Inflammatory host cell signalling of epithelial cells in response to Chlamydia trachomatis infection:
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| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Berlin
2013
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| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | IX, 184 S. Ill., graph. Darst. |
Internformat
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| 100 | 1 | |a Peucker, Manuela |d 1976- |e Verfasser |0 (DE-588)1052119484 |4 aut | |
| 245 | 1 | 0 | |a Inflammatory host cell signalling of epithelial cells in response to Chlamydia trachomatis infection |c von Manuela Peucker |
| 264 | 1 | |a Berlin |c 2013 | |
| 300 | |a IX, 184 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
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| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Berlin, Humboldt-Univ., Diss., 2013 | ||
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| 776 | 0 | 8 | |i Reproduziert als |a Peucker, Manuela |t Inflammatory host cell signalling of epithelial cells in response to Chlamydia trachomatis infection |d Berlin : MIK-Center GmbH, 2013 |h 2 Mikrofiches |w (DE-604)BV042058016 |
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Datensatz im Suchindex
| _version_ | 1804152274200559616 |
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| adam_text | TABLE OF CONTENTS
1 INTRODUCTION 11
1.1 CHLAMYDIAE 11
1.1.1 TAXONOMY O/CHLAMYDIAE 11
1.1.2 PATHOLOGY OF CHLAMYDIA (DISEASES ASSOCIATED WITH CHLAMYDIA) 12
1.1.2.1 INFECTIONS WITH OCULAR C. TRACHOMATIS 12
1.1.2.2 GENITAL TRACT INFECTION WITH C. TRACHOMATIS 13
1.1.2.3 THE ACUTE INFLAMMATORY RESPONSE TO GENITAL C. TRACHOMATIS 13
1.1.2.4 INFECTION WITH C. TRACHOMATIS SEROVARS LI, L2 AND L3 16
1.1.2.5 CHLAMYDOPHILA PNEUMONIAE INFECTION 16
1.1.3 DEVELOPMENTAL CYCLE OF CHLAMYDIA 17
1.1.3.1 ATTACHMENT AND ENTRY 18
1.1.3.2 PRIMARY DIFFERENTIATION (ELEMENTARY BODY TO RETICULATE BODY
TRANSITION) 19
1.1.3.3 CHLAMYDIAL INCLUSION: ESCAPE FROM THE ENDOCYTIC PATHWAY 20
1.1.3.4 PROTEINS IN THE CHLAMYDIAL INCLUSION MEMBRANE 20
1.1.3.5 TYPE III SECRETION SYSTEM 21
1.1.3.6 SUBVERSION OF MEMBRANE TRAFFICKING PATHWAYS 22
1.1.3.7 PERSISTENCE 22
1.2 CHLAMYDIA
AND HOST CELL SIGNALING PATHWAYS
23
1.2.1 THE NF-KB SIGNALING PATHWAY 23
1.2.2 MODULATION OF NF-KB SIGNALING BY C. TRACHOMATIS DEUBIQUITINATING
ENZYMES (CHLADUBS)24
1.2.3 MODULATION OF NF-KB SIGNALLING BY CLEAVAGE OFP65 (RELA) 25
1.3 CHLAMYDIA
AND THE INNATE
IMMUNE RESPONSE
26
1.3.1 SIGNALING VIA TOLL-LIKE RECEPTORS (TLRS) 27
1.3.2 SIGNALING VIA NOD-LIKE RECEPTORS (NLRS)
27
1.3.3 SIGNALING VIA THE INFLAMMASOME 28
