Characterization of the Coxiella burnetii effector protein AnkF and host cell vimentin during Coxiella containing vacuole biogenesis: = Charakterisierung des C. burnetii Effektorproteins AnkF und Wirtszell-Vimentin in Bezug auf die Reifung der Coxiella-enthaltenden Vakuole
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| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
Erlangen ; Nürnberg
2018
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Physical Description: | v, 143 Seiten Illustrationen, Diagramme 21 cm |
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| 100 | 1 | |a Pechstein, Julian |e Verfasser |0 (DE-588)1177419025 |4 aut | |
| 245 | 1 | 0 | |a Characterization of the Coxiella burnetii effector protein AnkF and host cell vimentin during Coxiella containing vacuole biogenesis |b = Charakterisierung des C. burnetii Effektorproteins AnkF und Wirtszell-Vimentin in Bezug auf die Reifung der Coxiella-enthaltenden Vakuole |c vorgelegt von Julian Pechstein |
| 246 | 1 | 3 | |a Charakterisierung des C. burnetii Effektorproteins AnkF und Wirtszell-Vimentin in Bezug auf die Reifung der Coxiella-enthaltenden Vakuole |
| 246 | 1 | 1 | |a Charakterisierung des C. burnetii Effektorproteins AnkF und Wirtszell-Vimentin in Bezug auf die Reifung der Coxiella-enthaltenden Vakuole |
| 264 | 1 | |a Erlangen ; Nürnberg |c 2018 | |
| 300 | |a v, 143 Seiten |b Illustrationen, Diagramme |c 21 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Friedrich-Alexander-Universität Erlangen-Nürnberg |d 2018 | ||
| 650 | 0 | 7 | |a Vermehrung |0 (DE-588)4137211-6 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Vimentin |0 (DE-588)4265164-5 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Coxiella burneti |0 (DE-588)4252060-5 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Vakuole |0 (DE-588)4187316-6 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 689 | 0 | 0 | |a Coxiella burneti |0 (DE-588)4252060-5 |D s |
| 689 | 0 | 1 | |a Vermehrung |0 (DE-588)4137211-6 |D s |
| 689 | 0 | 2 | |a Vakuole |0 (DE-588)4187316-6 |D s |
| 689 | 0 | 3 | |a Vimentin |0 (DE-588)4265164-5 |D s |
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| 999 | |a oai:aleph.bib-bvb.de:BVB01-030760687 | ||
Record in the Search Index
| _version_ | 1804179220147994624 |
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| adam_text | 1 INTRODUCTION
1
1.1 COXIELLA
BURNETII............................................................................................................
1
1.1.1. HISTORY AND
TAXONOMY.......................................................................................1
1.1.2.
BACTERIOLOGY......................................................................................................1
1.1.3.
EPIDEMIOLOGY....................................................................................................
2
1.1.4. Q
FEVER..............................................................................................................3
1.1.5. INTRACELLULAR LIFE CYCLE AND
COXIELLA CONTAINING VACUOLE BIOGENESIS
.................
4
1.1.6. C. BURNETII TYPE 4 B SECRETION
SYSTEM..........................................................10
1.1.7. EFFECTOR PROTEINS OF C. BURNETII
.......................................................................
12
1.1.8. THE C. BURNETII EFFECTOR ANKF
.........................................................................
14
1.2 THE INTERMEDIATE FILAMENT
VIMENTIN............................................................................
16
1.2.1. PHYSIOLOGICAL ROLE OF VIMENTIN
........................................................................
17
1.2.2. THE INFLUENCE OF VIMENTIN DURING INFECTION WITH
PATHOGENS............................17
1.3 AIM OF THIS
STUDY........................................................................................................
20
2 MATERIALS AND
METHODS..................................................................................................
22
2.1
MATERIALS......................................................................................................................22
2.1.1. PRIMARY
ANTIBODIES..........................................................................................22
2.1.2. SECONDARY
ANTIBODIES.....................................................................................22
2.1.3. BACTERIAL
STRAINS..............................................................................................23
2.1.4. BACTERIAL CULTURE MEDIA
..................................................................................
