Negative regulation between DAPK and STAT3 in intestinal epithelial cells under TNF-stimulus: = Negative Regulierung zwischen DAPK und STAT3 in intestinalen Epithelzellen unter TNF-Stimulus
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
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| Schlagworte: | |
| Online-Zugang: | Volltext Volltext Volltext Inhaltsverzeichnis |
| Beschreibung: | XVII, 122 S. Ill., graph. Darst. |
Internformat
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| 245 | 1 | 0 | |a Negative regulation between DAPK and STAT3 in intestinal epithelial cells under TNF-stimulus |b = Negative Regulierung zwischen DAPK und STAT3 in intestinalen Epithelzellen unter TNF-Stimulus |c von Saritha Chakilam |
| 246 | 1 | 1 | |a Negative Regulierung zwischen DAPK und STAT3 in intestinalen Epithelzellen unter TNF-Stimulus |
| 264 | 1 | |c 2013 | |
| 300 | |a XVII, 122 S. |b Ill., graph. Darst. | ||
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Datensatz im Suchindex
| _version_ | 1804150315249827840 |
|---|---|
| adam_text | IMAGE 1
INDEX
I N D E X
ABBREVIATIONS X
ABSTRACT XIV
ZUSAMMENFASSUNG XVI
1. INTRODUCTION 1
1.1 INTESTINAL EPITHELIUM 1
1.2 INTESTINAL INFLAMMATION 2
1.2.1 INFLAMMATORY BOWEL DISEASE (IBD) 3
1.2.2 ULCERATIVE COLITIS (UC) 4
1.3 TUMOUR NECROSIS FACTOR-A (TNF) 5
1.4 DEATH ASSOCIATED PROTEIN KINASE (DAPK) 6
1.4.1 DAPK STRUCTURE 7
1.4.2 DAPK SIGNAL TRANSDUCTION 7
1.4.3 DAPK S FUNCTIONS 8
1.4.3.1 DAPK IN INFLAMMATION 9
1.4.4 DAPK REGULATION 10
1.5 NUCLEAR FACTOR -KB ( N F K B ) 11
1.6 LNTERLEUKIN-6 (IL-6) 13
1.7 SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION (STAT3) 14
1.7.1 STAT3 STRUCTURE 14
1.7.2 STAT3 ACTIVATION 15
1.7.3 NUCLEAR IMPORT/EXPORT OF STAT3 16
1.7.4 NEGATIVE REGULATORS OF STAT3 17
1.7.5 STAT3 FUNCTIONS 17
1.7.5.1 STAT3 IN INFLAMMATION 19
1.8 LNTERLEUKIN-8 (IL-8) 19
2. AIMS OF THE WORK 21
3. MATERIALS AND METHODS 23
3.1 MATERIALS 23
3.1.1 CHEMICALS AND REAGENTS 23
3.1.2 CELL CULTURE 24
3.1.3 STIMULANTS AND INHIBITORS 24
III
HTTP://D-NB.INFO/1035381508
IMAGE 2
INDEX
3.1.4 BUFFERS 24
3.1.5 MEDIA 25
3.1.6 KITS AND OTHER RESEARCH PRODUCTS 26
3.1.7 ANTIBODIES 27
3.1.8 EQUIPMENT 28
3.1.9 CONSUMABLES 29
3.2 METHODS 31
3.2.1 CELL CULTURE 31
3.2.1.1 CULTURING OF HCEC CELLS 31
3.2.1.2 COUNTING OF CELLS 32
3.2.1.3 STIMULATION WITH TNF 32
3.2.1.4 COLLECTION OF SUPERNATANTS & CELL PELLETS 32
3.2.1.5 CHRONIC TREATMENT OF HCEC CELLS WITH TNF 33
3.2.1.6 FREEZING & THAWING OF HCEC CELLS 33
3.2.2 MOLECULAR BIOLOGY AND BIOCHEMICAL METHODS 34
3.2.2.1 ISOLATION OF RNA 34
3.2.2.2 CDNA SYNTHESIS 34
3.2.2.3 REAL-TIME RT-PCR 34
