Modulations of the CD83 molecule on mature dendritic cells by herpes virus infection: = Modulationen des CD83-Moleküls durch Infektion reifer dendritischer Zellen mit Herpesviren
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1. Verfasser: | |
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Format: | Abschlussarbeit Buch |
Sprache: | English German |
Veröffentlicht: |
2014
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Schlagworte: | |
Online-Zugang: | Inhaltsverzeichnis |
Beschreibung: | Zsfassung in engl. und dt. Sprache |
Beschreibung: | IV, 108 S. Ill., graph. Darst. |
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245 | 1 | 0 | |a Modulations of the CD83 molecule on mature dendritic cells by herpes virus infection |b = Modulationen des CD83-Moleküls durch Infektion reifer dendritischer Zellen mit Herpesviren |c vorgelegt von Christiane Silke Heilingloh |
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adam_text | MODULATIONS OF THE CD83 MOLECULE ON MATURE
DENDRITIC CELLS BY HERPES VIRUS INFECTION
MODULATIONEN DES CD83-MOLEKULS DURCH INFEKTION REIFER
DENDRITISCHER ZELLEN MIT HERPESVIREN
DER NATURWISSENSCHAFTLICHEN FAKULTAT
DER FRIEDRICH-ALEXANDER-UNIVERSITAT ERLANGEN-NURNBERG
ZUR
ERLANGUNG DES DOKTORGRADES DR. RER. NAT.
VORGELEGT VON
CHRISTIANE SILKE HEILINGLOH
AUS COBURG
HTTP://D-NB.INFO/1053635036
TABLE OF CONTENTS
TABLE OF CONTENTS
SUMMAIY 1
ZUSAMMENFASSUNG 3
1 INTRODUCTION 5
1.1 THE IMMUNE SYSTEM 5
1.2 DENDRITIC CELLS 8
1.3 THE CD83 MOLECULE 10
1.4 HERPES VIRUSES 12
1.4.1 HUMAN CYTOMEGALOVIRUS (HCMV) 14
1.4.2 HERPES SIMPLEX VIRUS TYPE 1 (HSV-1) 16
1.5 VIRAL IMMUNE EVASION STRATEGIES 18
1.6 UBIQUITIN PROTEASOME SYSTEM 19
1.7 OBJECTIVES 21
2 MATERIALS AND METHODS
22
2.1 MATERIALS 22
2.1.1 LABORATORY EQUIPMENT 22
2.1.2 CONSUMABLES 23
2.1.3 CHEMICALS 23
2.1.4 EUKARYOTIC CELL CULTURE 23
2.1.4.1 MEDIA AND ADDITIVES 23
2.1.4.2 EUKARYOTIC CELLS 23
2.1.5 HUMAN CYTOKINES 23
2.1.6 INHIBITORS 24
2.1.7 ANTIBODIES 24
2.1.8 VIRUSES 25
2.1.9 OLIGONUCLEOTIDES 25
2.1.10 COMMERCIALLY PURCHASED KITS 26
2.1.11 PLASMIDS 27
*2.1.12 BACTERIAL STRAINS 27
2.1.13 SOFTWARE AND WEBSITES 27
2.2 METHODS 28
2.2.1 CELL CULTURE 28
I
TABLE OF CONTENTS
2.2.1.1 CULTIVATION OF CELL LINES 28