23
2.1.5. BUFFERS AND
REAGENTS......................................................................................24
2.1.6. CELL CULTURE
MEDIA...........................................................................................27
2.1.7. CELL
LINES.........................................................................................................
27
2.1.8.
CHEMICALS......................................................................................................
28
2.1.9. COMMERCIALLY AVAILABLE KITS AND STANDARDS
...................................................
30
2.1.10.
CONSUMABLES.................................................................................................
30
2.1.11.
ENZYMES........................................................................................................
32
2.1.12.
EQUIPMENT......................................................................................................
32
2.1.13.
OLIGONUCLEOTIDES.............................................................................................34
2.1.14. PLASMIDS USED FOR CLONING
..............................................................................
35
2.1.15. CLONED
PLASMIDS..............................................................................................36
2.1.16. COMPUTER SOFTWARE
.........................................................................................
36
2.2 MICROBIOLOGICAL
METHODS............................................................................................
37
2.2.1. CULTIVATION OF E.
COLI........................................................................................
37
2.2.2. STORAGE OF E COLI................................................
37
2.2.3. PREPARATION OF CHEMICALLY-COMPETENT E.
COLI..................................................37
2.2.4. TRANSFORMATION OF CHEMICALLY-COMPETENT E. COLI
............................................
38
2.2.5. AXENIC CULTIVATION OF C.
BURNETII.......................................................................38
2.2.6. GENERATING RECOMBINANT C. BURNETII BY ELECTROPORATION
.................................
36
2.2.7. PLATING AND CLONAL ISOLATION OF C.
BURNETII.......................................................39
2.2.6. IPTG INDUCED PROTEIN EXPRESSION OF AXENICALLY-GROWN C. BURNETII
..............
40
2.2.9. LONG-TERM STORAGE OF AXENICALLY-GROWN C. BURNETII
........................................
40
2.2.10. ACCM-2 GROWTH
TEST.......................................................................................
40
2.3 MOLECULAR BIOLOGY
METHODS........................................................................................
41
2.3.1. POLYMERASE CHAIN REACTIONS
(PCR)................................................................ 41
2.3.2. REVERSE TRANSCRIPTION OF RNA INTO
CDNA.......................................................43
2.3.3. QUANTITATIVE REAL-TIME PCR
(QPCR)............................................................... 43
2.3.4. ANALYTICAL AGAROSE GEL
ELECTROPHORESIS..........................................................45
2.3.5. ISOLATION OF PLASMID DNA FROM E.
COLI..............................................................45
2.3.6. PURIFICATION OF RNA FROM SILICA MEMBRANES
....................................................
46
2.3.7. PURIFICATION OF DNA FROM SILICA MEMBRANES
....................................................
46
2.3.8. PURIFICATION OF DNA FROM AGAROSE
GELS...........................................................46
2.3.9. MEASUREMENT OF DNA AND RNA CONCENTRATION
.............................................
46
2.3.10. DIGESTION OF DNA WITH RESTRICTION ENDONUCLEASES
..........................................
46
2.3.11. LIGATION OF DIGESTED PCR FRAGMENTS AND CLONING VECTORS
..............
47
2.3.12. LIGATION OF NON-DIGESTED PCR FRAGMENTS AND DIGESTED CLONING
VECTORS
(GIBSON
ASSEMBLY)......................................................................................................47
2.3.13. SEQUENCING OF CLONED DNA
CONSTRUCTS..........................................................48
2.3.14. PREPARATION OF C. BURNETII NMII GDNA FROM AXENIC CULTURES
.........................
48
2.4 CELL CULTURE
METHODS..................................................................................................
49
2.4.1 CONTINUOUS PASSAGING OF CELL
LINES................................................................49
2.4.2 LONG-TERM STORAGE OF CELL LINES
......................................................................
