Suitability of N-terminal autophosphorylation of cGMP-dependent protein kinase type I as an in vivo molecular monitor of cGMP signaling:
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| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
2014
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | XIV, 92, XV S. Ill., graph. Darst. |
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| adam_text | SUITABILITY OF N-TERMINAL AUTOPHOSPHORYLATION
OF CGMP-DEPENDENT PROTEIN KINASE TYPE I AS
AN
IN VIVO
MOLECULAR MONITOR OF CGMP
SIGNALING
DISSERTATION
ZUR ERLANGUNG DES GRADES EINES
DOKTORS DER NATURWISSENSCHAFTEN
DER MATHEMATISCH-NATURWISSENSCHAFTLICHEN FAKULTAT
UND
DER MEDIZINISCHEN FAKULTAT
DER EBERHARD-KARLS-UNIVERSITAT TUBINGEN
VORGELEGT
VON
RAGHAVAN VALLUR
AUS KADAPA, INDIA
MARCH-2014
HTTP://D-NB.INFO/105242354X
TABLE OF CONTENTS
TABLE OF CONTENTS
LIST OF ABBREVIATIONS IV
LIST OF FIGURES VII
LIST OF TABLES IX
ZUSAMMENFASSUNG X
SUMMARY XII
1 INTRODUCTION 1
1.1 AN OVERVIEW OF CGMP SIGNALING 1
1.2 NO-CGMP-CGKJ SIGNALING 2
1.2.1 NO-INDUCED CGMP SIGNALING IN THE
CNS 3
1.2.2 CGKL-MEDIATED NO-CGMP SIGNALING IN THE BRAIN 4
1.3 NO RELEASE FROM P-GAL-NONOATE IN A CELL TYPE-SPECIFIC MANNER 8
1.4 MOLECULAR MONITOR FOR IN VIVO RELEASE OF NO IN THE BRAIN 10
1.5 CGMP-DEPENDENT PROTEIN KINASE TYPE I 12
1.5.1 STRUCTURE 12
1.5.2 FUNCTIONS OF THE DIFFERENT DOMAINS 12
1.5.3 CGKL AUTOPHOSPHOIYLATION 13
1.6. AIM OF THE THESIS 16
2 MATERIALS AND METHODS 17
2.1 REAGENTS 17
2.2 PEPTIDE SYNTHESIS AND ANTIBODY GENERATION 17
2.2.1 SELECTION OF PHOSPHORYLATABLE SITES IN THE CGKL N-TERMINUS USING
AN INSILICO
PROGRAM (GPS) AND IN VITRO ASSAYS FROM THE LITERATURE 17
2.2.2 SYNTHESIS OF PEPTIDES AND ANTISERUM COLLECTION 20
2.2.3 1 MMUNOAFFINITY CHROMATOGRAPHY 20
2.3 ELISA 21
2.4 MOUSE BREEDING AND GENOTYPING 22
2.4.1 DNA EXTRACTION FROM MOUSE TISSUES 24
2.4.2 PCR 25
2.4.3 AGAROSE GEL ELECTROPHORESIS 25
I
TABLE OF CONTENTS
2.5 CELL CULTURE 26
2.5.1 THAWING AND PASSAGING OF MEFS 27
2.5.2 FREEZING OF MEFS 28
2.5.3 CELL COUNTING 29
2.5.4 TRANSACTION OF COS-7 CELLS 29
2.5.5 GRIESS ASSAY FOR DETECTION OF NO 31
2.5.6 ISOLATION OF VSMCS 32
2.6 TISSUE ISOLATION AND STIMULATION 34
2.6.1 ISOLATION AND STIMULATION OF MOUSE PLATELETS 34
2.6.2 ISOLATION OF AOITA, BLADDER AND CEREBELLUM, AND TISSUE LYSIS 35
2.7 PROTEIN ANALYSIS 37
2.7.1 CELL LYSIS 37
2.7.2 PROTEIN QUANTIFICATION 38
2.7.3 PROTEIN PRECIPITATION 39
2.7.4 SDS-PAGE AND WESTERN BLOT 40
2.8 HISTOLOGICAL METHODS 42
2.8.1 X-GAL STAINING 42
2.8.2 CIYOSECTIONING ;. 43
2.8.3 IMMUNOFLUORESCENCE 44
2.8.4 IMMUNOHISTOCHEMISTIY 45
2.9 IN VITRO AUTOPHOSPHORVLATION
OF CGKJ 47
2.9.1 PHOSPHORYLATION OF PURIFIED CGKI ISOFORMS AND LAMBDA PROTEIN
PHOSPHATASE
ASSAY 47
2.9.2 PRE-INCUBATION OF PURIFIED PROTEINS AND BROKEN CELL EXTRACTS WITH