3.2.2.4 ISOLATION OF PROTEINS 35
3.2.2.5 DETERMINATION OF PROTEIN CONCENTRATION 36
3.2.2.6 WESTERN BLOTTING 36
3.2.2.7 IMMUNOPRECIPITATION (IP) 37
3.2.2.8 IL-6 AND IL-8 ELISA 38
3.2.2.9 SILENCING OF DAPK EXPRESSION BY SMALL-INTERFERING RNA (SIRNA)..
38
3.2.2.10 PREPARATION OF CYTOPLASMIC AND NUCLEAR LYSATES 39
3.2.2.11 ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA) 39
3.2.2.12 CHROMATIN IMMUNOPRECIPITATION (CHIP) 40
3.2.2.13 POLYMERASE CHAIN REACTION (PCR) 41
3.2.3 APOPTOSIS MEASUREMENT 41
3.2.3.1 ANNEXIN V/PROPIDIUM IODIDE (PI) STAINING 41
3.2.3.2 M30 CYTODEATH ASSAY 42
3.2.3.3 CELL CYCLE ANALYSIS 42
3.2.3.4 CRYSTAL VIOLET CELL VIABILITY ASSAY 42
IV
IMAGE 3
INDEX
3.2.4 IN VIVO TRANSLATION 43
3.2.4.1 PATIENT SAMPLES 43
3.2.4.2 IMMUNOHISTOCHEMISTRY (IHC) AND HISTOLOGICAL SCORE 43
3.2.4.3 ISOLATION AND ENRICHMENT OF EPITHELIAL CELLS FROM UC PATIENTS 44
3.2.4.4 IMMUNOCYTOCHEMISTRY 44
3.2.4.5 EXPERIMENTAL MOUSE MODELS AND IEC ISOLATION 45
3.2.4.5.1 IEC ISOLATION 45
3.2.4.5.2 EXPERIMENTAL MODEL OF INTESTINAL INFLAMMATION 45
3.2.4.5.3 EXPERIMENTAL MODEL OF COLON CARCINOGENESIS 45
3.2.5 STRUCTURAL ANALYSIS OF DAPK-STAT3 COMPLEX 46
3.2.6 STATISTICAL ANALYSIS 46
4. RESULTS 47
4.1 HUMAN COLON EPITHELIAL CELLS (HCEC) 47
4.1.1 CHARACTERISATION OF HCEC CELL LINE 47
4.1.2 HCEC CELLS EXPRESS CYTOKERATIN 47
4.1.3 HCEC CELLS DO NOT EXPRESS CD34 47
4.1.4 HCEC CELLS ARE NON-TURMOURIGENIC 47
4.2 TNF-INDUCED SIGNAL TRANSDUCTION IN HCEC 49
4.2.1 TNF INDUCED PHOSPHORYLATION OF MAP KINASES (MAPKS) 49
4.2.2 TNF INDUCED DAPK MRNA AND PROTEIN EXPRESSION 50
4.2.3 TNF INDUCED STAT3 ACTIVATION 51
4.2.3.1 TNF INDUCED STAT3 Y705 PHOSPHORYLATION 51
4.2.3.2 TNF-ACTIVATED STAT3 TRANSLOCATED INTO THE NUCLEUS 51
4.2.3.3 TNF INCREASED STAT3 DNA BINDING ACTIVITY 52
4.2.4 T N F INDUCED NF K B ACTIVATION 5 4
4.2.5 TNF INDUCED ATF-2 ACTIVATION 54
4.3 TNF-INDUCED CELLULAR FUNCTIONS IN HCEC 55
4.3.1 TNF ACTIVATED FUNCTIONALLY INFLAMMATORY PATHWAY IN HCEC 55
4.3.1.1 TNF INDUCED THE EXPRESSION OF IL-6 & IL-8 MRNA 55
4.3.1.2 TNF INDUCED THE SECRETION OF IL-6 & IL-8 56
4.3.1.3 TNF INDUCED THE PRO-INFLAMMATORY FUNCTION THROUGH
TNF-RI 57
V
IMAGE 4
INDEX
4.3.1.4 EXPRESSION OF TNF-RI & RECEPTOR ASSOCIATED ADAPTOR
MOLECULES 58
4.3.2 HCEC CELLS ARE RESISTANT TO TNF-INDUCED APOPTOSIS 59
4.3.3 TNF TREATMENT DID NOT INDUCE CELL CYCLE ARREST 60
4.3.4 TNF INDUCED THE DNA DAMAGE 60
4.3.5 TNF TREATMENT DID NOT MODULATE CELL VIABILITY 61
4.3.6 CHRONIC TNF TREATMENT 62
4.3.6.1 CHRONIC EXPOSURE OF HCEC CELLS TO TNF INDUCED
CELLULAR GROWTH 62
4.3.6.2 CHRONIC EXPOSURE OF HCEC CELLS TO TNF INDUCED CELL
PROLIFERATION 63
