Function of Lin9 in vivo and MAP3K4-p38 signaling regulates p53 mediated cell cycle arrest after defective mitosis:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
|
| Schlagworte: | |
| Online-Zugang: | Volltext Inhaltsverzeichnis |
| Beschreibung: | Online erschienen: 2013 |
| Beschreibung: | 106 Bl. Ill., graph. Darst. |
Internformat
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| 100 | 1 | |a Ulrich, Tanja |d 1979- |e Verfasser |0 (DE-588)104564630X |4 aut | |
| 245 | 1 | 0 | |a Function of Lin9 in vivo and MAP3K4-p38 signaling regulates p53 mediated cell cycle arrest after defective mitosis |c von Tanja Ulrich |
| 246 | 1 | 3 | |a Funktion von Lin9 in vivo und MAP3K4-p38 Signalweg reguliert einen p53-vermittelten Zellzyklus-Arrest nach fehlerhafte Mitose |
| 264 | 1 | |c 2012 | |
| 300 | |a 106 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a Online erschienen: 2013 | ||
| 502 | |a Würzburg, Univ., Diss., 2012 | ||
| 650 | 0 | 7 | |a Protein p53 |0 (DE-588)4176550-3 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a MAP-Kinase |0 (DE-588)4576144-9 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Mitose |0 (DE-588)4170185-9 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Aneuploidie |0 (DE-588)4112524-1 |2 gnd |9 rswk-swf |
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| 689 | 0 | 0 | |a Mitose |0 (DE-588)4170185-9 |D s |
| 689 | 0 | 1 | |a MAP-Kinase |0 (DE-588)4576144-9 |D s |
| 689 | 0 | 2 | |a Protein p53 |0 (DE-588)4176550-3 |D s |
| 689 | 0 | 3 | |a Aneuploidie |0 (DE-588)4112524-1 |D s |
| 689 | 0 | |5 DE-604 | |
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| 856 | 4 | 1 | |u https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-73975 |x Resolving-System |z kostenfrei |3 Volltext |
