Manipulation of host cell apoptosis by Coxiella burnetii type IV secretion system effector proteins CaeA and CaeB: = Manipulation der Wirtszellapoptose durch Coxiella burnetii Typ IV Sekretionssystem Effektorproteine CaeA und CaeB
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| Main Author: | |
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2013
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Item Description: | Zsfassung in engl. und dt. Sprache |
| Physical Description: | 113 S. Ill., graph. Darst. |
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| 100 | 1 | |a Klingenbeck, Leonie |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Manipulation of host cell apoptosis by Coxiella burnetii type IV secretion system effector proteins CaeA and CaeB |b = Manipulation der Wirtszellapoptose durch Coxiella burnetii Typ IV Sekretionssystem Effektorproteine CaeA und CaeB |c vorgelegt von Leonie Klingenbeck |
| 246 | 1 | 1 | |a Manipulation der Wirtszellapoptose durch Coxiella burnetii Typ IV Sekretionssystem Effektorproteine CaeA und CaeB |
| 264 | 1 | |c 2013 | |
| 300 | |a 113 S. |b Ill., graph. Darst. | ||
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| 500 | |a Zsfassung in engl. und dt. Sprache | ||
| 502 | |a Erlangen-Nürnberg, Univ., Diss., 2013 | ||
| 650 | 0 | 7 | |a Apoptosis |0 (DE-588)4291096-1 |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 Wirtszelle |0 (DE-588)4232591-2 |2 gnd |9 rswk-swf |
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Record in the Search Index
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| adam_text | MANIPULATION OF HOST CELL APOPTOSIS BY COXIELLA BURNETII
TYPE IV SECRETION SYSTEM EFFECTOR PROTEINS CAEA AND CAEB
MANIPULATION DER WIRTSZELLAPOPTOSE DURCH COXIELLA BURNETII
TYP IV SEKRETIONSSYSTEM EFFEKTORPROTEINE CAEA UND CAEB
DER NATURWISSENSCHAFTLICHEN FAKULTAT
DER FRIEDRICH-ALEXANDER-UNIVERSITAT ERLANGEN-NIIRNBERG
ZUR
ERLANGUNG DES DOKTORGRADES DR. RER. NAT.
VORGELEGT VON
LEONIE KLINGENBECK
AUS ERLANGEN
HTTP://D-NB.INFO/1053432836
TABLE OF CONTENTS
TABLE OF CONTENTS
1 ABSTRACT 1
2 ZUSAMMENFASSUNG 3
3 INTRODUCTION 5
3.1 Q-FEVER 5
3.2 COXIELLA BURNETII : 6
3.2.1 THE BACTERIUM 6
3.2.2 C. BURNETII INTRACELLULAR LIFESTYLE 7
3.3 TYPE IV SECRETION SYSTEM 9
3.3.1 C. BURNETII TYPE IV SECRETION SYSTEM 10
3.3.2 C. BURNETII EFFECTOR PROTEINS 10
3.4 APOPTOSIS 12
3.4.1 INITIATOR AND EFFECTOR CASPASES DURING APOPTOSIS 12
3.4.2 EXTRINSIC APOPTOSIS 13
3.4.3 INTRINSIC APOPTOSIS 13
3.4.4 INHIBITOR OF APOPTOSIS PROTEINS 16
3.5 C. BURNETII MODULATES HOST CELL APOPTOSIS 17
3.5.1 C. BURNETII INHIBITS INTRINSIC AND EXTRINSIC APOPTOSIS 17
3.5.2 C. BURNETII ANTI-APOPTOTIC EFFECTOR ANKG 18
3.6 C. BURNETII SUMMARY 18