49
2.4.3 SEEDING OF CELL LINES FOR
EXPERIMENTS.............................................................49
2.4.4 INDUCTION OF STABLE HELA-PWHE644/655-ANKF CELLS FOR ANKF
EXPRESSION ....50
2.4.5 HARVEST OF STABLE HELA-PWHE644/655-ANKF CELLS FOR ISOLATION OF
TOTAL RNA50
2.4.6 TRANSFECTION OF CELL LINES WITH PLASMID DMA AND PEI
.....................................
51
2.4.7 TRANSFECTION OF CELL LINES WITH PLASMID DMA AND X-TREMEGENE* 9
............
51
2.4.8 TRANSFECTION OF HELA CELLS WITH ON-TARGETPLUS HUMAN SIRNA POOLS
AND
DHARMAFECT TRANSFECTION
REAGENT................................................................................51
2.4.9 INFECTION OF CELL LINES WITH C. BURNETII
.............................................................
52
2.4.10 PROPAGATION AND HARVEST OF C. BURNETII FOR LONG-TERM
STORAGE........................52
2.4.11 LYSIS OF CELL LINES FOR WB
ANALYSIS.................................................................53
2.4.12 LYSIS OF CELL LINES FOR IMMUNOPRECIPITATION
.....................................................
53
2.4.13 HARVEST OF C. BURNETII FROM INFECTED CELLS FOR QPCR ANALYSIS
........................
53
2.5 PROTEIN BIOCHEMICAL METHODS
....................................................................................
54
2.5.1 IMMUNOPRECIPITATION AND SAMPLE PREPARATION FOR NANOLC-MS/MS
.............
54
2.5.2 PROCESSING OF LAEMMLI LYSATES AND SUBSEQUENT SDS-PAGE
.......................
54
2.5.3 WESTERN
BLOT...................................................................................................
54
2.5.4 IMMUNODETECTION OF PROTEINS ON PVDF MEMBRANES
......................................
55
2.6
IMMUNOCYTOCHEMISTRY................................................................................................56
2.6.1
FIXATION..........................................................................................................
56
2.6.2 INDIRECT IMMUNOFLUORESCENCE
ANALYSIS...........................................................56
2.6.3 INCUBATION OF CELLS WITH DQ-RED BSA
............................................................
56
2.6.4 DIFFERENTIAL
IMMUNOFLUORESCENCE...................................................................57
2.7 FLUORESCENCE MICROSCOPY
ANALYSES..........................................................................
57
3
RESULTS..............................................................................................................................
58
3.1 ECTOPIC ANKF MEDIATES RE-ARRANGEMENT OF VIMENTIN FILAMENTS AND
CO-LOCALIZATION.. 58
3.2 LOCALIZATION STUDIES OF THE C. BURNETII EFFECTOR ANKF
.................................................
59
3.3 VIMENTIN CLOSELY ASSOCIATES WITH THE CC
V................................................................61
3.4 VIMENTIN IS RECRUITED TO THE CCV IN A TIME-DEPENDENT MANNER
................................
63
3.5 VIMENTIN ASSOCIATES WITH THE CCV BY POLYMERIZATION ALONG THE CCV
SURFACE
.......
64
3.6 NEITHER ANKF NOR C. BURNETII INFECTION AFFECT VIMENTIN EXPRESSION
............................
65
3.7 THE C. BURNETII EFFECTOR ANKF IS ESSENTIAL FOR CCV
FORMATION...................................69
3.8 C. BURNETI HANKF::!N#1775 RESIDES IN A PHAGOLYSOSOMAL COMPARTMENT
...................
73
3.9 RESTORATION OF THE C. BURNETII-ANKF::TN#1775 GROWTH DEFECT DURING
INFECTION
..........
76
3.10 VIMENTIN IS DISPENSABLE FOR INTRACELLULAR REPLICATION OF C.
BURNETII.............................78
3.11 OTHER CYTOSKELETAL COMPONENTS ASSOCIATE WITH THE CC V
..........................................