1.3.4 CYTOKINE/CHEMOKINE INDUCTION UPON CHLAMYDIA INFECTION: THE ROLE
OFLL-8 29
1.4 HISTONE MODIFICATIONS IN
TRANSCRIPTIONAL REGULATION AND IMMUNITY
TO INTRACELLULAR PATHOGENS
31
1.4.1 NATURE AND FUNCTION OF HISTONE MODIFICATIONS 31
1.4.2 HISTONE MODIFICATIONS INDUCED BY INTRACELLULAR BACTERIA 32
1.5 REGULATORS OF NF-KB SIGNALING IN
INFLAMMATION 34
1.5.1 BCL-3 AS A MEMBER OF THE IKB
FAMILY OF NF-KB INHIBITORS 36
VI
HTTP://D-NB.INFO/1054238456
1.5.1.1 MOLECULAR FUNCTIONS OF BCL-3 36
1.5.1.2 POST-TRANSLATIONAL MODIFICATION OF BCL-3 37
1.5.1.3 TRANSCRIPTIONAL REGULATION OF THE BCL-3 GENE 38
1.5.1.4 BCL-3 TARGET GENES 39
1.5.2 TRANSDUCIN FI-LIKE 1 (TBLL)-RELATED PROTEIN (TBLR1) 40
1.5.3 C-TERMINAL BINDING PROTEIN (CTBP) 41
1.6 HTRA AS CHLAMYDIAL EFFECTOR PROTEIN 41
1.7 AIM OF THIS
STUDY 43
2 MATERIALS AND METHODS 45
2.1 MATERIALS AND GENERAL METHODS 45
2.1.1 CELL CULTURE
45
2.1.1.1 CELL LINES AND PROPAGATION OF CELLS 45
2.1.1.2 GENERATION OF CELL LINES STABLY EXPRESSING SHRNA (SHBCL-3) 45
2.1.1.3 INCUBATION WITH HISTONE DEACETYLASE INHIBITORS 47
2.1.1.4 INCUBATION WITH PROTEIN SYNTHESIS INHIBITOR 47
2.1.1.5 INCUBATION WITH HTRA SERINE PROTEASE INHIBITOR 48
2.1.2 BACTERIA 48
2.1.2.1 CHLAMYDIAL STRAINS AND INFECTION OF CELLS 48
2.1.2.2 PREPARATION OF CHLAMYDIAL STOCKS 48
2.1.2.3 DETERMINATION OF THE MOI 49
2.1.2.4 DETERMINATION OF INFECTIOUS PROGENY (INFECTIVITY ASSAY) 49
2.1.3 REAGENTS 50
2.1.4 ANTIBODIES 51
2.1.5 GENERATION OF THE EUKARYOTIC EXPRESSION PLASMIDFOR OVEREXPRESSION
OFC. TRACHOMATIS
SERINE PROTEASE HTRA PDZ DOMAINS FUSED TO GST (GST-HTRA
PDZ
) 51
2.1.6 OTHER EUKARYOTIC EXPRESSION PLASMIDS 53
2.1.7 STATISTICAL ANALYSIS 53
2.2 DNA
TECHNIQUES
54
2.2.1 SIRNA TRANSFECTION 54
2.2.2 TRANSFORMATION OFE. COLI CELLS AND PURIFICATION OFPLASMID DNA
55
2.2.3 PLASMID TRANSFECTION 56
2.2.4 DNA RESTRICTION 57
2.2.5 SEMIQUANTITATIVE PCR
57
2.2.6 PURIFICATION OF PCR PRODUCTS AND GEL EXTRACTION
57
VII
2.2.7 VISUALISATION OF PCR PRODUCTS AND DNA RESTRICTION PRODUCTS BY
AGAROSE GEL
ELECTROPHORESIS 58
2.2.8 RNA ISOLATION 58
2.2.9 QUANTITATIVE REALTIME PCR 58
2.2.10 CHROMATIN IMMUNOPRECIPITATION (CHIP) 60
2.3 PROTEIN TECHNIQUES 61
2.3.1 GEL ELECTROPHORESIS 61
2.3.2 MEMBRANE TRANSFER AND IMMUNOBLOT 62
2.3.3 COLLOIDAL COOMASSIE CBB-G250 STAINING 63
2.3.4 PROCESSING AND MALDI-MS/LS MASS SPECTROMETRY ANALYSIS OF PROTEIN
BANDS SEPARATED BY
SDS-PAGE. 64
2.3.5 ENZYMATIC ASSAY 65
2.3.6 CO-IMMUNOPRECIPITATION (CO-IP) WITH BCL-3-FLAG 65
2.3.7 OVEREXPRESSION AND PURIFICATION OF GST
FUSION PROTEIN 66
2.3.8 PULL DOWN ASSAY 68
2.4 MICROSCOPY 69
3 RESULTS 71
3.1 PART 1: REGULATION OF NF-KB-DEPENDENT INFLAMMATORY GENES
IN RESPONSE TO C.
TRACHOMATIS.