4 GOAL OF THE STUDY 20
5 RESULTS 21
5.1 C. BURNETII EFFECTOR PROTEINS INTERFERE WITH EXTRINSIC AND INTRINSIC
APOPTOSIS 21
5.2 STRUCTURAL FEATURES AND CELLULAR LOCALIZATION OF THE ANTI-APOPTOTIC
EFFECTOR PROTEINS.. 23
5.3 CAEA AND CAEB INHIBIT INTRINSIC APOPTOSIS DEPENDENT ON STIMULUS AND
CELL TYPE 27
5.4 CHARACTERIZATION OF CAEB MEDIATED INHIBITION OF INTRINSIC APOPTOSIS
30
5.4.1 ANTI-APOPTOTIC ACTIVITY OF CAEB IN DIFFERENT STABLE HEK293 CLONES
30
5.4.2 CAEB IS A POTENT APOPTOSIS INHIBITOR 31
5.4.3 STEADY STATE LEVEL OF APOPTOSIS REGULATORS IS NOT INFLUENCED BY
CAEB 32
5.4.4 CAEB BLOCKS INITIATOR AND EFFECTOR CASPASE ACTIVITY 34
5.4.5 NARROWING DOWN THE POINT OF CAEB ACTION 35
I
TABLE OF CONTENTS
5.4.6 CAEB DOES NOT INTERFERE WITH BAX RECRUITMENT TO THE MITOCHONDRIA
38
5.4.7 CAEB INHIBITS PERMEABILIZATION OF THE MITOCHONDRIAL MEMBRANE 39
5.4.8 CREB3 IDENTIFIED AS POTENTIAL CAEB BINDING PARTNER BY YEAST
TWO-HYBRID 41
5.4.9 CO-IMMUNOPRECIPITATION OF CAEB AND CREB3 43
5.5 CHARACTERIZATION OF CAEA MEDIATED INHIBITION OF INTRINSIC APOPTOSIS
44
5.5.1 CAEA MODULATES STEADY STATE LEVEL OF APOPTOSIS REGULATORS 44
5.5.2 CAEA BLOCKS EFFECTOR CASPASE ACTIVATION 45
5.5.3 CAEA INHIBITS PERMEABILIZATION OF THE MITOCHONDRIAL MEMBRANE 47
6 DISCUSSION 49
6.1 IDENTIFICATION OF NOVEL ANTI-APOPTOTIC C. BURNETII EFFECTOR PROTEINS
49
6.2 CHARACTERISTICS OF ANTI-APOPTOTIC EFFECTOR PROTEINS 51
6.3 DIFFERENTIAL MODULATION OF INTRINSIC APOPTOSIS BY CAEA AND CAEB 52
6.4 ANTI-APOPTOTIC MOLECULAR ACTIVITY OF CAEB 53
6.5 DOES CREB3 CONTRIBUTE TO APOPTOSIS INHIBITION BY CAEB ? 57
6.6 ANTI-APOPTOTIC MOLECULAR ACTIVITY OF CAEA 59
6.7 HOW DOES CAEA INHIBIT APOPTOSIS ? 60
6.8 PERSPECTIVES 62
6.9 CONCLUSION 63
7 MATERIAL AND METHODS 65
7.1 MATERIAL 65
7.1.1 ANTIBODIES 65
7.1.2 OLIGONUCLEOTIDES AND PLASMID EXPRESSION VECTORS 66
7.1.3 BUFFERS AND MEDIA 67
7.1.4 CELL LINES, BACTERIAL AND YEAST STRAINS 72
7.1.5 COMMERCIALIZED KITS AND ENZYMES 73
7.1.6 CHEMICALS AND REAGENTS 74
7.1.7 LABORATORY EQUIPMENT 76
7.1.8 SOFTWARE 77
7.2 METHODS 78
7.2.1 MOLECULAR BIOLOGY 78
7.2.1.1 CLONING STRATEGIES 78
7.2.1.2 POLYMERASE CHAIN REACTION 79
II
TABLE OF CONTENTS
7.2.1.3 AGAROSE GEL ELECTROPHORESIS 80
7.2.1.4 PURIFICATION OF DNA FRAGMENTS FROM AGAROSE GELS 80
7.2.1.5 PURIFICATION OF DNA FRAGMENTS FROM SILICA MEMBRANES 81
7.2.1.6 DIGESTION OF DNA WITH RESTRICTION ENZYMES 81
7.2.1.7 LIGATION OF DNA INSERTS INTO PLASMID CLONING VECTORS 82
7.2.1.8 PREPARATION OF CHEMICALLY COMPETENT E.COLI DH1 OP CELLS 82
7.2.1.9 TRANSFORMATION OF PLASMID DNA INTO COMPETENT E.COLI DH 1 OP
CELLS 82
7.2.1.10 SCREENING FOR POSITIVE CLONES 83
7.2.1. U SMALL-SCALE PREPARATION OF PLASMID DNA (MINIPREP) 83
7.2.1.12 LARGE-SCALE PREPARATION OF PLASMID DNA (MIDIPREP) 84