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| 912 | |a ebook | ||
| 999 | |a oai:aleph.bib-bvb.de:BVB01-026928488 | ||
Datensatz im Suchindex
| _version_ | 1804151626492018688 |
|---|---|
| adam_text | CONTENTS
1 INTRODUCTION 1
1.1 THE HUMAN CELL CYCLE 1
1.1.1 CELL CYCLE REGULATION 1
1.1.2 MITOSIS AND CYTOKINESIS 4
1.1.2.1 THE SPINDLE ASSEMBLY CHECKPOINT 4
1.1.2.2 AURORA
KINASES 5
1.1.3 ANEUPLOIDY AND
TUMORIGENESIS 6
1.2 CELLULAR STRESS RESPONSE PATHWAYS 7
1.2.1 MAP KINASE STRESS SIGNALING PATHWAYS 7
1.2.2 THE P53 TUMOR SUPPRESSOR PATHWAY 9
1.2.3 SENESCENCE AS CONSEQUENCE OF CELLULAR STRESS 11
1.3 THE DREAM COMPLEX 12
1.4 AIM OF THIS PROJECT 14
2 MATERIALS AND
METHODS 15
2.1 MATERIALS 15
2.1.1 CHEMICAL STOCKS AND REAGENTS 15
2.1.2 BUFFERS 16
2.1.2.1 GENERAL
BUFFERS 16
2.1.2.2 BUFFERS FOR WHOLE CELL
LYSATES 16
2.1.2.3 BUFFERS FOR IMMUNOBLOT 17
2.1.2.4 BUFFERS FOR CHROMATIN
IMMUNOPRECIPITATION
(CHIP) 18
2.1.2.5 BUFFERS FOR LUCIFERASE
ASSAY 19
2.1.2.6 BUFFERS FOR FLOW CYTOMETRY
(FACS) 19
2.1.2.7 BUFFERS FOR IMMUNOHISTOCHEMISTRY . . . , 20
2.1.2.8 BUFFERS FOR IMMUNOFLUORESCENCE 20
2.1.2.9 BUFFERS FOR GENOMIC
DNA EXTRACTION 20
2.1.2.10 BUFFERS FOR CENTROMERE FLUORESCENCE IN SITU HYBRIDISATION
(C-FISH) 21
2.1.3 ENZYMES 21
2.1.4 ANTIBIOTICS 21
2.1.5 STAINING SOLUTIONS 21
2.1.6 ANTIBODIES 22
2.1.6.1 PRIMARY ANTIBODIES 22
2.1.6.2 SECONDARY ANTIBODIES 23
2.1.7 PRIMERS 23
HTTP://D-NB.INFO/1047474190
2.1.7.1 PRIMEIS FOR QUALITATIVE REAL TIME PCR 23
2.1.7.2 PRIMERS FOR CHROMATIN IMMUNOPRECIPITATION 25
2.1.7.3 PRIMEIS FOR MOUSE GENOTYPING 25
2.1.8 PLASMIDS 25
2.1.9 SIRNA SEQUENCES 26
2.1.10 MARKERS 26
2.1.11 CELL CULTURE MEDIUM AND CELL LINES 27
2.1.12 MOUSE STRAINS 27
2.2 METHODS 28
2.2.1 CELL CULTURE METHODS 28
2.2.1.1 PASSAGING CELTS 28
2.2.1.2 FREEZING AND THAWING CELLS 28
2.2.1.3 COUNTING CELLS 28
2.2.1.4 CELL TREATMENT WITH DIFFERENT REAGENTS 28
2.2.1.5 GENERATION OF MEFS 29
2.2.1.6 TRANSIENT TRANSFECTION 29
2.2.1.6.1 PLASMID TRANSFECTION WITH CALCIUM PHOSPHATE ... 29
2.2.1.6.2 PLASMID TRANSFECTION WITH JETPEI 29
2.2.1.6.3 SIRNA TRANSFECTION WITH DHARMAFECT 30
2.2.1.6.4 REVERSE SIRNA TRANSFECTION
WITH DHARMAFECT ... 30
2.2.1.7 CELL INFECTION 30
2.2.1.8 IMMUNOFLUERESCENCE STAINING 30
2.2.1.9 SIRNA SCREEN 31
2.2.1.10 ROW CYTOMETRY (FACS) 31
2.2.1.10.1 PROPIDIUM IODIDE (PI) FACS 31
2.2.1.10.2 ANNEXIN V FACS 32
2.2.1.11 CENTROMERE FLUORESCENCE
IN SITU HYBRIDISATION (C-FISH) . . 32
2.2.1.12 LUCIFERASE REPORTER
ASSAY 32
2.2.1.13 SA-0-GALACTOSIDASE STAINING 32
2.2.1.14 COLONY FARMING ASSAY 33
2.2.2 MOLECULAR METHODS 33
2.2.2.1 RNA ISOLATION 33
2.2.2.2 REVERSE TRANSCRIPTION (RT) 33
2.2.2.3 QUANTITATIVE REAL-TIME PCR (QPCR) 34
2.2.2.4 MICROARRAY ANALYSIS 34
2.2.2.5 GENOMIC DNA
PCR 35
2.2.3 BIOCHEMICAL METHODS 35
2.2.3.1 WHOLE CELL LYSATES 35
2.2.3.2 DETERMINATION OF PROTEIN CONCENTRATION (BRADFORD) 35
2.2.3.3 SDS POLYACRYIAMIDE GEL ELECTROPHORESIS
(SDS-PAGE).... 36
2.2.3.4 IMMUNOBLOTTING 36
2.2.3.5 CHROMATIN IMMMUNOPRECIPITATION (CHIP) 37
2.2.4 IMMUNOHISTOCHEMICAL METHODS 37
2.2.4.1 PREPARATION OF PARAFFIN SECTIONS 37
2.2.4.2 H&E STAINING 38
2.2.4.3 ANTIBODY STAINING 38
2.2.5 MOUSE INTRAPERITONAL INJECTION 38
3 RESULTS 39
3.1 SENESCENCE PHENOTYPE AFTER LIN9 DELETION IN MEFS 39