81
3.12 IDENTIFICATION OF NEW BINDING PARTNERS FOR
ANKF.........................................................83
4
DISCUSSION..........................................................................................................................85
4.1 THE INTERACTION BETWEEN ANKF AND
VIMENTIN.............................................................85
4.2 VIMENTIN ALONE IS REDUNDANT FOR CCV
BIOGENESIS......................................................89
4.3 OTHER HOST CELL PROTEINS AND PROCESSES COULD SUPPORT CCV BIOGENESIS
IN CONJUNCTION
WITH
VIMENTIN........................................................................................................................
91
4.4 THE CONTRIBUTION OF VIMENTIN TO HOST CELL INVASION BY C.
BURNETII.................................93
4.5 METHODOLOGICAL ISSUES ON SIRNA-MEDIATED KNOCK-DOWN OF VIMENTIN
.......................
94
4.6 THE ROLE OF THE EFFECTOR PROTEIN ANKF DURING CCV BIOGENESIS
....................................
95
4.7 NEW BINDING PARTNERS FOR ANKF INVOLVED IN HOST CELL METABOLISM
...............................
99
5
CONCLUSION........................................................................................................................101
6
SUMMARY...........................................................................................................................
103
7
ZUSAMMENFASSUNG..........................................................................................................
105
8
INDEX..................................................................................................................................
107
8.1 LIST OF
FIGURES............................................................................................................
107
8.2 LIST OF
TABLES.............................................................................................................
108
8.3 LIST OF
ABBREVIATIONS....................................................................................
109
9
REFERENCES........................................................................................................................116
10
PUBLICATIONS......................................................................................................................140
11 CONFERENCE
CONTRIBUTIONS...............................................................................................140
10.1
TALKS..........................................................................................................................140
10.2
POSTER........................................................................................................................140
10.3
AWARDS..................................................................................................................141
12
DANKSAGUNG................................................................................................................
142
|
| any_adam_object | 1 |
| author | Pechstein, Julian |
| author_GND | (DE-588)1177419025 |
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| building | Verbundindex |
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| ctrlnum | (OCoLC)1090102785 (DE-599)DNB1171495579 |
| discipline | Biologie |
| format | Thesis Book |
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| language | English |
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| spelling | Pechstein, Julian Verfasser (DE-588)1177419025 aut Characterization of the Coxiella burnetii effector protein AnkF and host cell vimentin during Coxiella containing vacuole biogenesis = Charakterisierung des C. burnetii Effektorproteins AnkF und Wirtszell-Vimentin in Bezug auf die Reifung der Coxiella-enthaltenden Vakuole vorgelegt von Julian Pechstein Charakterisierung des C. burnetii Effektorproteins AnkF und Wirtszell-Vimentin in Bezug auf die Reifung der Coxiella-enthaltenden Vakuole Erlangen ; Nürnberg 2018 v, 143 Seiten Illustrationen, Diagramme 21 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Friedrich-Alexander-Universität Erlangen-Nürnberg 2018 Vermehrung (DE-588)4137211-6 gnd rswk-swf Vimentin (DE-588)4265164-5 gnd rswk-swf Coxiella burneti (DE-588)4252060-5 gnd rswk-swf Vakuole (DE-588)4187316-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Coxiella