Role of the Cullin-RING E3 ubiquitin ligase 7 in oncogenic transformation by simian virus 40 large T-antigen:
Gespeichert in:
| Beteilige Person: | |
|---|---|
| Format: | Hochschulschrift/Dissertation Buch |
| Sprache: | Englisch |
| Veröffentlicht: |
2014
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| Schlagwörter: | |
| Links: | https://mediatum.ub.tum.de/node?id=1192165 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20140702-1192165-0-2 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027417697&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| Umfang: | 69 Bl. Ill., graph. Darst. |
Internformat
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| 245 | 1 | 0 | |a Role of the Cullin-RING E3 ubiquitin ligase 7 in oncogenic transformation by simian virus 40 large T-antigen |c Thomas Hartmann |
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| 912 | |a ebook | ||
| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-027417697 | |
Datensatz im Suchindex
| DE-BY-TUM_call_number | 0001 DM 32230 |
|---|---|
| DE-BY-TUM_katkey | 2013096 |
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| adam_text | FAKULTAT FUR MEDIZIN
INSTITUT FUR PHARMAKOLOGIE UND TOXIKOLOGIE
ROLE OF THE CULLIN-RING E3 UBIQUITIN LIGASE 7 IN ONCOGENIC
TRANSFORMATION BY SIMIAN VIRUS 40 LARGE T-ANTIGEN
THOMAS HARTMANN
VOLLSTANDIGER ABDRUCK DER VON DER FAKULTAT FUR MEDIZIN DER TECHNISCHEN
UNIVERSITAT
MUNCHEN ZUR ERLANGUNG DES AKADEMISCHEN GRADES EINES DOKTOR DER
NATURWISSENSCHAFTEN
(DR. RER.
NAT.) GENEHMIGTEN DISSERTATION.
VORSITZENDER: UNIV.-PROF. DR. THOMAS MISGELD
PRUFER DER DISSERTATION:
1. UNIV.-PROF. DR. DR. STEFAN
ENGELHARDT
2. UNIV.-PROF. DR. CLAUS SCHWECHHEIMER
DIE DISSERTATION WURDE AM 19. FEBRUAR 2014 BEI DER TECHNISCHEN
UNIVERSITAT MUNCHEN
EINGEREICHT UND DURCH DIE FAKULTAT FUR MEDIZIN AM 04. JUNI 2014
ANGENOMMEN.
HTTP://D-NB.INFO/1055962042
TABLE OF CONTENTS
2
TABLE OF CONTENTS
1 INTRODUCTION 4
1.1 LT-ANTIGEN OF SV40 5
1.1.1 DOMAIN ORGANIZATION AND INTERACTION PARTNERS OF LT-ANTIGEN 5
1.1.2 FUNCTIONS OF LT-ANTIGEN I 7
1.2 THE UBIQUITIN-PROTEASOME SYSTEM (UPS) 8
1.3 E3 UBIQUITIN
LIGASES 9
1.4 CULLIN-RING E3 UBIQUITIN LIGASE 7 12
1.4.1 CUL7 BINDING PARTNERS 12
1.4.2 CRL7 SUBSTRATES 13
1.4.3 BIOLOGICAL SIGNIFICANCE OF CRL7 13
1.5 AIM OF THIS STUDY 15
2 MATERIALS AND METHODS 16
2.1 CHEMICALS 16
2.2 ANTIBODIES 16
2.3 OLIGONUCLEOTIDES 17
2.4 PLASMIDS 18
2.5 SMALL INTERFERING
RNA (SIRNA)... 18
2.6 ENZYMES 19
2.7 BACTERIA 19
2.8 EUKARYOTIC CELL
LINES 19
2.9 MOUSE LINES 19
2.10 MOLECULAR BIOLOGY 20
2.10.1 POLYMERASE CHAIN
REACTION (PCR) 20
2.10.2 RNA ISOLATION 21
2.10.3 REVERSE TRANSCRIPTION 21
2.10.4 QUANTITATIVE REAL-TIME PCR (QRT-PCR) 22
2.10.5 ISOLATION OF MURINE DNA FOR GENOTYPING 22
2.10.6 AGAROSE GEL
ELECTROPHORESIS 23
2.10.7 DNA GEL EXTRACTION 23
2.10.8 RESTRICTION OF VECTOR DNA AND PCR PRODUCTS 23
2.10.9 DE-PHOSPHORYLATION OF DNA 24
2.10.10 LIGATION OF DNA FRAGMENTS 24
2.10.11 TRANSFORMATION OF ELECTROCOMPETENT DH10B BACTERIA 24
2.10.12 BACTERIAL MINI CULTURES AND MINI PLASMID PREPARATION 25
2.10.13 BACTERIAL MIDI CULTURES AND MIDI PLASMID PREPARATION 26
2.10.14 SEQUENCING OF PLASMID DNA 26
2.11 CELL CULTURE 26
2.11.1 CULTIVATION OF EUKARYOTIC CELL LINES 26
2.11.2 THAWING AND FREEZING OF CELL ALIQUOTS 27
2.11.3 TRANSIENT TRANSFECTION OF CELLS USING LIPOFECTAMINE 2000 27
2.11.4 GENERATION OF RETROVIRUSES 28
2.11.5 RETROVIRAL INFECTION OF CELLS 28
2.11.6 CRISTAL VIOLET STAINING FOR CELL DENSITY 28