..71
3.1.1 EXPRESSION AND SUBCELLULAR DISTRIBUTION OFBCL-3 IN C.
TRACHOMATIS-INFECTED HELA CELLS 71
3.1.2 C. TRACHOMATIS-/NDUCED EXPRESSION OF NF-KB-DEPENDENT GENES IN
BCL-3-DEPLETED CELLS.... 77
3.1.3 BCL-3 ASSOCIATES WITH TRANSCRIPTIONAL COREGULATORS UPON C.
TRACHOMATIS INFECTION 81
3.1.4 TBLR1 DEPLETION DOES NOT INFLUENCE C. TRACHOMATIS-INDUCED
UPREGULATION OF BCL-3 85
3.1.5 EXPRESSION OF BCL-3-REGULATED GENES IN TBLRL-DEPLETED CELLS UPON
C. TRACHOMATIS INFECTION
88
3.1.6 EXPRESSION OF BCL-3-REGULATED GENES IN DOUBLE KNOCKDOWN OF BCL-3
AND TBLR1 89
3.1.7 CTBP CONTRIBUTES TO STABILITY OFC. TRACHOMATIS-//? C/UC/B/E BCL-3
91
3.1.8 CTBP IS REQUIRED
FOR THE REPRESSIVE ABILITIES OF BCL-3 92
3.1.9 NF-KBL/P50 REGULATES THE TRANSCRIPTIONAL POTENTIAL OF BCL-3 IN
ATTENUATING C.
