTION 15
1.1 CARDIAC HYPERTROPHY 15
1.2 NEGATIVE REGULATION O F CARDIAC HYPERTROPHY BY CLASS I LA HDACS 15
1.2.1 HISTONE ACETYLTRARISFERASES AND HISTONE DEACETYLASES CAN REGULATE
GENE TRANSCRIPTION 15
1.2.2 HDACS CLASSIFICATION 16
1.2.3 CLASS ILA H D A C S REPRESS MEF2 ACTIVITY AND CARDIAC HYPERTROPHY
18
1.2.4 SIGNAL-RESPONSIVE REGULATION O F CLASS MA H D A C S 19
1.3 CAMKLI CAN SPECIFICALLY ACTIVATE H D A C 4 23
1.3.1 CAMK.II STRUCTURE AND ITS ACTIVATION 23
1.3.2 CAMKII ISOFORMS AND FUNCTIONS 24
1.3.3 CAMKII CAN SPECIFICALLY PHOPHORYLATE H D A C 4 25
1.4 REGULATION O F CLASS ILA H D A C S BY C A M P 2 7
1.5 AIM O F THIS WORK 2 8
2. MATRERIALS AND METHODS 30
2.1 MATERIALS 3 0
2.1.1 LABORATORY APPARATUS 3 0
2.1.2 CONSUMABLE LABORATORY SUPPLIES 31
2.1.3 KITS 32
2.1.4 CHEMICALS 33
2.1.5 ENZYMES, ENZYME BUFFERS AND LIGANDS 33
2.1.6 ANTIBODIES 33
2.1.7 BACTERIA, CELL LINES 3 4
2.1.8 VECTORS AND OLIGOS 34
2.2 MOLECULAR BIOLOGICAL METHODS 3 7
2.2.1 QUIKCHANGE - SITE DIRECTED MUTAGENESIS 3 7
2.2.1.1 QUIKCHANGE PRINCIPLE 3 7
2.2.1.2 PROCEDURE O F QUIKCHANGE 3 8
2.2.1.2.1 PREPARATION O F PRIMERS (FROM N W G ) 3 9
2.2.1.2.2 PCR 3 9
HTTP://D-NB.INFO/1025558855
IMAGE 2
2.2.1.2.3 DPNL DIGESTION AND TRANSFORMATION 3 9
2.2.1.2.4 MINI PREPARATION 4 0
2.2.1.2.5 RECLONING O F D N A INTO ORIGINAL VECTOR 4 0
2.2.1.2.5.1 DIGESTION O F VECTOR AND INSERT WITH RESTRICTION
ENDONUCLEASES 40
2.2.1.2.5.2 ISOLATION O F D N A FRAGMENT FROM AGAROSE GEL AND
PURIFICATION 4 0
2.2.1.2.5.3 LIGATION AND TRANSFORMATION 41
2.2.2 G S T FUSION PROTEIN RECOMBINANT EXPRESSION IN BL21 41
2.2.2.1 CLONING O F G S T H D A C 4 FRAGMENT 41
2.2.2.2 TRANSFORMATION O F G S T H D A C 4 FRAGMENT D N A INTO BL21 41
2.2.2.3 RECOMBINATION EXPRESSION O F G S T H D A C 4 FUSION PROTEIN 4 2
2.2.3 ADENOVIRUS PRODUCTION 4 3
2.2.4 RNA ISOLATION AND RT-PCR 4 4
2.3 PROTEINCHEMICAL UND IMMUNOLOGICAL METHODS 4 5
2.3.1 SDS-PAGE AND WESTERN BLOT ANALYSIS 4 5
2.3.1.1 SDS-PAGE 4 5
2.3.1.2 COOMASSIE STAINING AND DESTAINING 4 7
2.3.1.3 WESTERN BLOT ANALYSIS 4 7
2.3.2 G S T PULL-DOWN ASSAY 4 8
2.3.3 I N VITRO TRANSLATION 4 8
2.3.4 IMMUNOPRECIPITATION 4 9
2.3.5 IMMUNOSTAINING 50
2.3.6 PROTEIN DETERMINATION THROUGH B C A 51
2.3.7 LUCIFERASE ASSAY 51
2.3.8 IN GEL DIGESTION 52
2.4 CELL BIOLOGICAL METHODS 53
2.4.1 CELL CULTIVATION, TRANSFECTION AND HARVESTING 53
2.4.2 NRVMS ISOLATION _ . 54
2.4.3 INFECTION O F ADENOVIRUS IN N R V M S AND H9C2 CELL 55
2.4.4 INDUCTION O F HYPERTROPHY IN N R V M S 55
2.4.5 PE, ET-1 AND FCS INDUCED G F P H D A C 5 TRANSLOCATION IN N R V M
S 56
2.4.6 TREATMENT O F PROTEIN PHOSPHATASE INHIBITORS AND STIMULATION WITH
C A M P
IN NRVMS 56
2.4.7 SIRNA TRANSFECTION IN H9C2 CELLS 56
2.5 GENERATION O F H D A C 4 R 6 0 I F KNOCK-IN MOUSE 57
2.5.1 CLONING O F TARGETING CONSTRUCT 57
2.5.1.1 GENOMIC D N A ISOLATION FROM MOUSE TAIL BIOPSIES 57
2.5.1.2 FISHING LONG ARM, KNOCKOUT ARM AND SHORT ARM D N A FROM MICE
TAIL
GENOMIC DNA THROUGH P C R 58
2.5.1.3 CLONING O F TARGETING CONSTRUCT 59
2.5.1.4 PREPARATION O F TARGETING CONSTRUCT DNA FOR ELECTROPORATION 61
2.5.2 ELECTROPORATION O F TARGETING CONSTRUCT INTO ES CELL 61
2.5.3 ES CELL CLONES PICKING AND CULTIVATION 6 2
IMAGE 3