4.4 NEGATIVE CORRELATION OF DAPK & PSTATS* 705 IN VIVO 64
4.4.1 EXPRESSION OF DAPK, PSTAT3 Y705 & TNF IS AUGMENTED IN IEC
OF UC & UCC 64
4.4.2 DIVERGENT EXPRESSION PATTERN OF DAPK, PSTAT3 Y705 & TNF ALONG THE
SINGLE STEPS OF UC CARCINOGENESIS 66
4.4.3 THE EXPRESSION PATTERN OF DAPK & PSTAT3 Y705 IS HETEROGENEOUS IN
DALM SPECIMENS 66
4.4.4 INVERSE EXPRESSION OF DAPK & PSTATS 7705 IN IEC OF UC & UCC... 67
4.4.4.1 PURITY OF EPITHELIAL CELLS 67
4.4.4.2 EXPRESSION OF DAPK & PSTAT3 Y705 IN IEC OF UC & UCC.. 68
4.4.5 DAPK EXPRESSION IS ENHANCED IN STAT3 L6&KO MICE 69
4.4.6 EXPRESSION/ACTIVATION OF DAPK/STAT3 IS MODULATED DURING
COLITISASSOCIATED CARCINOGENESIS 70
4.5 DIVERGENT EXPRESSION OF DAPK AND PSTAT3 Y705 IN HCEC CELLS
UNDER CHRONIC INFLAMMATION 72
4.6 ROLE OF DAPK IN TNF-INDUCED INFLAMMATION IN HCEC 73
4.6.1 EFFICIENCY OF DAPK KNOCKDOWN 73
4.6.2 DAPK IS A NOVEL NEGATIVE REGULATOR OF IL-6/STAT3 74
4.6.2.1 DAPK ATTENUATES TNF-INDUCED STAT3 Y705 PHOSPHORYLATION 74
4.6.2.2 DAPK SUPPRESSES THE EXPRESSION/SECRETION OF IL-6 74
4.6.2.3 DAPK DEPLETION DID NOT MODULATE TNF-INDUCED IL-8
SECRETION 76
4.6.3 ROLE OF KINASE ACTIVITY 77
VI
IMAGE 5
INDEX
4.6.4 DAPK AND STAT3 INTERACTION IS INCREASED UNDER TNF TREATMENT... 77
4.6.5 3D-STRUCTURAL MODELLING OF DAPK-STAT3 COMPLEX 78
4.6.5.1 DAPK INDUCED CONFORMATIONAL CHANGES IN STAT3 DIMER... 78
4.6.5.2 DAPK MIGHT COMPETE WITH JAK TO BIND TO STAT3 81
4.6.6 D A P K ATTENUATES TNF-INDUCED NF K B ACTIVATION 8 2
4.7 ROLE OF STAT3 IN TNF-INDUCED INFLAMMATION IN HCEC 83
4.7.1 TNF INDUCED A POSITIVE FEED BACK LOOP BETWEEN IL-6 & STAT3 83
4.7.1.1 TNF-INDUCED STAT3 V705 PHOSPHORYLATION IS MEDIATED
BY IL-6 83
4.7.1.2 TNF-INDUCED IL-6 EXPRESSION IS POSITIVELY REGULATED
BY STAT3 84
4.7.2 TNF-INDUCED IL-8 EXPRESSION IS STAT3 INDEPENDENT 85
4.7.3 DAPK IS A NOVEL TRANSCRIPTIONAL TARGET OF STAT3 86
4.7.3.1 SCHEMATIC REPRESENTATION OF DAPK PROMOTER 86
4.7.3.2 TNF AUGMENTED STAT3 BINDING TO THE DAPK PROMOTER.... 86
4.7.3.3 DAPK EXPRESSION IS NEGATIVELY REGULATED BY STAT3 87
4.7.3.3.1 STAT3 INHIBITION BY AG490 ENHANCED TNF-INDUCED
DAPK EXPRESSION 88
4.7.3.3.2 STAT3 INHIBITION BY STATTIC ENHANCED TNF-INDUCED
DAPK EXPRESSION 88
5. DISCUSSION 90
5.1 TNF-INDUCED SIGNALLING AND FUNCTIONS IN HCEC 90
5.1.1 CELLULAR TURNOVER OF HCEC IN THE INFLAMMATORY MILIEU 90
5.2 DAPK CONTROLS IL-6/STAT3 ACTIVITY IN HCEC THROUGH ITS KINASE
ACTIVITY AND
PROTEIN-PROTEIN INTERACTION 91
5.3 EXPRESSION OF IL-6 AND DAPK ARE REGULATED BY STAT3 IN HCEC 94
5.4 NEGATIVE REGULATION BETWEEN DAPK AND STAT3 IN HCEC 96
5.5 SCHEMATIC OVERVIEW OF TNF-INDUCED SIGNALLING/FUNCTIONS IN HCEC 96