7.2.1.13 DETERMINATION OF DNA CONCENTRATION 85
7.2.1.14 DIGESTION OF CONSTRUCTED PLASMID DNA WITH RESTRICTION ENZYMES
85
7.2.1.15 DNA SEQUENCING 85
7.2.1.16 PREPARATION OF BACTERIAL SAFETY STOCKS 86
7.2.2 CELL CULTURE 86
7.2.2.1 CELL LINES 86
7.2.2.2 CELL SEEDING 87
7.2.2.3 TRANSFECTION OF CELLS WITH FUGENE 6 TRANSFECTION REAGENT 87
7.2.2.4 TRANSFECTION OF CELLS WITH POLYETHYLENIMINE 88
7.2.2.5 GENERATION OF STABLE HEK293 CELLS 88
7.2.3 MICROSCOPIC ANALYSIS 88
7.2.3.1 MOUNTING LIVE CELLS FOR MICROSCOPY 88
7.2.3.2 IMMUNOFLUORESCENCE STAINING 89
7.2.4 APOPTOSIS ASSAYS 89
7.2.4.1 NUCLEAR FRAGMENTATION ASSAY 89
7.2.4.2 TUNEL ASSAY 90
7.2.4.3 PREPARATION OF IMMUNOBLOT LYSATES AFTER APOPTOSIS INDUCTION 90
7.2.4.4 IMMUNOBLOTTING 91
7.2.4.5 TET-ON EXPRESSION SYSTEM 92
7.2.4.6 MITOCHONDRIAL OUTER MEMBRANE PERMEABILIZATION 92
7.2.4.7 ISOLATION OF MITOCHONDRIA 93
7.2.5 YEAST TWO-HYBRID 93
7.2.5.1 TRANSFORMATION OF CAEB IN Y187 94
7.2.5.2 CDNA HELA LIBRARY IN AH109 94
111
TABLE OF CONTENTS
7.2.5.3 TWO-HYBRID MATING OF YEAST STRAINS Y187 AND AH 109 95
7.2.5.4 LACZ FILTER ASSAY 95
7.2.5.5 PLASMID ISOLATION FROM YEAST 96
7.2.5.6 PREPARATION OF ELECTROCOMPETENT E .COLI KC8 CELLS 96
7.2.5.7 TRANSFORMATION OF PLASMID DNA INTO ELECTROCOMPETENT E.COLI KC8
CELLS. 97
7.2.5.8 ISOLATION OF BAIT AND PREY PLASMIDS 97
7.2.5.9 ENZYMATIC DIGEST OF DNA WITH RESTRICTION ENZYMES 97
7.2.5.10 DNA SEQUENCING AND BLAST ANALYSIS 97
7.2.5.11 CO-IMMUNOPRECIPITATION 98
8 ABBREVIATIONS 99
9 REFERENCES 103
10 PUBLICATIONS ILL
11 DANKSAGUNG 112
IV
|
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| spelling | Klingenbeck, Leonie Verfasser aut Manipulation of host cell apoptosis by Coxiella burnetii type IV secretion system effector proteins CaeA and CaeB = Manipulation der Wirtszellapoptose durch Coxiella burnetii Typ IV Sekretionssystem Effektorproteine CaeA und CaeB vorgelegt von Leonie Klingenbeck Manipulation der Wirtszellapoptose durch Coxiella burnetii Typ IV Sekretionssystem Effektorproteine CaeA und CaeB 2013 113 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in engl. und dt. Sprache Erlangen-Nürnberg, Univ., Diss., 2013 Apoptosis (DE-588)4291096-1 gnd rswk-swf Coxiella burneti (DE-588)4252060-5 gnd rswk-swf Wirtszelle (DE-588)4232591-2 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Wirtszelle (DE-588)4232591-2 s Apoptosis (DE-588)4291096-1 s Coxiella burneti (DE-588)4252060-5 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027176882&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Klingenbeck, Leonie Manipulation of host cell apoptosis by Coxiella burnetii type IV secretion system effector proteins CaeA and CaeB = Manipulation der Wirtszellapoptose durch Coxiella burnetii Typ IV Sekretionssystem Effektorproteine CaeA und CaeB Apoptosis (DE-588)4291096-1 gnd Coxiella burneti (DE-588)4252060-5 gnd Wirtszelle (DE-588)4232591-2 gnd |