2.5.4 GENOMIC D N A ISOLATION FROM ES CELL 63
2.5.5 SCREENING O F ES CELL THROUGH SOUTHERN BLOT 63
2.5.5.1 FISHING D N A PROBES FOR SOUTHERN BLOT ANALYSIS 63
2.5.5.2 SOUTHERN BLOT ANALYSIS 64
3. RESULTS 66
3.1 CHARACTERIZATION O F CAMKLL BINDING DOMAIN ON HDAC4 66
3.1.1 IN VITRO IDENTIFICATION O F ESSENTIAL AMINO ACIDS IN THE REGION O
F CAMKII
PSEUDO SUBSTRATE SEQUENCE 67
3.1.1.1 QUIKCHANGE IN THE PSEUDO SUBSTRATE SEQUENCE REGION ON HDAC4 67
3.1.1.2 RECOMBINANT EXPRESSION AND PURIFICATION O F GST-HDAC4 FRAGMENT
FUSION
PROTEIN IN BL21 67
3.1.1.3 GST PULLDOWN O F PURIFIED GST-HDAC4 FRAGMENTS WITH CAMKLL 68
3.1.2 INTERACTION O F HDAC4 WT AND VARIOUS MUTANTS TO CAMKLL IN COSL
CELL 70
3.1.2.1 CO-IMMUNOPRECIPITATION O F HDAC4 W T AND MUTANTS TO CAMKLL IN
COSL CELL 70
3.1.2.2 CO-LOCALIZATION O F HDAC4 WT AND MUTANTS WITH CAMKLL IN COSL
CELL 71
3.2 IDENTIFICATION O F AMINO ACIDS ON CAMKLL ESSENTIAL FOR INTERACTION
WITH HDAC4 73
3.2.1 INTERACTION O F CAMKLL WT AND VARIOUS MUTANTS WITH HDAC4 73
3.2.1.1 IN VITRO TRANSLATION O F CAMKLL WT AND VARIOUS MUTANTS 73
3.2.1.2 GST PULLDOWN O F GST HDAC4 419-670 WITH IN VITRO TRANSLATED
CAMKLL 74
3.2.2 INTERACTION O F CAMKLL WT AND ITS TRUNCATED FORM WITH HDAC4 74
3.2.2.1 IN VITRO TRANSLATION O F CAMKLL WT AND ITS TRUNCATED FORMS 74
3.2.2.2 GST PULLDOWN O F GST HDAC4 419-670 WITH IN VITRO TRANSLATED
CAMKLL 75
3.3 IDENTIFICATION O F SMALL PEPTIDE TO INHIBIT HDAC4 CAMKLL INTERACTION
76
3.3.1 IDENTIFICATION O F THE SMALLEST HDAC4 PEPTIDE RESPONDS TO CAMKII
BINDING 76
3.3.1.1 CLONING O F VARIOUS GST HDAC4 FRAGMENT 76
3.3.1.2 EXPRESSION AND PURIFICATION O F GST HDAC4 FRAGMENT IN BL21 76
3.3.1.3 GST PULLDOWN O F GST HDAC4 FRAGMENT WITH HIS-CAMKH 76
3.3.2 GST HDAC4 FRAGMENT INHIBITS THE INTERACTION O F HDAC4 WITH CAMKII
IN COSL CELL 77
3.3.3 INTERACTION O F GFP HDAC4 FRAGMENT WITH CAMKII IN COSL CELL 78
3.3.4 GFP HDAC4 FRAGMENT INHIBITS INTERACTION O F HDAC4 WITH CAMKII
IN COSL CELL 80
3.3.5 INHIBITION O F CHEMICAL SYNTHESIZED PEPTIDE TO INTERACTION O F
HDAC4 WITH
CAMKII IN VITRO 82
3.4 GENERATION O F CAMKII-UNRESPONSIVE HDAC4 MUTANT MICE BY THE USE O F
GENE
TARGETING (KNOCK-IN MOUSE) 83
3.4.1 STRATEGY O F HDAC4 R598F KNOCKIN MOUSE 84
3.4.2 STRATEGY O F SCREENING POSITIVE ES CELLS 85
3.4.3 SCREENING O F POSITIVE ES CELL USING SOUTHERN BLOT ANALYSIS 86
3.4.3.1 TEST O F PROBES FOR SOUTHERN BLOT 86
3.4.3.2 SCREENING O F POSITIVE ES CELL 87
IMAGE 4
3.5 CAMP DEPENDENT REGULATION O F HDAC5 NUCLEAR IMPORT 89
3.5.1 CAMP CAN INDUCE HDAC5 DEPHOSPHORYLATION BUT NOT HDAC4 IN NRVMS 89
3.5.2 CAMP CAN INDUCE DEPHOSPHORYLATION O F HDAC5 SPECIFICALLY IN
CARDIOMYOCYTES
BUT NOT IN OTHER CELL TYPES 90
3.5.3 CAMP CAN INDUCE DECREASED BINDING O F 14-3-3 TO HDAC5 BUT NOT TO
HDAC4
IN NRVMS 91
3.5.4 CAMP CAN INDUCE NUCLEAR REIMPORT O F HDAC5 IN NRVMS 92
3.5.5 CAMP CAN PREVENT HYPERTROPHY IN NRVMS 94
3.5.5.1 CAMP CAN PREVENT ET-1, PE AND FCS INDUCED HYPERTROPHY IN NRVMS
94
3.5.5.2 CAMP CAN INHIBIT ET-1 INDUCED MEF2 ACTIVATION IN NRVMS 95
3.5.6 CAMP RESPONDSE DOMAIN ON HDAC5 96
3.5.7 SCREENING O F IMPORTANT PROTEINS RESPONDING TO CAMP INDUCED HDAC5
DEPHOSPHORYLATION. 97
3.5.7.1 PP2A IS THE KEY MOLECULE RESPONSE FOR CAMP INDUCED HDAC5