2.2.1.2 GENERATION OF DENDRITIC CELLS 28
2.2.1.3 CRYOPRESERVATION OF PBMCS, CELL LINES AND MDCS 29
2.2.1.4 THAWING OF CELLS 29
2.2.1.5 PROPAGATION AND STORAGE OF HCMV STRAINS 29
2.2.1.6 TITRATION OF HCMV STOCKS 30
2.2.1.7 INFECTION OF MDCS WITH HCMV 30
2.2.1.8 CYDOHEXIMIDE (CHX) - ACTINOMYCIN D (ACTD)-CHASE ASSAY 30
2.2.1.9 LUCIFERASE ASSAY 31
2.2.1.10 PROPAGATION AND STORAGE OF HSV-1 STRAINS 31
2.2.1.11 TITRATION OF HSV-1 STOCKS 31
2.2.1.12 INFECTION OF MDCS WITH HSV-1 32
2.2.1.13 ELECTROPORATION OF MDCS WITH DNA 32
2.2.1.14 UV-INACTIVATION OF VIRUSES 33
2.2.1.15 CO-CULTURE OF HSV-1-INFECTED MDCS WITH UNINFECTED MDCS 33
2.2.1.16 TRANSWELL ASSAY 33
2.2.1.17 ISOLATION OF H- AND L-PARTICLES USING A FICOLL 400 GRADIENT 33
2.2.1.18 INCUBATION OF MDCS WITH L-PARTIDES 34
2.2.2 ELECTRON MICROSCOPY 34
2.2.3 FLOW CYTOMETRIC ANALYSES (FACS) AND CELL SORTING 34
2.2.4 BIOPHYSICAL AND BIOCHEMICAL METHODS 35
2.2.4.1 GENERATION OF CELL LYSATES FOR SDS POLYACRYLAMIDE GEL
ELECTROPHORESIS
(SDS-PAGE) 35
2.2.4.2 BRADFORD ASSAY 35
2.2.4.3 DISCONTINUOUS SDS-PAGE (LAEMMLI METHOD) 36
2.2.4.4 WESTERN BLOT ANALYSES 36
2.2.5 MOLECULAR BIOLOGICAL METHODS 37
2.2.5.1 CLONING OF CD83 WT AND CD83 STOP MUTANT 37
2.2.5.1.1 POLYMERASE CHAIN REACTION (PCR) OF CD83 WT AND CD83 STOP
CDS... 37
2.2.5.1.2 AGAROSE GEL ELECTROPHORESIS 37
2.2.5.1.3 PURIFICATION OF DNA FRAGMENTS FROM AGAROSE GELS 38
2.2.5.1.4 TOPO-DONING 38
2.2.5.1.5 TRANSFORMATION OF PLASMID-DNA IN CHEMICAL COMPETENT BACTERIA
38
II
TABTE OF CONTENTS
2.2.5.1.6 ISOLATION OF PLASMID-DNA FOR ANALYTIC PURPOSES 38
2.2.5.1.7 QUANTIFICATION OF NUCLEIC ACIDS 38
2.2.5.1.8 DIGESTION OF PLASMID-DNA 39
2.2.5.1.9 SEQUENCING 39
2.2.5.1.10 ISOLATION OF PLASMID-DNA FOR QUANTITATIVE PURPOSES 39
2.2.5.1.11 DIGESTION OF THE CODING SEQUENCES OF CD83 CONSTRUCTS AND
LIGATION INTO
PBSPGKP+T 39
2.2.5.2 CLONING OF CD83 K177/192/201R AND CD83 K TO R 40
2.2.5.3 TRANSIENT TRANSFECTION USING JETPRIME 40
2.2.5.4 REVERSE TRANSCRIPTASE QUANTITATIVE PCR (RT QPCR) 40
2.2.5.5 FLUORESCENCE IN SITU HYBRIDIZATION (FISH) 41
2.2.6 STATISTICAL ANALYSES 42
3 RESULTS. 43
3.1 GENERATION OF MONOCYTE-DERIVED DENDRITIC CELLS 43
3.2 HCMV-MEDIATED LOSS OF CD83 FROM MDCS 44
