Regulation of the cytosolic stress response: identification of positive and negative modulators by a Genome-Wide RNA interference screen
Gespeichert in:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2014
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| Schlagworte: | |
| Online-Zugang: | kostenfrei kostenfrei kostenfrei Inhaltsverzeichnis |
| Beschreibung: | IV, 172 S. Ill., graph. Darst. |
| Format: | Langzeitarchivierung gewährleistet, LZA |
Internformat
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| 100 | 1 | |a Löw, Christian Frank |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Regulation of the cytosolic stress response |b identification of positive and negative modulators by a Genome-Wide RNA interference screen |c Christian Frank Löw |
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| 502 | |a München, Univ., Diss., 2014 | ||
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Datensatz im Suchindex
| _version_ | 1804152287217582080 |
|---|---|
| adam_text | TABLE OF CONTENTS I
TABLE OF CONTENTS
1 SUMMARY 1
2 INTRODUCTION 3
2.1 PROTEOSTASIS 3
2.2 PROTEIN FOLDING 4
2.2.1 FOLDING VERSUS AGGREGATION 4
2.2.2 PROTEIN FOLDING IN VIVO AND THE CONCEPT OF MOLECULAR CHAPERONES 7
2.2.3 MAJOR CHAPERONE CLASSES 8
2.3 PROTEIN DEGRADATION SYSTEMS 15
2.3.1 THE UBIQUITIN-PROTEASOME SYSTEM 15
2.3.2 AUTOPHAGY 20
2.3.3 INTERCONNECTION BETWEEN PROTEIN DEGRADATION PATHWAYS 22
2.4 STRESS-RESPONSIVE PATHWAYS IN PROTEOSTASIS 23
2.4.1 CHALLENGERS OF PROTEOSTASIS 23
2.4.2 THE CYTOSOLIC STRESS RESPONSE 25
2.4.3 ORGANELLE-SPECIFIC STRESS RESPONSE PATHWAYS 31
2.5 INTEGRATION OF THE PROTEOSTASIS NETWORK AND ITS RELATION TO AGING
AND DISEASE 34
2.6 AIM OF THE STUDY 37
3 MATERIAL AND METHODS 39
3.1 CHEMICALS AND BIOCHEMICALS 39
3.2 ANTIBODIES 43
3.3 MEDIA AND BUFFERS 44
3.3.1 MEDIA 44
3.3.2 BUFFERS AND STANDARD SOLUTIONS 44
3.4 MATERIALS AND INSTRUMENTS 47
HTTP://D-NB.INFO/1051111587
TABLE OF CONTENTS II
3.5 MARKER, KITS, AND ENZYMES 52
3.5.1 MARKER AND LOADING DYES 52
3.5.2 KITS 52
3.5.3 ENZYMES 52
3.6 STRAINS AND VECTORS 53
3.6.1 BACTERIAL STRAINS 53
3.6.2 VECTORS 53
3.7 MOLECULAR BIOLOGICAL METHODS 53
3.7.1 DNA ANALYTICAL METHODS 53
3.7.2 PURIFICATION OF PLASMID DNA AND DNA FRAGMENTS 54
3.7.3 GENERATION OF EXPRESSION CONSTRUCTS 55
3.7.4 POLYMERASE CHAIN REACTION 55
3.7.5 RESTRICTION ENDONUCLEASE DIGESTION AND DNA LIGATION 58
3.7.6 PREPARATION AND TRANSFORMATION OF COMPETENT E. COLI DH5A CELLS 58
3.8 PROTEIN BIOCHEMICAL METHODS 59
3.8.1 PROTEIN QUANTIFICATION 59
3.8.2 PREPARATION OF CELL EXTRACTS 59
3.8.3 SDS-PAGE 59
3.8.4 WESTERN BLOTTING 60
3.9 CELL BIOLOGICAL METHODS 61
3.9.1 BASIC CELL CULTURE TECHNIQUES 61
3.9.2 MANIPULATION OF CULTURED CELLS 61
3.9.3 CELL BIOLOGICAL ASSAYS 63
3.10 GENOME-SCALE ESIRNA SCREEN 66
3.10.1 GENOME-SCALE ESIRNA SCREENING PROTOCOL 66
3.10.2 COMPUTATIONAL AND BIOINFORMATIC ANALYSIS 67
3.11 MASS SPECTROMETRY (MS) 67
3.11.1 SILAC MEDIUM AND SAMPLE PREPARATION 67
TABLE OF CONTENTS HI
3.11.2 SDS-PAGE 68
3.11.3 LC-MS/MS 68
4 RESULTS 70
4.1 GENERATION AND VALIDATION OF LUCIFERASE REPORTER CELL LINE 70
4.2 GENOME-SCALE RNAI SCREEN FOR MODULATORS OF THE HEAT-SHOCK RESPONSE
73
4.3 BIOCHEMICAL VALIDATION OF HSP70 MRNA LEVEL AFTER THERMAL STRESS 76
4.4 OVERVIEW OF THE SCREENING RESULTS AND FUNCTIONAL VALIDATION 78
4.4.1 INFLUENCE OF HSR MODULATORS ON CELLULAR PROTEOSTASIS 80
4.4.2 INFLUENCE OF HSR MODULATORS ON FORMATION OF NUCLEAR STRESS BODIES