2.12 PROTEIN BIOCHEMISTRY 28
2.12.1 LYSIS OF CELL LINES 28
TABLE OF CONTENTS
3
2.12.2 (CO-)LMMUNOPRECIPITATION 29
2.12.3 MEASUREMENT OF PROTEIN CONCENTRATION 30
2.12.4 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) 30
2.12.5 WESTERN BLOT 31
2.12.6 IMMUNODETECTION 31
2.12.7
35
S PULSE-CHASE 32
2.12.8 IN VITRO UBIQUITINATION 33
2.12.9 DE-PHOSPHORYLATION OF V5-IRS1 33
2.13 MICROSCOPY 34
2.13.1 IMMUNOFLUORESCENCE 34
2.13.2 IMMUNOHISTOCHEMISTRY 34
2.13.3 QUANTIFICATION OF AND STAINING INTENSITIES 34
2.14 STATISTICS ! 35
3 RESULTS 36
3.1 SV40 LT-ANTIGEN IMPAIRS CRL7 FUNCTION 36
3.1.1 LT-ANTIGEN EXPRESSION RESULTS IN POSTTRANSLATIONAL STABILIZATION
OF ECTOPIC IRS1 * ....36
3.1.2 DEGRADATION OF ENDOGENOUS IRS1 39
3.1.3 LT-ANTIGEN IMPAIRS IRS1 IN VITRO UBIQUITINATION BY CRL7 * 41
3.2 THE BIOLOGICAL RELEVANCE OF LT-ANTIGEN BINDING TO CUL7 42
3.2.1 LT-ANTIGEN ENHANCES ACTIVATION OF IRS1 DOWNSTREAM SIGNALING
PATHWAYS * 42
3.2.2 STABLE LT-ANTIGEN EXPRESSION RESULTS IN INCREASED PROLIFERATION OF
U2-OS CELLS * 45
3.2.3 LT-ANTIGEN POSITIVE CARCINOMA DISPLAY ELEVATED IRS1 PROTEIN LEVEL
AND SIGNALING IN
VIVO * 45
3.3 MOLECULAR MECHANISM OF CRL7 INHIBITION BY LT-ANTIGEN * 48
4 DISCUSSION 50
4.1 SV40 LT-ANTIGEN IMPAIRS CRL7 FUNCTION 50
4.2 THE BIOLOGICAL
RELEVANCE OF LT-ANTIGEN BINDING TO CUL7 51
4.3 MOLECULAR MECHANISM OF CRL7 INHIBITION BY LT-ANTIGEN 53
5 SUMMARY 55
6 REFERENCES 56
7 PUBLICATIONS 66
8 CONGRESS CONTRIBUTIONS 66
9 APPENDIX 67
9.1 ABBREVIATIONS 67
9.2 ACKNOWLEDGEMENTS 69
|
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| indexdate | 2024-12-20T16:59:12Z |
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| language | English |
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| oclc_num | 884479661 |
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| spellingShingle | Hartmann, Thomas Role of the Cullin-RING E3 ubiquitin ligase 7 in oncogenic transformation by simian virus 40 large T-antigen |
| subject_GND | (DE-588)4113937-9 |
| title | Role of the Cullin-RING E3 ubiquitin ligase 7 in oncogenic transformation by simian virus 40 large T-antigen |
| title_auth | Role of the Cullin-RING E3 ubiquitin ligase 7 in oncogenic transformation by simian virus 40 large T-antigen |
| title_exact_search | Role of the Cullin-RING E3 ubiquitin ligase 7 in oncogenic transformation by simian virus 40 large T-antigen |
| title_full | Role of the Cullin-RING E3 ubiquitin ligase 7 in oncogenic transformation by simian virus 40 large T-antigen Thomas Hartmann |
| title_fullStr | Role of the Cullin-RING E3 ubiquitin ligase 7 in oncogenic transformation by simian virus 40 large T-antigen Thomas Hartmann |
| title_full_unstemmed | Role of the Cullin-RING E3 ubiquitin ligase 7 in oncogenic transformation by simian virus 40 large T-antigen Thomas Hartmann |
| title_short | Role of the Cullin-RING E3 ubiquitin ligase 7 in oncogenic transformation by simian virus 40 large T-antigen |
| title_sort | role of the cullin ring e3 ubiquitin ligase 7 in oncogenic transformation by simian virus 40 large t antigen |
| topic_facet | Hochschulschrift |
| url | https://mediatum.ub.tum.de/node?id=1192165 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20140702-1192165-0-2 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027417697&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT hartmannthomas roleofthecullinringe3ubiquitinligase7inoncogenictransformationbysimianvirus40largetantigen |
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