3.2.1 MATURE DCS LOOSE CD83 FROM THE CELL SURFACE UPON HCMV INFECTION 44
3.2.2 CD83 IS ALSO LOST INTRACELLULARTY 45
3.2.3 CD83 IS DEGRADED BY THE PROTEASOME 46
3.2.4 AN IMMEDIATE EARLY GENE PRODUCT IS RESPONSIBLE FOR HCMV-MEDIATED
CD83
DEGRADATION 48
3.3 HSV-1 MEDIATED CD83 DEGRADATION 52
3.3.1 HSV-1 MEDIATED CD83 DEGRADATION IS PROTEASOME-DEPENDENT 53
3.3.2 ICP0 IS SUFFICIENT TO SPECIFICALLY INDUCES CD83 DEGRADATION IN
MDCS 54
3.3.3 ICPO-INDUCED CD83 DEGRADATION IS SPECIFIC. 55
3.3.4 LYSINE RESIDUES ARE DISPENSABLE FOR ICPO-INDUCED CD83 DEGRADATION
57
3.3.5 E3 UBIQUITIN LIGASE ACTIVITY OF ICP0 IS NOT ESSENTIAL FOR CD83
DEGRADATION 59
3.3.6 ICPO-INDUCED CD83 DEGRADATION IS UBIQUITIN-INDEPENDENT 61
3.4 REDUCTION OF CD83 IN UNINFECTED BYSTANDER DCS 64
3.4.1 GFP NEGATIVE BYSTANDER DCS LOOSE CD83 AFTER HSV-1 INFECTION 64
3.4.2 LOSS OF CD83 IN BYSTANDER DCS IS TIMELY DELAYED 66
3.4.3 BYSTANDER DCS ARE NOT SECONDARILY INFECTED 66
3.4.4 CD83 LOSS ON BYSTANDER DCS CAN BE TRANSMITTED AND IS
CELL-CELL-CONTACT
DEPENDENT 69
3.4.5 GFP NEGATIVE MDCS CONTAIN VIRAL ENVELOPE PROTEINS BUT NO CAPSID
PROTEINS... 71
III
TABLE OF CONTENTS
3.4.6 UV-INACTIVATED VIRUS LEADS TO REDUCED CD83 LEVELS ON MDCS 72
3.4.7 CD83 LOSS ON BYSTANDER DCS IS TRANSMITTED BY L-PARTICLES 73
4 DISCUSSION 76
4.1 DCS AS A TARGET FOR VIRAL IMMUNE ESCAPE 76
4.2 HCMV-INDUCED CD83 DOWN-MODULATION 77
4.2.1 CD83 IS SPECIFICALLY REDUCED WITH FAST KINETICS 77
4.2.2 INHIBITION OF THE PROTEASOME IS ABLE TO RESCUE CD83 78
4.2.3 AN IMMEDIATE EARLY GENE PRODUCT IS RESPONSIBLE FOR CD83 REDUCTION
79
4.3 HSV-1 MEDIATED CD83 DEGRADATION 80
4.3.1 ICPO INDUCES CD83 DOWN-MODULATION INDEPENDENTLY OF LYSINE RESIDUES
80
4.3.2 C083 DEGRADATION OCCURS INDEPENDENTLY OF E3 UBIQUITIN LIGASE
ACTIVITY OF ICPO
AND OF UBIQUITINATION 81
4.3.3 POSSIBLE UBIQUITIN-INDEPENDENT MECHANISMS FOR CD83 DEGRADATION 81
4.4 L-PARTIDE-MEDIATED TRANSMISSION OF ICPO IN HSV-1 NEGATIVE BYSTANDER
DCS 83
4.4.1 REDUCTION OF CD83 IN UNINFECTED BYSTANDER DCS IS
CELL-CELL-CONTACT-DEPENDENT..
83
4.4.2 L-PARTICLES ARE ABLE TO TRANSMIT FUNCTIONAL ICPO INDUCING CD83
DEGRADATION.... 84
4.5 OUTLOOK
5 REFERENCES..
6 ABBREVIATIONS
7 INDICES
7.1 LIST OF FIGURES
7.2 LIST OF TABLES
PUBLICATIONS AND PRESENTATIONS...
