Analysis of host-pathogen interactions during infection of human endothelial and epithelial cells by pathogenic Neisseria species:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Berlin
Mensch & Buch Verl.
2014
|
| Schriftenreihe: | Biology
|
| Schlagworte: | |
| Online-Zugang: | Inhaltstext Inhaltsverzeichnis |
| Beschreibung: | XII, 155 S. Ill., graph. Darst. |
| ISBN: | 9783863874810 |
Internformat
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| 264 | 1 | |a Berlin |b Mensch & Buch Verl. |c 2014 | |
| 300 | |a XII, 155 S. |b Ill., graph. Darst. | ||
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Datensatz im Suchindex
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TABLE OF CONTENTS
I SUMMARY 1
ZUSAMMENFASSUNG 3
II INTRODUCTION
1. THE GENUS NEISSERIA 5
1.1. PATHOGENESIS, CLINICAL MANIFESTATIONS AND MEDICAL IMPACT 5
1.1.1. GONOCOCCAL INFECTIONS 5
1.1.2. MENINGOCOCCAL INFECTIONS 6
1.2. VIRULENCE DETERMINANTS 8
1.2.1. TYPE IV PILI (TFP) 8
1.2.2. OPA AND OPC PROTEINS 10
1.2.3. PORINS 11
1.2.4. SURFACE GLYCANS 12
1.2.5. ADDITIONAL FACTORS MEDIATING VIRULENCE 14
1.3. NEISSERIA-HOST CELL INTERPLAY 15
1.3.1. TFP MEDIATED RECEPTOR INTERACTIONS 15
1.3.2. OPAAA AND OPA
H
S MEDIATED
RECEPTOR INTERACTIONS 16
1.3.3. COURSE OF INFECTION IN NONPHAGOCYTIC CELLS ^ 17
2. NEISSERIA AND BARRIER CROSSING 19
2.1. GENERAL ASPECTS OF EPITHELIAL AND ENDOTHELIAL CELL BARRIERS 19
2.1.1. MOLECULAR STRUCTURE 19
2.1.2. REGULATION OF BARRIER FUNCTION 21
2.1.3. THE BLOOD-BRAIN BARRIER 22
2.1.4. IN VITRO MODELS 22
2.2. TRAVERSAL OF EPITHELIAL CELL BARRIERS BY PATHOGENIC NEISSERIA 23
2.3. INTERACTION OF PATHOGENIC NEISSERIA WITH ENDOTHELIAL CELL BARRIERS
24
3. FUNCTIONAL STUDIES OF NEISSERIA-HOST CELL INTERPLAY 26
3.1. LOSS-OF-FUNCTION SCREENING BASED ON RNA INTERFERENCE 26
3.1.1. RNA INTERFERENCE (RNAI) 26
3.1.2. MIGH-THROUGHPUT SCREENING AND HIGH CONTENT SCREENING 26
3.2. ANALYSIS OF PATHOGEN-HOST CELL INTERPLAY BY RNAI SCREENING 28
4. OBJECTIVES 29
HTTP://D-NB.INFO/1051271959
III MATERIALS AND METHODS
1. EQUIPMENT AND CONSUMABLES 30
1.1. ANTIBODIES 30
1.2. SMALL CHEMICAL COMPOUNDS, INHIBITORS AND ENZYMES. 31
2. BACTERIAL CULTURE 32
2.1. BACTERIA MAINTENANCE AND GROWTH 32
2.2. INFECTION ASSAY 32
3. CELL CULTURE 34
3.1. ISOLATION OF PRIMARY HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVEC)
34
