Identification of novel p73 target genes by means of genomewide expression profiling:
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2014
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | VIII, 90 S. graph. Darst. 21 cm |
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| adam_text | AUS DER KLINIK FUR INNERE MEDIZIN, SCHWERPUNKT
HAMATOLOGIE, ONKOLOGIE UND IMMUNOLOGIE
DIREKTOR: PROF. DR. ANDREAS NEUBAUER
DES FACHBEREICHS MEDIZIN DER PHILIPPS-UNIVERSITAT MARBURG
IDENTIFICATION OF NOVEL P73 TARGET GENES BY MEANS OF
GENOMEWIDE EXPRESSION PROFILING
INAUGURAL-DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES
DER GESAMTEN HUMANMEDIZIN
DEM FACHBEREICH MEDIZIN DER PHILIPPS-UNIVERSITAT
MARBURG VORGELEGT VON
JAN ULRICH HERBERT FORSTER AUS HANAU
MARBURG, 2014
HTTP://D-NB.INFO/1060273438
INDEX
ZUSAMMENFASSUNG V
SUMMARY VII
1 INTRODUCTION 1
1.1 CANCER 1
1.2 ONCOGENES AND THE E2F FAMILY
OF TRANSCRIPTION FACTORS 2
1.2.1 ONCOGENES 2
1.2.2 E2F1 3
1.3 TUMOR SUPPRESSORS AND THE P53 FAMILY 4
1.3.1 TUMOR SUPPRESSOR GENES 4
1.3.2 THE P53 FAMILY
OF TRANSCRIPTION FACTORS 5
1.3.3 P53 - THE GUARDIAN
OF THE GENOME 5
1.3.4 P73. A SOPHISTICATED MEMBER OF
THE P53 FAMILY 7
1.4 IMMUNE SYSTEM 10
1.4.1 TUMORIGENESIS AND INFLAMMATION 10
1.4.2 IFN SIGNALING 12
1.4.3 P53 FAMILY AND IFN 14
1.5 AIMS OF THIS
STUDY 16
2 MATERIAL AND METHODS 17
2.1 CELL CULTURE 18
2.1.1 CELL LINES 18
2.1.2 MATERIAL FOR CELL CULTURE 18
2.1.3 PREPARATION OF PRIMARY
MOUSE EMBRYONIC FIBROBLASTS 19
2.1.4 CULTIVATION 19
2.1.5 INCUBATION OF CELLS WITH INTERFERON-K
AND INTERFERON-Y 19
2.1.6 HARVESTING OF CELLS 19
2.1.7 FREEZING AND DEFROSTING
OF CELLS 20
2.2 ANALYSIS OF DNA 20
2.2.1 ISOLATION OF GENOMIC DNA 20
2.2.1.1
FROM THE PREPARATION
OF PRIMARY MOUSE EMBRYONIC FIBROBLASTS
20
2.2.1.2
FROM CULTIVATED CELLS
20
2.2.2 DETERMINATION OF DNA CONCENTRATION 21
2.2.3
DNA
AMPLIFICATION VIA POLYMERASE CHAIN
REACTION (PCR) 21
2.2.4
D
ETECTION OF
AMPLIFIED
DNA
(PCR PRODUCTS) BY AGAROSE-GEL
ELECTROPHORESIS 25
2.3 ANALYSIS OF RNA 26
2.3.1 ISOLATION OF CELLULAR RNA 26
2.3.2 DETERMINATION OF
RNA
CONCENTRATION
AND QUALITY 26
2.3.3 TRANSCRIPTION OF
RNA
TO COMPLEMENTARY
DNA (CDNA)
27
2.3.4 MEASURING THE LEVEL
OF TRANSCRIPTION OF TARGET GENES
VIA REAL TIME PCR /
QUANTITATIVE QPCR 27
2.4 ANALYSIS OF PROTEINS 31
2.4.1 PREPARATION OF
CELL LYSATES 31
2.4.2 DETERMINATION OF PROTEIN
CONCENTRATION 31
2.4.3 SDS-PAGE - SODIUM DODECYL
SULFATE POLYACRYLAMIDE GEL
ELECTROPHORESIS 32
2.4.4 WESTERN BLOTTING /
IMMUNOBLOT 33
2.4.4.1
TRANSFERRING THE PROTEINS FROM
THE POLYACRYLAMIDE GEL
TO THE
NITROCELLULOSE
MEMBRANE
33
2.4.4.2
IMMUNODETECTION
34
2.5
MLCROARRAY ANALYSES OF PRIMARY FIBROBLASTS
37
III
3 RESULTS
38
3.1 ESTABLISHMENT OF PRIMARY
MOUSE EMBRYONIC FIBROBLASTS (MEF) 38
3.2 MICROARRAY ANALYSES OF PRIMARY FIBROBLASTS
REVEAL GENE SPECIFIC
PATHWAYS 39
3.2.1 EXPRESSION DATA ANALYSIS WITH
MSIGDB AND GSEA 39