burneti (DE-588)4252060-5 s Vermehrung (DE-588)4137211-6 s Vakuole (DE-588)4187316-6 s Vimentin (DE-588)4265164-5 s DE-604 B:DE-101 application/pdf http://d-nb.info/1171495579/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030760687&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Pechstein, Julian Characterization of the Coxiella burnetii effector protein AnkF and host cell vimentin during Coxiella containing vacuole biogenesis = Charakterisierung des C. burnetii Effektorproteins AnkF und Wirtszell-Vimentin in Bezug auf die Reifung der Coxiella-enthaltenden Vakuole Vermehrung (DE-588)4137211-6 gnd Vimentin (DE-588)4265164-5 gnd Coxiella burneti (DE-588)4252060-5 gnd Vakuole (DE-588)4187316-6 gnd |
| subject_GND | (DE-588)4137211-6 (DE-588)4265164-5 (DE-588)4252060-5 (DE-588)4187316-6 (DE-588)4113937-9 |
| title | Characterization of the Coxiella burnetii effector protein AnkF and host cell vimentin during Coxiella containing vacuole biogenesis = Charakterisierung des C. burnetii Effektorproteins AnkF und Wirtszell-Vimentin in Bezug auf die Reifung der Coxiella-enthaltenden Vakuole |
| title_alt | Charakterisierung des C. burnetii Effektorproteins AnkF und Wirtszell-Vimentin in Bezug auf die Reifung der Coxiella-enthaltenden Vakuole |
| title_auth | Characterization of the Coxiella burnetii effector protein AnkF and host cell vimentin during Coxiella containing vacuole biogenesis = Charakterisierung des C. burnetii Effektorproteins AnkF und Wirtszell-Vimentin in Bezug auf die Reifung der Coxiella-enthaltenden Vakuole |
| title_exact_search | Characterization of the Coxiella burnetii effector protein AnkF and host cell vimentin during Coxiella containing vacuole biogenesis = Charakterisierung des C. burnetii Effektorproteins AnkF und Wirtszell-Vimentin in Bezug auf die Reifung der Coxiella-enthaltenden Vakuole |
| title_full | Characterization of the Coxiella burnetii effector protein AnkF and host cell vimentin during Coxiella containing vacuole biogenesis = Charakterisierung des C. burnetii Effektorproteins AnkF und Wirtszell-Vimentin in Bezug auf die Reifung der Coxiella-enthaltenden Vakuole vorgelegt von Julian Pechstein |
| title_fullStr | Characterization of the Coxiella burnetii effector protein AnkF and host cell vimentin during Coxiella containing vacuole biogenesis = Charakterisierung des C. burnetii Effektorproteins AnkF und Wirtszell-Vimentin in Bezug auf die Reifung der Coxiella-enthaltenden Vakuole vorgelegt von Julian Pechstein |
| title_full_unstemmed | Characterization of the Coxiella burnetii effector protein AnkF and host cell vimentin during Coxiella containing vacuole biogenesis = Charakterisierung des C. burnetii Effektorproteins AnkF und Wirtszell-Vimentin in Bezug auf die Reifung der Coxiella-enthaltenden Vakuole vorgelegt von Julian Pechstein |
| title_short | Characterization of the Coxiella burnetii effector protein AnkF and host cell vimentin during Coxiella containing vacuole biogenesis |
| title_sort | characterization of the coxiella burnetii effector protein ankf and host cell vimentin during coxiella containing vacuole biogenesis charakterisierung des c burnetii effektorproteins ankf und wirtszell vimentin in bezug auf die reifung der coxiella enthaltenden vakuole |
| title_sub | = Charakterisierung des C. burnetii Effektorproteins AnkF und Wirtszell-Vimentin in Bezug auf die Reifung der Coxiella-enthaltenden Vakuole |
| topic | Vermehrung (DE-588)4137211-6 gnd Vimentin (DE-588)4265164-5 gnd Coxiella burneti (DE-588)4252060-5 gnd Vakuole (DE-588)4187316-6 gnd |
| topic_facet | Vermehrung Vimentin Coxiella burneti Vakuole Hochschulschrift |
| url | http://d-nb.info/1171495579/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030760687&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT pechsteinjulian characterizationofthecoxiellaburnetiieffectorproteinankfandhostcellvimentinduringcoxiellacontainingvacuolebiogenesischarakterisierungdescburnetiieffektorproteinsankfundwirtszellvimentininbezugaufdiereifungdercoxiellaenthaltendenvakuole AT pechsteinjulian charakterisierungdescburnetiieffektorproteinsankfundwirtszellvimentininbezugaufdiereifungdercoxiellaenthaltendenvakuole |
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