DEPHOSPHORYLATION 97
3.5.7.2 CAMP INDUCED HDAC5 DEPHOSPHORYLATION IS A PKA PARTIAL DEPENDENT
EVENT 9 9
3.5.7.3 PP2A SUBUNITS B56 CANNOT SELECTIVELY BIND TO HDAC5 9 9
3.5.7.4 SCREENING O F HDAC5 SPECIFIC BINDING PARTNERS THROUGH PROTEOMIC
ANALYSIS 102
3.5.8 EFFECT O F PP2A B55 ALPHA AND DELTA ISOFORMS ON CAMP INDUCED
HDAC5 DEPHOSPHORYLATION IN H9C2 CELLS 103
4. DISCUSSION 105
4.1 DOES THE INTERACTION BETWEEN CAMKII AND HDAC4 PLAY A ROLE FOR
CARDIAC HYPERTROPHY? 105
4.2 IDENTIFICATION O F PEPTIDE THAT COMPETES WITH HDAC4 FOR CAMKII
BINDING 108
4.3 GENERATION O F A CAMKII RESISTANT HDAC4 R601F KNOCK-IN MOUSE MODEL
109
4.4 CAMP INDUCED HDAC5 DEPHOSPHORYLATION IS A CARDIOMYOCYTES SPECIFIC
EVENT 110
4.5 AMINO ACIDS 299-659 ON HDAC5 ARE REQUIRED FOR CAMP INDUCED
HDAC5 DEPHOSPHORYLATION 111
4.6 CAMP CAN REPRESS CARDIOMYOCYTEAC HYPERTROPHY AFTER VARIOUS STIMULI
THROUGH
PP2A-MEDIATED HDAC5 DEPHOSPHORYLATION 112
4.7 CLINICAL IMPLICATIONS 114
5. REFERENCES 116
6. ABBREVIATIONS 123
S:
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| indexdate | 2024-07-10T00:22:36Z |
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| language | English |
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| spelling | Chen, Lan Verfasser (DE-588)1024916790 aut Regulation of cardiac hypertrophy through histone deacetylases from molecular characterization to novel therapeutic strategies [presented by Lan Chen] 2012 124 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in engl. und dt. Sprache Heidelberg, Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025220071&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Chen, Lan Regulation of cardiac hypertrophy through histone deacetylases from molecular characterization to novel therapeutic strategies |
| subject_GND | (DE-588)4113937-9 |
| title | Regulation of cardiac hypertrophy through histone deacetylases from molecular characterization to novel therapeutic strategies |
| title_auth | Regulation of cardiac hypertrophy through histone deacetylases from molecular characterization to novel therapeutic strategies |
| title_exact_search | Regulation of cardiac hypertrophy through histone deacetylases from molecular characterization to novel therapeutic strategies |
| title_full | Regulation of cardiac hypertrophy through histone deacetylases from molecular characterization to novel therapeutic strategies [presented by Lan Chen] |
| title_fullStr | Regulation of cardiac hypertrophy through histone deacetylases from molecular characterization to novel therapeutic strategies [presented by Lan Chen] |
| title_full_unstemmed | Regulation of cardiac hypertrophy through histone deacetylases from molecular characterization to novel therapeutic strategies [presented by Lan Chen] |
| title_short | Regulation of cardiac hypertrophy through histone deacetylases |
| title_sort | regulation of cardiac hypertrophy through histone deacetylases from molecular characterization to novel therapeutic strategies |
| title_sub | from molecular characterization to novel therapeutic strategies |
| topic_facet | Hochschulschrift |
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