3.1.1 PL6 AND P21 ARE UPREGULATED AFTER LESS OF LIN9 39
3.1.2 SENESCENCE CAN BE INDEPENDENTLY INDUCED BY THE P53 OR THE PRB
TUMOR SUPPRESSOR PATHWAY 40
3.2 FUNCTION OF LIN9
IN VIVO
43
3.2.1 EFFICIENCY OF LIN9 KNOCKOUT IN DIFFERENT
TISSUES 43
3.2.2 LOSS OF LIN9
IN VIVO
CAUSES ATROPHY OF THE GASTROINTESTINAL EPITHELIUM 43
3.2.3 PROLIFERATION IN THE INTESTINAL CRYPTS IS IMPAIRED UPON DELETION
OF LIN9 45
3.2.4 REDUCED MITOTIC GENE EXPRESSION AND BINUCLEATED CELLS IN THE SMALL
INTESTINE AFTER LIN9 KNOCKOUT 46
3.2.5 ATROPHIC INTESTINAL EPITHELIUM AFTER LIN9 DELETION IN PL6 KNOCKOUT
MICE 47
3.3 AURORA B INHIBITION
IN U20S CELLS - PRETESTS FOR SIRNA SCREEN 49
3.3.1 U20S CELLS BECOME POLYPLOID AFTER
AURORA B INHIBITION 49
3.3.2 P53 DEPENDENT UPREGULATION OF P21 IN RESPONSE TO AURORA B
INHIBITION 50
3.3.3 IMMUNOFLUORESCENCE
STAINING OF P21 IS SUITABLE AS READOUT FOR THE
SIRNA SCREEN 52
3.4 HIGH
THROUGHPUT SIRNA SCREENING IDENTIFIES
MAP3K4 AS A KINASE ACTING IN
MITOTIC STRESS SIGNALING PATHWAYS 53
3.5 ANALYSIS OF DOWNSTREAM
KINASES OF MAP3K4 55
3.5.1 THE STRESS KINASES P38 AND JNK ARE ACTIVATED UPON AURORA B
INHIBITION 55
3.5.2 P380, NOT P38A, IS REQUIRED FOR P21 ACTIVATION AFTER AURORA B
INHIBITION 56
3.6 REGULATION OF P53 ACTIVITY BY P38 AFTER AURORA B INHIBITION 56
3.6.1 THE MAP3K4-P38 PATHWAY IS NOT REQUIRED FOR P53 STABILIZATION UPON
AURORA B INHIBITION 57
3.6.2 P38 IS NOT RESPONSIBLE FOR P53 PHOSPHORYLATION IN RESPONSE TO
AURORA B
INHIBITION 58
3.6.3 P38 IS REQUIRED FOR TRANSCRIPTIONAL ACTIVITY
OF P53
AFTER AURORA B INHIBITION
59
T
3.6.4 INHIBITION
OF P38 DOES NOT ALTER THE NUCLEAR LOCALIZATION AND P21-
PROMOTER BINDING OF P53 UPON AURORA B INHIBITION 59
3.7 P38 DEPENDENT AND INDEPENDENT GENE REGULATION AFTER AURORA B
INHIBITION . . 61
3.8 CELLULAR OUTCOME IN RESPONSE TO PARTIAL AURORA B INHIBITION 63
3.8.1 TETRAPLOIDIZATION IS NOT NECESSARY TO INDUCE CELL CYCLE ARREST VIA
THE
MAP3K4-P38 PATHWAY 63
3.8.2 INCREASE IN CHROMOSOME MISSEGREGATION AFTER PARTIAL AURORA B
INHIBI
TION 65
3.9 CONSEQUENCES OF AURORA B AND
P38 CO-INHIBITION 66
3.9.1 P38 INHIBITION OVERCOMES THE P21 INDUCED G1 ARREST UPON AURORA B
INHIBITION 66
3.9.2 IMPAIRED CELL PROLIFERATION
IN RESPONSE TO AURORA B AND P38 CO-INHIBITION
69
3.10 LIN9 DEPLETION IN U20S CELLS ACTIVATES P38 AND JNK TO INDUCE CELL
CYCLE ARREST 70
4 DISCUSSION 72
4.1 ANALYSIS OF THE SENESCENCE PHENOTYPE AFTER LIN9 KNOCKOUT 72
4.2 FUNCTION OF LIN9
IN VIVO
73
4.3 ANALYSIS OF PATHWAYS ACTIVATING P53 AFTER CYTOKINESIS FAILURES 74
4.3.1 MAP3K4-P38 REGULATES ACTIVATION OF P21 UPON AURORA B INHIBITION .
. 75
4.3.2 REGULATION OF P53 ACTIVITY BY P38 AFTER AURORA B INHIBITION 77
4.3.3 CHROMOSOMAL MISSEGREGATION IS SUFFICIENT TO ACTIVATE THE MAP3K4-
P38-P53 PATHWAY UPON PARTIAL AURORA B INHIBITION 79
4.3.4 CONSEQUENCES OF AURORA B AND P38 CO-INHIBITION 80
4.3.5 MAP3K4-P38-P53 - A COMMON PATHWAY ACTIVATED AFTER CYTOKINESIS
FAILURE? 81
4.3.6 MODEL OF THE MITOTIC STRESS INDUCED PATHWAY 82
5 SUMMARY 84
6 ZUSAMMENFASSUNG 85
7 REFERENCES 86
8 APPENDIX 99
8.1 LIST OF FIGURES 99
8.2 ABBREVIATIONS 101
8.3 OWN PUBLICATIONS 103
8.4 LEBENSLAUF 104
8.5 DANKSAGUNG 105
8.6 EIDESSTATTLICHE ERKLARUNG 106
|
