Functional analysis of the core autophagy protein ATG12 in Dictyostelium discoideum:
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| 1. Verfasser: | |
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| Format: | Abschlussarbeit Mikrofilm Buch |
| Sprache: | English |
| Veröffentlicht: |
Köln
2018
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Beschreibung: | 2 Mikrofiches (VI, 115 Seiten) Illustrationen |
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| 245 | 1 | 0 | |a Functional analysis of the core autophagy protein ATG12 in Dictyostelium discoideum |c vorgelegt von Sarah Fischer |
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Datensatz im Suchindex
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| adam_text | TABLE OF CONTENTS
TABLE OF
CONTENTS......................................................................................................
IV
1
INTRODUCTION...........................................................................................................
1
1.1 THE MODEL ORGANISM DICTYOSTELIUM
DISCOIDEUM......................................................1
1.2 PROTEIN TURNOVER
MECHANISMS...................................................................................3
1.2.1 THE UBIQUITIN-PROTEASOME SYSTEM
(UPS)........................................................3
1.2.2
AUTOPHAGY........................................................................................................
5
1.2.3 CROSSTALK BETWEEN AUTOPHAGY AND THE
UPS..................................................14
1.3 KEY AIMS OF THIS
WORK..............................................................................................
16
1.4
PUBLICATIONS.............................................................................................................16
2
RESULTS..................................................................................................................17
2.1 GENERATION AND VERIFICATION OF ATG12* AND ATG12716 KNOCK-OUT
STRAINS
........
17
2.2 GENERATION OF ATG 12-SPECIFIC MONOCLONAL
ANTIBODIES.........................................19
2.2.1 PURIFICATION OF RECOMBINANT GST-TAGGED
ATG12.........................................19
2.2.2 THE IMMUNISATION OF MICE WITH RECOMBINANT ATG12 RESULTED IN
SPECIFIC
ANTIBODIES.......................................................................................................20
2.3 MUTANT STRAINS SHOW SEVERE DEVELOPMENTAL
DEFECTS.............................................23
2.4 CELL AND SPORE VIABILITY ARE DECREASED IN ATG 12 AND ATG 16
DEFICIENT CELLS
.....
25
2.5 GROWTH BEHAVIOUR IS ALTERED IN ATG12 AND ATG16 KNOCK-OUT STRAINS
................
26
2.5.1 CELL GROWTH IN LIQUID CULTURE IS SLOWED DOWN IN STRAINS LACKING
ATG12 AND
ATG
16............................................................................................................
26
2.5.2 GROWTH ON K. AEROGENES IS OPPOSITELY AFFECTED IN ATG12* AND ATG16*
CELLS................................................................................................................27
2.6 AUTOLYSOSOME FORMATION IS DISTURBED IN MUTANT STRAINS
......................................
28
2.7 PHAGOCYTOSIS OF TRITC-LABELLED YEAST IS LESS EFFICIENT IN MUTANT
STRAINS
...........
30
2.8 ATG12 AND ATG16 DEFICIENCY RESULTS IN IMPAIRED MACROPINOCYTOSIS
................
31
2.9 PROTEIN HOMEOSTASIS IS DISTURBED IN KNOCK-OUT
STRAINS.........................................32
2.9.1 GLOBAL PROTEIN UBIQUITINATION AND PROTEIN AGGREGATES ARE INCREASED
IN
ATG 12 AND ATG 16 DEFICIENT
CELLS............................................................... 32
2.9.2 PROTEASOMAL ACTIVITY IS SIGNIFICANTLY DECREASED IN MUTANT STRAINS
..............
34
2.10
RN ASEQ
ANALYSIS OF AX2, ATG 12*, ATG 16* AND ATG 12716* CELLS
.....................
35
2.10.1 MASSIVE TRANSCRIPTIONAL CHANGES OCCUR IN MUTANT STRAINS
..........................
35
2.10.2 ATG12 AND ATG16 DEFICIENCY RESULTS IN UP-REGULATION OF OTHER CORE
AUTOPHAGY
GENES...........................................................................................
41
2.10.3 GENE ONTOLOGY ANALYSIS REVEALS DIFFERENTIALLY REGULATED ENRICHED
GENE SETS
IN ATG12 ATG16 AND ATG12716* STRAINS
..............................................
43
2.11 SUMMARY OF ANALYSED CELLULAR PROCESSES IN KNOCK-OUT
STRAINS.............................45
3
DISCUSSION.........................................................................................................