81
4.5 POSITIVE MODULATORS OF THE HSR 84
4.5.1 REGULATION OF THE HSR BY MULTIPLE NUCLEAR PROTEINS 84
4.5.2 HSR MODULATORS IN THE CYTOSOL AND ORGANELLES 85
4.5.3 ROLE OF THE CELL MEMBRANE IN STRESS SENSING 86
4.6 NEGATIVE MODULATORS OF THE HSR 86
4.7 NUCLEAR PROTEIN NETWORK REGULATING THE HSR 87
4.8 SPECIFIC ROLE OF EP300 IN HSF1 REGULATION 89
4.9 REORGANIZATION OF THE NUCLEAR PROTEOME DURING HEAT STRESS 94
4.10 ROLE OF THE PROTEASOME IN ATTENUATION OF THE HSR 100
4.11 HSR REGULATION AFTER HEAT-SHOCK AND OTHER STRESSES 106
5 DISCUSSION 110
5.1 IDENTIFICATION OF NOVEL HSR MODULATORS ILL
5.2 ROLE OF EP300 IN REGULATING THE HSR 112
5.3 REORGANIZATION OF THE NUCLEAR PROTEOME DURING HEAT STRESS 113
5.4 ATTENUATION OF THE HSR BY THE PROTEASOME 115
5.5 COMPARING THE REGULATORY NETWORKS OF HEAT-SHOCK AND OTHER
PROTEOTOXIC STRESSES .115
5.6 IMPLICATIONS OF THE HSR IN AGING AND DISEASE 117
6 REFERENCES 119
7 APPENDICES 140
TABLE OF CONTENTS IV
7.1 TABLES : 140
7.2 LIST OF ABBREVIATIONS 168
|
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| spelling | Löw, Christian Frank Verfasser aut Regulation of the cytosolic stress response identification of positive and negative modulators by a Genome-Wide RNA interference screen Christian Frank Löw 2014 IV, 172 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Univ., Diss., 2014 Langzeitarchivierung gewährleistet, LZA (DE-588)4113937-9 Hochschulschrift gnd-content Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:19-168322 https://nbn-resolving.org/urn:nbn:de:bvb:19-168322 Resolving-System kostenfrei Volltext http://d-nb.info/1049647998/34 Langzeitarchivierung Nationalbibliothek kostenfrei http://edoc.ub.uni-muenchen.de/16832/ Verlag kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027362940&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Löw, Christian Frank Regulation of the cytosolic stress response identification of positive and negative modulators by a Genome-Wide RNA interference screen |
| subject_GND | (DE-588)4113937-9 |
| title | Regulation of the cytosolic stress response identification of positive and negative modulators by a Genome-Wide RNA interference screen |
| title_auth | Regulation of the cytosolic stress response identification of positive and negative modulators by a Genome-Wide RNA interference screen |
| title_exact_search | Regulation of the cytosolic stress response identification of positive and negative modulators by a Genome-Wide RNA interference screen |
| title_full | Regulation of the cytosolic stress response identification of positive and negative modulators by a Genome-Wide RNA interference screen Christian Frank Löw |
| title_fullStr | Regulation of the cytosolic stress response identification of positive and negative modulators by a Genome-Wide RNA interference screen Christian Frank Löw |
| title_full_unstemmed | Regulation of the cytosolic stress response identification of positive and negative modulators by a Genome-Wide RNA interference screen Christian Frank Löw |
| title_short | Regulation of the cytosolic stress response |
| title_sort | regulation of the cytosolic stress response identification of positive and negative modulators by a genome wide rna interference screen |
| title_sub | identification of positive and negative modulators by a Genome-Wide RNA interference screen |
| topic_facet | Hochschulschrift |
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