3.2. CELL PROPAGATION, MAINTENANCE AND SEEDING 34
4. GENERAL MOLECULAR BIOLOGICAL METHODS 36
4.1. DISCONTINUOUS SODIUM DODECYL SULPHATE-POLYACRYLAMIDE GEL
ELECTRO-PHORESIS (SDS-
PAGE) 36
4.2. IMMUNOBLOTTING 36
4.3. MICROSCOPY
37
4.3.1. CONFOCAL IMMUNOFLUORESCENCE MICROCOPY (IFM) 37
4.3.2. TRANSMISSION ELECTRON MICROSCOPY (TEM) 37
5. IN VITRO MODEL OF CELL MONOLAYERS: REAL-TIME MONITORING OF BARRIER
STABILITY 38
5.1. CELL SEEDING
AND CULTURE CONDITIONS 38
5.2. REAL-TIME MEASUREMENT OF BARRIER INTEGRITY BY CELLZSCOPE* 38
5.3. INFECTION PROCEDURE FORTRANSWELL CULTURE 39
5.4. RHOA ACTIVATION ASSAY 41
5.5. DATA ANALYSIS AND STATISTICS 41
6. ARRAYED RNAI SCREENING 42
6.1. GENERATION OF REPORTER CELL LINE ME-180ACTRFP03 42
6.2. AUTOMATED SCREENING PROCEDURE: TRANSIENT SIRNATRANSFECTION AND
INFECTION ASSAY
.43
6.3. AUTOMATED MICROSCOPY 44
6.4. DATA ANALYSIS AND STATISTICS 45
IV RESULTS AND DISCUSSION - PART A 'NEISSERIA AND BARRIER CROSSING'
1. RESULTS 49
1.1. IN VITRO MODEL TO STUDY THE BARRIER INTEGRITY OF ENDOTHELIAL CELL
MONOLAYERS 49
1.1.1. CHOICE OF CELL SYSTEM 49
1.1.2. REAL-TIME MEASUREMENT OF THE BARRIER INTEGRITY BY CELLZSCOPE* 55
1.2. EFFECTS OF NEISSERIA
ON ENDOTHELIAL CELL BARRIERS 56
1.2.1. N. MENINGITIDIS AS WELL AS N. GONORRHOEAE STRAINS CAUSE A
SPECIFIC TRANSIENT
DECREASE OF BARRIER INTEGRITY 56
1.2.2. DETERMINATION OF THE BACTERIAL FACTOR INDUCING THE TEMPORARY
DECREASE IN TER
OBSERVED IN HUVEC
MONOLAYERS UPON NEISSERIA INFECTION 61
1.2.2.1. ROLE
OF MENINGOCOCCAL CAPSULE 61
1.2.2.2. ROLE
OF TYPE IV PILI 62
1.2.2.3. ROLE
OF OPA PROTEINS 64
1.2.3. INVOLVEMENT OF HOST CELL SIGNALLING
NETWORKS 68
1.2.3.1. INFLUENCE ON RHOA-RACL EQUILIBRIUM 68
1.2.3.2. ROLE
OF BETA-ADRENERGIC RECEPTORS 71
1.3. SUMMARY OF RESULTS 73
2. DISCUSSION 74
2.1. ESTABLISHMENT OF AN IN VITRO MODEL FOR ENDOTHELIAL CELL BARRIERS 74
2.2. NEISSERIA AND ENDOTHELIAL CELL BARRIER FUNCTION 76
2.2.1. N. MENINGITDIS AND N. GONORRHOEAE CAUSED A TRANSIENT IMPAIRMENT
OF BARRIER
INTEGRITY 77
2.2.2. REDUNDANT FUNCTION OF TFP AND
OPA PROTEINS 80
2.2.3. ENGAGEMENT OF CELLULAR SIGNALLING PATHWAYS 82
2.3. CONCLUDING REMARKS 84
V RESULTS AND DISCUSSION - PART B 'FUNCTIONAL STUDIES OF NEISSERIA-HOST
CEIL INTERPLAY'
1. RESULTS 86
1.1. ASSAY DESIGN 86
1.1.1. EXPERIMENTAL SET-UP 86
1.1.2. QUANTITATIVE IMAGE ANALYSIS 89
1.1.2.1. SCAN
A
R
ASSAY I - QUANTIFICATION OF MC 89
1.1.2.2.