| subject_GND | (DE-588)4291096-1 (DE-588)4252060-5 (DE-588)4232591-2 (DE-588)4113937-9 |
| title | Manipulation of host cell apoptosis by Coxiella burnetii type IV secretion system effector proteins CaeA and CaeB = Manipulation der Wirtszellapoptose durch Coxiella burnetii Typ IV Sekretionssystem Effektorproteine CaeA und CaeB |
| title_alt | Manipulation der Wirtszellapoptose durch Coxiella burnetii Typ IV Sekretionssystem Effektorproteine CaeA und CaeB |
| title_auth | Manipulation of host cell apoptosis by Coxiella burnetii type IV secretion system effector proteins CaeA and CaeB = Manipulation der Wirtszellapoptose durch Coxiella burnetii Typ IV Sekretionssystem Effektorproteine CaeA und CaeB |
| title_exact_search | Manipulation of host cell apoptosis by Coxiella burnetii type IV secretion system effector proteins CaeA and CaeB = Manipulation der Wirtszellapoptose durch Coxiella burnetii Typ IV Sekretionssystem Effektorproteine CaeA und CaeB |
| title_full | Manipulation of host cell apoptosis by Coxiella burnetii type IV secretion system effector proteins CaeA and CaeB = Manipulation der Wirtszellapoptose durch Coxiella burnetii Typ IV Sekretionssystem Effektorproteine CaeA und CaeB vorgelegt von Leonie Klingenbeck |
| title_fullStr | Manipulation of host cell apoptosis by Coxiella burnetii type IV secretion system effector proteins CaeA and CaeB = Manipulation der Wirtszellapoptose durch Coxiella burnetii Typ IV Sekretionssystem Effektorproteine CaeA und CaeB vorgelegt von Leonie Klingenbeck |
| title_full_unstemmed | Manipulation of host cell apoptosis by Coxiella burnetii type IV secretion system effector proteins CaeA and CaeB = Manipulation der Wirtszellapoptose durch Coxiella burnetii Typ IV Sekretionssystem Effektorproteine CaeA und CaeB vorgelegt von Leonie Klingenbeck |
| title_short | Manipulation of host cell apoptosis by Coxiella burnetii type IV secretion system effector proteins CaeA and CaeB |
| title_sort | manipulation of host cell apoptosis by coxiella burnetii type iv secretion system effector proteins caea and caeb manipulation der wirtszellapoptose durch coxiella burnetii typ iv sekretionssystem effektorproteine caea und caeb |
| title_sub | = Manipulation der Wirtszellapoptose durch Coxiella burnetii Typ IV Sekretionssystem Effektorproteine CaeA und CaeB |
| topic | Apoptosis (DE-588)4291096-1 gnd Coxiella burneti (DE-588)4252060-5 gnd Wirtszelle (DE-588)4232591-2 gnd |
| topic_facet | Apoptosis Coxiella burneti Wirtszelle Hochschulschrift |
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