3.2.2 COMPARISON OF DIFFERENCES
IN OVERALL GENE
EXPRESSION 41
3.2.3 E2F1
STIMULATED VERSUS
UNSTIMULATED CONDITIONS
42
3.2.4 P53
WILDTYPE VERSUS
P53
KNOCKOUT
48
3.2.5 P73
WILDTYPE VERSUS
P73
KNOCKOUT INDUCED
GENES
54
3.2.6 P73
WILDTYPE VERSUS
P73
KNOCKOUT
REPRESSED GENES
59
3.3 DIFFERENCES IN INTERFERON SIGNALING
PROTEIN LEVELS AND PHOSPHORYLATION
PATTERNS BETWEEN P73 WILD
TYPE AND P73 KNOCKOUT
CELLS 63
3.4 RT-QPCR ANALYSIS OF JAK-STAT PATHWAY
TARGET GENES IN P73WT AND
P73KO CELLS 66
3.4.1 EXPRESSION OF IFN
TARGET GENES IN
MOUSE EMBRYONIC FIBROBLASTS
66
3.4.2 EXPRESSION ANALYSIS OF
IFN TARGET GENES
IN MOUSE EMBRYONIC
FIBROBLASTS
WITH WILDTYPE
P53 68
3.4.2.1 EXPRESSION ANALYSIS AFTER
INTERFERON ALPHA
STIMULATION 68
3.4.2.2 EXPRESSION ANALYSIS
AFTER INTERFERON GAMMA
STIMULATION 71
4 DISCUSSION 72
4.1 THE PERFORMED MICROARRAY EXPERIMENT
PROVIDES RESULTS THAT ARE IN
AGREEMENT WITH LITERATURE FINDINGS 73
4.2 MICROARRAY EVALUATION DEMONSTRATES THAT P73
MAY BE INVOLVED
IN IFN
SIGNALING 75
5 REFERENCES 80
6 APPENDIX 86
6.1 LIST OF ABBREVIATIONS 86
6.2 VERZEICHNIS DER AKADEMISCHEN LEHRER 89
6.3 DANKSAGUNG 90
|
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| author | Förster, Jan Ulrich Herbert |
| author_facet | Förster, Jan Ulrich Herbert |
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| spelling | Förster, Jan Ulrich Herbert Verfasser aut Identification of novel p73 target genes by means of genomewide expression profiling vorgelegt von Jan Ulrich Förster 2014 VIII, 90 S. graph. Darst. 21 cm txt rdacontent n rdamedia nc rdacarrier Marburg, Univ., Diss., 2014 (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=027837124&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Förster, Jan Ulrich Herbert Identification of novel p73 target genes by means of genomewide expression profiling |
| subject_GND | (DE-588)4113937-9 |
| title | Identification of novel p73 target genes by means of genomewide expression profiling |
| title_auth | Identification of novel p73 target genes by means of genomewide expression profiling |
| title_exact_search | Identification of novel p73 target genes by means of genomewide expression profiling |
| title_full | Identification of novel p73 target genes by means of genomewide expression profiling vorgelegt von Jan Ulrich Förster |
| title_fullStr | Identification of novel p73 target genes by means of genomewide expression profiling vorgelegt von Jan Ulrich Förster |
| title_full_unstemmed | Identification of novel p73 target genes by means of genomewide expression profiling vorgelegt von Jan Ulrich Förster |
| title_short | Identification of novel p73 target genes by means of genomewide expression profiling |
| title_sort | identification of novel p73 target genes by means of genomewide expression profiling |
| topic_facet | Hochschulschrift |
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