SCAN
A
R ASSAY II
- QUANTIFICATION OF ACTIN RECRUITMENT 91
1.1.3. SET-UP OF DATA ANALYSIS PIPELINES 93
1.2. IMPACT OF THE HUMAN KINOME ON N. GONORRHOEAE INFECTION OF
EPITHELIAL CELLS 95
1.2.1. PRIMARY SCREEN 95
1.2.1.1. SCREENING
PERFORMANCE 95
1.2.1.2. PRELIMINARY HITS AND SELECTION OF GENES FOR HIT VALIDATION 97
1.2.2. SECONDARY SCREEN 100
1.2.2.1. SCREENING
PERFORMANCE 101
1.2.2.2. DETERMINATION OF FINAL HITS 103
1.2.3. IDENTIFICATION OF SIGNALLING PATHWAYS 104
1.2.3.1. ACTIN CYTOSKELETON SIGNALLING 106
1.2.3.2. SIGNAL TRANSDUCTION 108
1.3. SUMMARY OF RESULTS ILL
2. DISCUSSION 112
2.1. ARRAYED RNAI SCREENING
APPROACH 112
2.1.1. ASSAY ESTABLISHMENT 112
2.1.2. SCREENING PERFORMANCE 115
2.2. THE ROLE OF THE HUMAN KINOME DURING N. GONORRHOEAE INFECTION OF
EPITHELIAL CELLS. 116
2.2.1. INVOLVEMENT OF SIGNAL TRANSDUCTION 116
2.2.2. INFLUENCE ON THE ACTIN CYTOSKELETON 119
2.3. CONCLUDING REMARKS 121
VI APPENDIX
1. MATERIALS
122
2. PART A - NEISSERIA AND BARRIER CROSSING I
27
3. PART B - FUNCTIONAL STUDIES OF WE/SSERIO-HOST
CELL INTERPLAY 130
VII REFERENCES 132
LIST OF PUBLICATIONS
155 |
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| dewey-ones | 616 - Diseases |
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| dewey-search | 616.9201 |
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| discipline | Biologie Medizin |
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| spelling | Lange, Claudia 1983- Verfasser (DE-588)1052331424 aut Analysis of host-pathogen interactions during infection of human endothelial and epithelial cells by pathogenic Neisseria species Claudia Lange Berlin Mensch & Buch Verl. 2014 XII, 155 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Biology Zugl.: Berlin, Humboldt-Univ., Diss., 2014 Zellkultur (DE-588)4067547-6 gnd rswk-swf Endothelzelle (DE-588)4152192-4 gnd rswk-swf Wirtszelle (DE-588)4232591-2 gnd rswk-swf Epithelzelle (DE-588)4259648-8 gnd rswk-swf Wechselwirkung (DE-588)4064937-4 gnd rswk-swf Molekularpathologie (DE-588)4170396-0 gnd rswk-swf Neisseria (DE-588)4263608-5 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Neisseria (DE-588)4263608-5 s Molekularpathologie (DE-588)4170396-0 s Wechselwirkung (DE-588)4064937-4 s Wirtszelle (DE-588)4232591-2 s Epithelzelle (DE-588)4259648-8 s Endothelzelle (DE-588)4152192-4 s Zellkultur (DE-588)4067547-6 s DE-604 X:MVB text/html http://deposit.dnb.de/cgi-bin/dokserv?id=4672639&prov=M&dok_var=1&dok_ext=htm Inhaltstext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027368341&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Lange, Claudia 1983- Analysis of host-pathogen interactions during infection of human endothelial and epithelial cells by pathogenic Neisseria species Zellkultur (DE-588)4067547-6 gnd Endothelzelle (DE-588)4152192-4 gnd Wirtszelle (DE-588)4232591-2 gnd Epithelzelle (DE-588)4259648-8 gnd Wechselwirkung (DE-588)4064937-4 gnd Molekularpathologie (DE-588)4170396-0 gnd Neisseria (DE-588)4263608-5 gnd |
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| title | Analysis of host-pathogen interactions during infection of human endothelial and epithelial cells by pathogenic Neisseria species |
| title_auth | Analysis of host-pathogen interactions during infection of human endothelial and epithelial cells by pathogenic Neisseria species |
| title_exact_search | Analysis of host-pathogen interactions during infection of human endothelial and epithelial cells by pathogenic Neisseria species |
| title_full | Analysis of host-pathogen interactions during infection of human endothelial and epithelial cells by pathogenic Neisseria species Claudia Lange |
| title_fullStr | Analysis of host-pathogen interactions during infection of human endothelial and epithelial cells by pathogenic Neisseria species Claudia Lange |
| title_full_unstemmed | Analysis of host-pathogen interactions during infection of human endothelial and epithelial cells by pathogenic Neisseria species Claudia Lange |
| title_short | Analysis of host-pathogen interactions during infection of human endothelial and epithelial cells by pathogenic Neisseria species |
| title_sort | analysis of host pathogen interactions during infection of human endothelial and epithelial cells by pathogenic neisseria species |
| topic | Zellkultur (DE-588)4067547-6 gnd Endothelzelle (DE-588)4152192-4 gnd Wirtszelle (DE-588)4232591-2 gnd Epithelzelle (DE-588)4259648-8 gnd Wechselwirkung (DE-588)4064937-4 gnd Molekularpathologie (DE-588)4170396-0 gnd Neisseria (DE-588)4263608-5 gnd |
| topic_facet | Zellkultur Endothelzelle Wirtszelle Epithelzelle Wechselwirkung Molekularpathologie Neisseria Hochschulschrift |
| url | http://deposit.dnb.de/cgi-bin/dokserv?id=4672639&prov=M&dok_var=1&dok_ext=htm http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027368341&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT langeclaudia analysisofhostpathogeninteractionsduringinfectionofhumanendothelialandepithelialcellsbypathogenicneisseriaspecies |