TRACHOMATIS-INDUCED CHEMOKINE GENE EXPRESSION 94
3.1.10 THE ROLE OFHISTONE DEACETYLASES IN TRANSCRIPTIONAL REGULATION
OFC. TRACHOMATIS-
DEPENDENT GENES 97
3.1.10.1 INHIBITION OF HDACS SYNERGISTICALLY ENHANCES C.
TRACHOMATIS-INDUCED IL-8 GENE
EXPRESSION 99
3.1.10.2
INHIBITION OF HDACS CAUSES A TEMPORAL SHIFT IN C. TRACHOMATIS-RELATED IL
-8
VIII
EXPRESSION TOWARDS EARLIER STAGES IN THE INFECTIOUS CYCLE 100
3.1.10.3 HDAC INHIBITION REGULATES THE EXPRESSION LEVEL OF
NF-KB
FAMILY MEMBERS 105
3.1.10.4 INHIBITION OF HISTONE DEACETYLASES DURING ONGOING CHLAMYDIA
INFECTION DOES NOT
ENHANCE INFECTION-INDUCED IL-8 TRANSCRIPTION ILL
3.1.11 BCL-3 DEPLETION AND HDAC INHIBITION SYNERGISTICALLY ENHANCE C.
TRACHOMATIS-DEPENDENT
IL-8 EXPRESSION 112
3.1.12 RECRUITMENT OF BCL-3 AND HDAC3 AT THE IL-8 GENE PROMOTER IN C.
TRACHOMATIS INFECTED
HELA CELLS 114
3.2 THE ROLE
OF A CHLAMYDIAL EFFECTOR PROTEIN
IN PATHOGENESIS AND INTRACELLULAR SURVIVAL
117
3.2.1 THE C. TRACHOMATIS PERIPLASMIC STRESS RESPONSE SERINE PROTEASE
CTRHTRA IS A POTENTIAL
VIRULENCE FACTOR 117
3.2.2 INHIBITION OF C. TRACHOMATIS HTRA NEGATIVELY AFFECTS PATHOGEN
REPLICATION 119
3.2.3 C. TRACHOMATIS HTRA LOCALIZES OUTSIDE OF REPLICATING BACTERIA TO
THE INCLUSION LUMEN 120
3.2.4 IDENTIFICATION OF HOST CELL INTERACTION PARTNERS OF CHLAMYDIAL
HTRA 121
3.2.4.1 CLONING AND PURIFICATION OF THE TWO PDZ DOMAINS OF C.
TRACHOMATIS HTRA AS GST
FUSION PROTEIN 122
3.2.4.2 GST
PULLDOWN WITH HELA CELL LYSATE USING C. TRACHOMATIS HTRA PDZ DOMAINS AS
BAIT
AND SUBSEQUENT MASS SPECTROMETRY ANALYSIS OF INTERACTION PARTNERS 123
3.2.5 THE C. TRACHOMATIS HTRA SERINE PROTEASE INTERACTS WITH THE HOST
CELL DEAD BOX RNA
HELICASE DDX5/P68 IN VITRO 128
3.2.6 THE DEAD BOX RNA HELICASE DDX5/P68 CO-LOCALIZES WITH CHLAMYDIAL
INCLUSIONS 129
4 DISCUSSION AND OUTLOOK 131
4.1 PART 1: REGULATION OF INFLAMMATORY GENE EXPRESSION BY EPITHELIAL
CELLS IN
RESPONSE TO
C.TRACHOMATIS
131
4.1.1 BCL-3 IS UPREGULATED IN RESPONSE TO C. TRACHOMATIS 133
4.1.2 BCL-3 AS TRANSCRIPTIONAL REPRESSOR OF CB AMYD A-INDUCED
INFLAMMATORY GENES 135
4.1.3 BCL-3 INDUCTION -HOST CELL MECHANISM OR IMMUNE EVASION STRATEGY OF
C. TRACHOMATIS? .136
4.1.4 INTERACTION PARTNERS OF BCL-3 IN THE CHLAMYDIA-INFECTED EPITHELIAL
CELL 138
4.1.5 TBLR1 AND CTBP AS TRANSCRIPTIONAL REPRESSORS OF C.
TRACHOMATIS-RELATED GENES 139
4.1.6 NF-KBL/P50 DEPLETION MIMICS THE EFFECT OF BCL-3 KNOCKDOWN 141
4.1.7 THE IMPACT OF HDAC INHIBITION ON C. TRACHOMATIS-DEPENDENT IL-8
INDUCTION 141
4.1.8 C. TRACHOMATIS-INDUCED HISTONE MODIFICATION AT THE IL-8 PROMOTER
142
4.1.9 CONTRIBUTION OF INDIVIDUAL HDACS TO TRANSCRIPTIONAL REPRESSION OF
IL-8 143
4.1.10 ELUCIDATING THE MECHANISM OF HDAC-DEPENDENT REPRESSION OF IL-8
INDUCTION 145
IX
4.1.11 REGULATION OFHDAC ACTIVITY BY INTERACTING CO-FACTORS 146
4.1.12 IMPACT OFHDACS ON THE EXPRESSION LEVEL OF NF-KB AND IKB FAMILY
MEMBERS IN C.
TRACHOMATIS-INFECTED EPITHELIAL CELLS 147
4.1.13 MODEL OF INFLAMMATORY GENE REGULATION BY CONCERTED ACTION OF
MULTIPLE TRANSCRIPTIONAL
REPRESSORS IN RESPONSE TO C. TRACHOMATIS 148
4.2 PART 2: THE ROLE OF THE
C.
TRACHOMATIS
HTRA SERINE PROTEASE IN
CHLAMYDIA
PATHOGENESIS AND
INTRACELLULAR SURVIVAL 149
5 REFERENCES 153
6 APPENDIX 176
6.1 ABBREVIATIONS 176
6.2 PUBLICATIONS AND POSTERS 181
6.3 CURRICULUM VITAE 182
6.4 ACKNOWLEDGEMENT 183
6.5 STATEMENT OF AUTHORSHIP (SELBSTSTANDIGKEITSERKLARUNG) 184
X
|
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| dewey-ones | 610 - Medicine and health |
| dewey-raw | 610 |
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| spelling | Peucker, Manuela 1976- Verfasser (DE-588)1052119484 aut Inflammatory host cell signalling of epithelial cells in response to Chlamydia trachomatis infection von Manuela Peucker Berlin 2013 IX, 184 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Berlin, Humboldt-Univ., Diss., 2013 (DE-588)4113937-9 Hochschulschrift gnd-content Reproduziert als Peucker, Manuela Inflammatory host cell signalling of epithelial cells in response to Chlamydia trachomatis infection Berlin : MIK-Center GmbH, 2013 2 Mikrofiches (DE-604)BV042058016 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027355100&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Peucker, Manuela 1976- Inflammatory host cell signalling of epithelial cells in response to Chlamydia trachomatis infection |
| subject_GND | (DE-588)4113937-9 |
| title | Inflammatory host cell signalling of epithelial cells in response to Chlamydia trachomatis infection |
| title_auth | Inflammatory host cell signalling of epithelial cells in response to Chlamydia trachomatis infection |
| title_exact_search | Inflammatory host cell signalling of epithelial cells in response to Chlamydia trachomatis infection |
| title_full | Inflammatory host cell signalling of epithelial cells in response to Chlamydia trachomatis infection von Manuela Peucker |
| title_fullStr | Inflammatory host cell signalling of epithelial cells in response to Chlamydia trachomatis infection von Manuela Peucker |
| title_full_unstemmed | Inflammatory host cell signalling of epithelial cells in response to Chlamydia trachomatis infection von Manuela Peucker |
| title_short | Inflammatory host cell signalling of epithelial cells in response to Chlamydia trachomatis infection |
| title_sort | inflammatory host cell signalling of epithelial cells in response to chlamydia trachomatis infection |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027355100&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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