5.6 NEGATIVE CORRELATION BETWEEN DAPK AND STAT3 IN VIVO 97
5.7 CONCLUSION AND OUTLOOK 100
6. REFERENCES 101
VII
IMAGE 6
INDEX
7. CURRICULUM VITAE 119
7.1 LIST OF ORIGINAL RESEARCH PUBLICATIONS 119
7.2 ORAL PRESENTATIONS 120
7.3 POSTER PRESENTATIONS 121
VIII
|
| any_adam_object | 1 |
| author | Chakilam, Saritha |
| author_facet | Chakilam, Saritha |
| author_role | aut |
| author_sort | Chakilam, Saritha |
| author_variant | s c sc |
| building | Verbundindex |
| bvnumber | BV040992019 |
| collection | ebook |
| ctrlnum | (OCoLC)844104914 (DE-599)DNB1033029866 |
| dewey-full | 570 610 |
| dewey-hundreds | 500 - Natural sciences and mathematics 600 - Technology (Applied sciences) |
| dewey-ones | 570 - Biology 610 - Medicine and health |
| dewey-raw | 570 610 |
| dewey-search | 570 610 |
| dewey-sort | 3570 |
| dewey-tens | 570 - Biology 610 - Medicine and health |
| discipline | Biologie Medizin |
| format | Thesis Book |
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| publishDateSort | 2013 |
| record_format | marc |
| spelling | Chakilam, Saritha Verfasser aut Negative regulation between DAPK and STAT3 in intestinal epithelial cells under TNF-stimulus = Negative Regulierung zwischen DAPK und STAT3 in intestinalen Epithelzellen unter TNF-Stimulus von Saritha Chakilam Negative Regulierung zwischen DAPK und STAT3 in intestinalen Epithelzellen unter TNF-Stimulus 2013 XVII, 122 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Erlangen-Nürnberg, Univ., Diss., 2013 Tumor-Nekrose-Faktor (DE-588)4202932-6 gnd rswk-swf Hemmung (DE-588)4024371-0 gnd rswk-swf STAT (DE-588)4790744-7 gnd rswk-swf Proteinkinasen (DE-588)4175996-5 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content STAT (DE-588)4790744-7 s Tumor-Nekrose-Faktor (DE-588)4202932-6 s Hemmung (DE-588)4024371-0 s DE-604 Proteinkinasen (DE-588)4175996-5 s https://open.fau.de/handle/openfau/3237 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-44690 Resolving-System kostenfrei Volltext http://d-nb.info/1033029866/34 Langzeitarchivierung Nationalbibliothek kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025969801&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Chakilam, Saritha Negative regulation between DAPK and STAT3 in intestinal epithelial cells under TNF-stimulus = Negative Regulierung zwischen DAPK und STAT3 in intestinalen Epithelzellen unter TNF-Stimulus Tumor-Nekrose-Faktor (DE-588)4202932-6 gnd Hemmung (DE-588)4024371-0 gnd STAT (DE-588)4790744-7 gnd Proteinkinasen (DE-588)4175996-5 gnd |
| subject_GND | (DE-588)4202932-6 (DE-588)4024371-0 (DE-588)4790744-7 (DE-588)4175996-5 (DE-588)4113937-9 |