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| spelling | Ulrich, Tanja 1979- Verfasser (DE-588)104564630X aut Function of Lin9 in vivo and MAP3K4-p38 signaling regulates p53 mediated cell cycle arrest after defective mitosis von Tanja Ulrich Funktion von Lin9 in vivo und MAP3K4-p38 Signalweg reguliert einen p53-vermittelten Zellzyklus-Arrest nach fehlerhafte Mitose 2012 106 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Online erschienen: 2013 Würzburg, Univ., Diss., 2012 Protein p53 (DE-588)4176550-3 gnd rswk-swf MAP-Kinase (DE-588)4576144-9 gnd rswk-swf Mitose (DE-588)4170185-9 gnd rswk-swf Aneuploidie (DE-588)4112524-1 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Mitose (DE-588)4170185-9 s MAP-Kinase (DE-588)4576144-9 s Protein p53 (DE-588)4176550-3 s Aneuploidie (DE-588)4112524-1 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-73975 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-73975 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026928488&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Ulrich, Tanja 1979- Function of Lin9 in vivo and MAP3K4-p38 signaling regulates p53 mediated cell cycle arrest after defective mitosis Protein p53 (DE-588)4176550-3 gnd MAP-Kinase (DE-588)4576144-9 gnd Mitose (DE-588)4170185-9 gnd Aneuploidie (DE-588)4112524-1 gnd |
| subject_GND | (DE-588)4176550-3 (DE-588)4576144-9 (DE-588)4170185-9 (DE-588)4112524-1 (DE-588)4113937-9 |
| title | Function of Lin9 in vivo and MAP3K4-p38 signaling regulates p53 mediated cell cycle arrest after defective mitosis |
| title_alt | Funktion von Lin9 in vivo und MAP3K4-p38 Signalweg reguliert einen p53-vermittelten Zellzyklus-Arrest nach fehlerhafte Mitose |
| title_auth | Function of Lin9 in vivo and MAP3K4-p38 signaling regulates p53 mediated cell cycle arrest after defective mitosis |
| title_exact_search | Function of Lin9 in vivo and MAP3K4-p38 signaling regulates p53 mediated cell cycle arrest after defective mitosis |
| title_full | Function of Lin9 in vivo and MAP3K4-p38 signaling regulates p53 mediated cell cycle arrest after defective mitosis von Tanja Ulrich |
| title_fullStr | Function of Lin9 in vivo and MAP3K4-p38 signaling regulates p53 mediated cell cycle arrest after defective mitosis von Tanja Ulrich |
| title_full_unstemmed | Function of Lin9 in vivo and MAP3K4-p38 signaling regulates p53 mediated cell cycle arrest after defective mitosis von Tanja Ulrich |
| title_short | Function of Lin9 in vivo and MAP3K4-p38 signaling regulates p53 mediated cell cycle arrest after defective mitosis |
| title_sort | function of lin9 in vivo and map3k4 p38 signaling regulates p53 mediated cell cycle arrest after defective mitosis |
| topic | Protein p53 (DE-588)4176550-3 gnd MAP-Kinase (DE-588)4576144-9 gnd Mitose (DE-588)4170185-9 gnd Aneuploidie (DE-588)4112524-1 gnd |
| topic_facet | Protein p53 MAP-Kinase Mitose Aneuploidie Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-73975 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026928488&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT ulrichtanja functionoflin9invivoandmap3k4p38signalingregulatesp53mediatedcellcyclearrestafterdefectivemitosis AT ulrichtanja funktionvonlin9invivoundmap3k4p38signalwegregulierteinenp53vermitteltenzellzyklusarrestnachfehlerhaftemitose |