ACKNOWIEDGEMENTS/DANKSAGUNG
..86
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104
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108
|
any_adam_object | 1 |
author | Heilingloh, Christiane Silke |
author_GND | (DE-588)1053092474 |
author_facet | Heilingloh, Christiane Silke |
author_role | aut |
author_sort | Heilingloh, Christiane Silke |
author_variant | c s h cs csh |
building | Verbundindex |
bvnumber | BV041953083 |
ctrlnum | (OCoLC)883093565 (DE-599)BVBBV041953083 |
dewey-full | 610 570 |
dewey-hundreds | 600 - Technology (Applied sciences) 500 - Natural sciences and mathematics |
dewey-ones | 610 - Medicine and health 570 - Biology |
dewey-raw | 610 570 |
dewey-search | 610 570 |
dewey-sort | 3610 |
dewey-tens | 610 - Medicine and health 570 - Biology |
discipline | Biologie Medizin |
format | Thesis Book |
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spelling | Heilingloh, Christiane Silke Verfasser (DE-588)1053092474 aut Modulations of the CD83 molecule on mature dendritic cells by herpes virus infection = Modulationen des CD83-Moleküls durch Infektion reifer dendritischer Zellen mit Herpesviren vorgelegt von Christiane Silke Heilingloh Modulationen des CD83-Moleküls durch Infektion reifer dendritischer Zellen mit Herpesviren 2014 IV, 108 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in engl. und dt. Sprache Erlangen-Nürnberg, Univ., Diss., 2014 Reduktion (DE-588)4177306-8 gnd rswk-swf Antigen CD83 (DE-588)4767577-9 gnd rswk-swf Herpesviren (DE-588)4159627-4 gnd rswk-swf Dendritische Zelle (DE-588)4313846-9 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Herpesviren (DE-588)4159627-4 s Dendritische Zelle (DE-588)4313846-9 s Antigen CD83 (DE-588)4767577-9 s Reduktion (DE-588)4177306-8 s DE-188 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027396010&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
spellingShingle | Heilingloh, Christiane Silke Modulations of the CD83 molecule on mature dendritic cells by herpes virus infection = Modulationen des CD83-Moleküls durch Infektion reifer dendritischer Zellen mit Herpesviren Reduktion (DE-588)4177306-8 gnd Antigen CD83 (DE-588)4767577-9 gnd Herpesviren (DE-588)4159627-4 gnd Dendritische Zelle (DE-588)4313846-9 gnd |
subject_GND | (DE-588)4177306-8 (DE-588)4767577-9 (DE-588)4159627-4 (DE-588)4313846-9 (DE-588)4113937-9 |
title | Modulations of the CD83 molecule on mature dendritic cells by herpes virus infection = Modulationen des CD83-Moleküls durch Infektion reifer dendritischer Zellen mit Herpesviren |
title_alt | Modulationen des CD83-Moleküls durch Infektion reifer dendritischer Zellen mit Herpesviren |
title_auth | Modulations of the CD83 molecule on mature dendritic cells by herpes virus infection = Modulationen des CD83-Moleküls durch Infektion reifer dendritischer Zellen mit Herpesviren |
title_exact_search | Modulations of the CD83 molecule on mature dendritic cells by herpes virus infection = Modulationen des CD83-Moleküls durch Infektion reifer dendritischer Zellen mit Herpesviren |
title_full | Modulations of the CD83 molecule on mature dendritic cells by herpes virus infection = Modulationen des CD83-Moleküls durch Infektion reifer dendritischer Zellen mit Herpesviren vorgelegt von Christiane Silke Heilingloh |
title_fullStr | Modulations of the CD83 molecule on mature dendritic cells by herpes virus infection = Modulationen des CD83-Moleküls durch Infektion reifer dendritischer Zellen mit Herpesviren vorgelegt von Christiane Silke Heilingloh |
title_full_unstemmed | Modulations of the CD83 molecule on mature dendritic cells by herpes virus infection = Modulationen des CD83-Moleküls durch Infektion reifer dendritischer Zellen mit Herpesviren vorgelegt von Christiane Silke Heilingloh |
title_short | Modulations of the CD83 molecule on mature dendritic cells by herpes virus infection |
title_sort | modulations of the cd83 molecule on mature dendritic cells by herpes virus infection modulationen des cd83 molekuls durch infektion reifer dendritischer zellen mit herpesviren |
title_sub | = Modulationen des CD83-Moleküls durch Infektion reifer dendritischer Zellen mit Herpesviren |
topic | Reduktion (DE-588)4177306-8 gnd Antigen CD83 (DE-588)4767577-9 gnd Herpesviren (DE-588)4159627-4 gnd Dendritische Zelle (DE-588)4313846-9 gnd |
topic_facet | Reduktion Antigen CD83 Herpesviren Dendritische Zelle Hochschulschrift |
url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027396010&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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