CGMP 50
2.10 STATISTICAL ANALYSIS 50
3 RESULTS 51
3.1 DETECTION OFNO RELEASE FROM P-GAL-NONOATE IN COS-7 CELLS 51
3.2 ESTABLISHING A MOLECULAR MONITOR FOR NO RELEASE FROM P-GAL-NONOATE
IN VIVO IN A
CELL TYPE-SPECIFIC MANNER IN THE BRAIN 52
3.3 SELECTION OF CGKI AUTOPHOSPHORVLATION SITES AND GENERATION OF
PHOSPHO-CGKL ANTISERA
55
3.4 VALIDATION OF ANTISERA 56
3.4.1 SCREENING OF ANTISERA FOR THEIR SPECIFICITY BY USING IN VITRO
PHOSPHORYLATED CGKI
56
II
TABLE OF CONTENTS
3.4.2 ELISA ANALYSIS OF ANTISERA USING PHOSPHOIYLATED AND
NON-PHOSPHOIYLATED
PEPTIDES 58
3.4.3 WESTERN BLOT ANALYSIS OF ANTISERA USING IN VITRO PHOSPHOIYLATED
PURIFIED CGKL
ISOZYMES TREATED WITH LAMBDA PROTEIN PHOSPHATASE 63
3.5 CGKI AUTOPHOSPHORYLATION IN VIVO 64
3.5.1 ANALYSIS OF CGKI AUTOPHOSPHORYLATION IN INTACT CELLS 64
3.5.2 ANALYSIS OF CGKI AUTOPHOSPHOIYLATION IN MOUSE TISSUES 69
3.6 EFFECT OF CGMP PRE-INCUBATION ON PURIFIED AND ENDOGENOUS CGKI IN
VITRO 71
4 DISCUSSION 73
4.1 PHOSPHORYLATION OF PDE5 AS AN IN VIVO MONITOR FOR ACTIVATION OF THE
NO-CGMP-CGKI
PATHWAY IN THE CEREBELLUM 73
4.2 N-TEIMINAL AUTOPHOSPHORYLATION OF CGKI AS AN IN VIVO MONITOR FOR
ACTIVATION OF THE
NO-CGMP-CGKI PATHWAY 75
4.3 OUTLOOK 80
5 REFERENCES 82
6 PUBLICATIONS 91
7 CURRICULUM VITAE 92
8 APPENDIX I
8.1 STOCKS OF CHEMICALS AND CONSUMABLES I
8.2 BUFFERS AND SOLUTIONS II
8.2.1 COMMON BUFFERS II
8.2.2 MEDIA AND SOLUTIONS USED FOR CELL CULTURE III
8.2.3 SOLUTIONS USED FOR SDS-PAGE AND WESTERN BLOTTING IV
8.2.4 SOLUTIONS USED FOR IMMUNOFLUORESCENCE VII
8.2.5 SOLUTIONS USED FOR IMMUNOHISTOCHEMISTRY VIII
8.2.6 SOLUTIONS USED FOR PCR IX
8.3 PRIMERS IX
8.4 PCR-BASED GENOTYPING FRAGMENTS X
8.5 ANTIBODY DILUTIONS XI
8.6 SUPPLEMENTARY FIGURES XII
8.7 PROTEIN SEQUENCE OF MURINE (MAS MUSCULUS) CGKJ ISOFOIMS XV
CGMP-DEPENDENT PROTEIN KINASE 1 ALPHA ISOFOMI XV
CGMP-DEPENDENT PROTEIN KINASE 1 BETA ISOFORM XV
III
|
| any_adam_object | 1 |
| author | Vallur, Raghavan 1986- |
| author_GND | (DE-588)1051283795 |
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| dewey-full | 570 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 570 - Biology |
| dewey-raw | 570 |
| dewey-search | 570 |
| dewey-sort | 3570 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| format | Thesis Book |
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| spelling | Vallur, Raghavan 1986- Verfasser (DE-588)1051283795 aut Suitability of N-terminal autophosphorylation of cGMP-dependent protein kinase type I as an in vivo molecular monitor of cGMP signaling vorgelegt von Raghavan Vallur 2014 XIV, 92, XV S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Tübingen, Univ., Diss., 2014 Mit einer Zssfassung in dt. Sprache Zentralnervensystem (DE-588)4067637-7 gnd rswk-swf Cyclo-GMP (DE-588)4318174-0 