46
3.1 IN WILD-TYPE CELLS ONLY ATG12-ATG5 CONJUGATES, BUT NO ATG12 MONOMERS
COULD
BE
DETECTED.............................................................................................................
46
3.2 ATG12 IS PRESENT IN VESICULAR STRUCTURES THROUGHOUT THE CYTOPLASM
...................
47
3.3 ATG12 AND/OR ATG16 DEFICIENCY CAUSED DISTINCT
PHENOTYPES............................49
3.4 ATG12 AND ATG16 ARE REQUIRED FOR EFFICIENT AUTOPHAGOSOME FORMATION
AND
MATURATION...............................................................................................................
51
3.5 PROTEIN HOMEOSTASIS IS IMPAIRED IN ATG12 AND ATG16 DEFICIENT STRAINS
...........
52
3.6 MASSIVE TRANSCRIPTIONAL CHANGES OCCURRED IN ATG12 AND/OR ATG16
DEFICIENT
STRAINS......................................................................................................................54
4 MATERIALS AND
METHODS.....................................................................................56
4.1
MATERIALS..................................................................................................................56
4.1.1
STRAINS............................................................................................................
56
4.1.2
ANTIBODIES......................................................................................................57
4.1.3
PLASMIDS........................................................................................................
57
4.1.4
ANTIBIOTICS.......................................................................................................57
4.1.5
OLIGONUCLEOTIDES............................................................................................58
4.1.6
CHEMICALS......................................................................................................58
4.1.7
ENZYMES........................................................................................................
59
4.1.8
KITS.................................................................................................................
60
4.1.9 MEDIA, BUFFERS AND AGAR
PLATES......................................................................60
4.2
METHODS..................................................................................................................
62
4.2.1 MICROBIOLOGICAL
METHODS................................................................................
62
4.2.2 CELL BIOLOGICAL
METHODS.................................................................................64
4.2.3 MOLECULAR BIOLOGICAL
METHODS.......................................................................
68
4.2.4 BIOCHEMICAL AND IMMUNOLOGICAL METHODS
...................................................
78
4.2.5 IMMUNOFLUORESCENCE
STUDIES........................................................................
85
4.2.6 STATISTICS AND REPRODUCIBILITY
.........................................................................
87
5
REFERENCES..........................................................................................................88
6
ABBREVIATIONS....................................................................................................104
7 LIST OF
FIGURES....................................................................................................107
8 LIST OF
TABLES......................................................................................................109
9
SUMMARY...........................................................................................................110
10
ZUSAMMENFASSUNG...........................................................................................I
L L
11
APPENDIX...........................................................................................................112
12
ERKLAERUNG...........................................................................................................114
13 CURRICULUM
VITAE..............................................................................................115
|
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| author | Fischer, Sarah 1990- |
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| bvnumber | BV045280235 |
| ctrlnum | (OCoLC)1136260263 (DE-599)DNB1163746835 |
| format | Thesis Microfilm Book |
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| institution | BVB |
| language | English |
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| spelling | Fischer, Sarah 1990- Verfasser (DE-588)1162524367 aut Functional analysis of the core autophagy protein ATG12 in Dictyostelium discoideum vorgelegt von Sarah Fischer Köln 2018 2 Mikrofiches (VI, 115 Seiten) Illustrationen txt rdacontent h rdamedia he rdacarrier 24x Dissertation Universität zu Köln 2018 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1163746835/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030667822&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Fischer, Sarah 1990- Functional analysis of the core autophagy protein ATG12 in Dictyostelium discoideum |
| subject_GND | (DE-588)4113937-9 |
| title | Functional analysis of the core autophagy protein ATG12 in Dictyostelium discoideum |
| title_auth | Functional analysis of the core autophagy protein ATG12 in Dictyostelium discoideum |
| title_exact_search | Functional analysis of the core autophagy protein ATG12 in Dictyostelium discoideum |
| title_full | Functional analysis of the core autophagy protein ATG12 in Dictyostelium discoideum vorgelegt von Sarah Fischer |
| title_fullStr | Functional analysis of the core autophagy protein ATG12 in Dictyostelium discoideum vorgelegt von Sarah Fischer |
| title_full_unstemmed | Functional analysis of the core autophagy protein ATG12 in Dictyostelium discoideum vorgelegt von Sarah Fischer |
| title_short | Functional analysis of the core autophagy protein ATG12 in Dictyostelium discoideum |
| title_sort | functional analysis of the core autophagy protein atg12 in dictyostelium discoideum |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1163746835/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030667822&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT fischersarah functionalanalysisofthecoreautophagyproteinatg12indictyosteliumdiscoideum |
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