| title | Negative regulation between DAPK and STAT3 in intestinal epithelial cells under TNF-stimulus = Negative Regulierung zwischen DAPK und STAT3 in intestinalen Epithelzellen unter TNF-Stimulus |
| title_alt | Negative Regulierung zwischen DAPK und STAT3 in intestinalen Epithelzellen unter TNF-Stimulus |
| title_auth | Negative regulation between DAPK and STAT3 in intestinal epithelial cells under TNF-stimulus = Negative Regulierung zwischen DAPK und STAT3 in intestinalen Epithelzellen unter TNF-Stimulus |
| title_exact_search | Negative regulation between DAPK and STAT3 in intestinal epithelial cells under TNF-stimulus = Negative Regulierung zwischen DAPK und STAT3 in intestinalen Epithelzellen unter TNF-Stimulus |
| title_full | Negative regulation between DAPK and STAT3 in intestinal epithelial cells under TNF-stimulus = Negative Regulierung zwischen DAPK und STAT3 in intestinalen Epithelzellen unter TNF-Stimulus von Saritha Chakilam |
| title_fullStr | Negative regulation between DAPK and STAT3 in intestinal epithelial cells under TNF-stimulus = Negative Regulierung zwischen DAPK und STAT3 in intestinalen Epithelzellen unter TNF-Stimulus von Saritha Chakilam |
| title_full_unstemmed | Negative regulation between DAPK and STAT3 in intestinal epithelial cells under TNF-stimulus = Negative Regulierung zwischen DAPK und STAT3 in intestinalen Epithelzellen unter TNF-Stimulus von Saritha Chakilam |
| title_short | Negative regulation between DAPK and STAT3 in intestinal epithelial cells under TNF-stimulus |
| title_sort | negative regulation between dapk and stat3 in intestinal epithelial cells under tnf stimulus negative regulierung zwischen dapk und stat3 in intestinalen epithelzellen unter tnf stimulus |
| title_sub | = Negative Regulierung zwischen DAPK und STAT3 in intestinalen Epithelzellen unter TNF-Stimulus |
| topic | Tumor-Nekrose-Faktor (DE-588)4202932-6 gnd Hemmung (DE-588)4024371-0 gnd STAT (DE-588)4790744-7 gnd Proteinkinasen (DE-588)4175996-5 gnd |
| topic_facet | Tumor-Nekrose-Faktor Hemmung STAT Proteinkinasen Hochschulschrift |
| url | https://open.fau.de/handle/openfau/3237 https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-44690 http://d-nb.info/1033029866/34 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025969801&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT chakilamsaritha negativeregulationbetweendapkandstat3inintestinalepithelialcellsundertnfstimulusnegativeregulierungzwischendapkundstat3inintestinalenepithelzellenuntertnfstimulus AT chakilamsaritha negativeregulierungzwischendapkundstat3inintestinalenepithelzellenuntertnfstimulus |