gnd rswk-swf Tiermodell (DE-588)4140660-6 gnd rswk-swf Signaltransduktion (DE-588)4318717-1 gnd rswk-swf Stickstoffmonoxid (DE-588)4183282-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Signaltransduktion (DE-588)4318717-1 s Stickstoffmonoxid (DE-588)4183282-6 s Cyclo-GMP (DE-588)4318174-0 s Zentralnervensystem (DE-588)4067637-7 s Tiermodell (DE-588)4140660-6 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027340972&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Vallur, Raghavan 1986- Suitability of N-terminal autophosphorylation of cGMP-dependent protein kinase type I as an in vivo molecular monitor of cGMP signaling Zentralnervensystem (DE-588)4067637-7 gnd Cyclo-GMP (DE-588)4318174-0 gnd Tiermodell (DE-588)4140660-6 gnd Signaltransduktion (DE-588)4318717-1 gnd Stickstoffmonoxid (DE-588)4183282-6 gnd |
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| title | Suitability of N-terminal autophosphorylation of cGMP-dependent protein kinase type I as an in vivo molecular monitor of cGMP signaling |
| title_auth | Suitability of N-terminal autophosphorylation of cGMP-dependent protein kinase type I as an in vivo molecular monitor of cGMP signaling |
| title_exact_search | Suitability of N-terminal autophosphorylation of cGMP-dependent protein kinase type I as an in vivo molecular monitor of cGMP signaling |
| title_full | Suitability of N-terminal autophosphorylation of cGMP-dependent protein kinase type I as an in vivo molecular monitor of cGMP signaling vorgelegt von Raghavan Vallur |
| title_fullStr | Suitability of N-terminal autophosphorylation of cGMP-dependent protein kinase type I as an in vivo molecular monitor of cGMP signaling vorgelegt von Raghavan Vallur |
| title_full_unstemmed | Suitability of N-terminal autophosphorylation of cGMP-dependent protein kinase type I as an in vivo molecular monitor of cGMP signaling vorgelegt von Raghavan Vallur |
| title_short | Suitability of N-terminal autophosphorylation of cGMP-dependent protein kinase type I as an in vivo molecular monitor of cGMP signaling |
| title_sort | suitability of n terminal autophosphorylation of cgmp dependent protein kinase type i as an in vivo molecular monitor of cgmp signaling |
| topic | Zentralnervensystem (DE-588)4067637-7 gnd Cyclo-GMP (DE-588)4318174-0 gnd Tiermodell (DE-588)4140660-6 gnd Signaltransduktion (DE-588)4318717-1 gnd Stickstoffmonoxid (DE-588)4183282-6 gnd |
| topic_facet | Zentralnervensystem Cyclo-GMP Tiermodell Signaltransduktion Stickstoffmonoxid Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027340972&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT vallurraghavan suitabilityofnterminalautophosphorylationofcgmpdependentproteinkinasetypeiasaninvivomolecularmonitorofcgmpsignaling |