The role of DUSP16 and DUSP9 in the innate immune system: = Die Funktion von DUSP16 und DUSP9 im angeborenen Immunsystem
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2013
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | XI, 166 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a The role of DUSP16 and DUSP9 in the innate immune system |b = Die Funktion von DUSP16 und DUSP9 im angeborenen Immunsystem |c vorgelegt von Magdalena Ewa Niedzielska |
| 246 | 1 | 1 | |a Die Funktion von DUSP16 und DUSP9 im angeborenen Immunsystem |
| 264 | 1 | |c 2013 | |
| 300 | |a XI, 166 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
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| 502 | |a Erlangen-Nürnberg, Univ., Diss., 2013 | ||
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| adam_text | CONTENTS L
CONTENTS
INDEX OF FIGURES V
INDEX OF TABLES VI
ABBREVIATIONS VII
1 INTRODUCTION 1
1.1 THE IMMUNE SYSTEM 1
1.2 THE MONONUCLEAR PHAGOCYTE SYSTEM (MPS) 2
1.2.1 MONOCYTES 3
1.2.2 MACROPHAGES 4
1.2.3 DENDRITIC CELLS 6
1.2.3.1 CONVENTIONAL DENDRITIC CELLS (CDCS) 6
1.2.3.2 PLASMACYTOID DENDRITIC CELLS (PDCS) 7
1.3 PATTERN RECOGNITION RECEPTORS (PRR) 8
1.3.1 TOLL-LIKE RECEPTORS (TLRS) 8
1.3.1.1 TLR SIGNALING 10
1.3.1.1.1 THE MYD88-DEPENDENT SIGNALING PATHWAY 10
1.3.1.1.2 TRIF-DEPENDENT PATHWAY 11
1.4 NEGATIVE REGULATION OF IMMUNE RESPONSES - MULTIPLE MECHANISMS 13
1.4.1 EXTRACELLULAR REGULATION - SOLUBE
FACTORS, HORMONES AND CYTOKINES 13
1.4.2 TRANSMEMBRANE REGULATORS 14
1.4.3 INTRACELLULAR NEGATIVE REGULATORS 14
1.4.3.1 CYTOPLASMIC PROTEINS 14
1.4.3.2 TRANSCRIPTIONAL REGULATION 15
1.4.3.3 PHOSPHATASES 15
1.4.3.3.1 DUAL SPECIFICITY PHOSPHATASES (DUSP) 16
1.4.3.3.2 MAPK PHOSPHATASES * REGULATING THE IMMUNE RESPONSE 17
1.4.3.3.2.1 DUSP16 19
1.4.3.3.2.2 DUSP9 21
2 AIM OF THE STUDY 22
3 MATERIALS 23
3.1 ANESTHETICS 23
3.2 ANTIBIOTICS 23
3.3 CYTOKINES 23
3.4 CHEMICALS AND REAGENTS 23
3.5 ENZYMES 25
3.6 KITS AND SETS 25
3.7 MEDIA AND MEDIA SUPPLEMENTS 26
3.8 PATTERN RECOGNITION RECEPTOR (PRR) AGONISTS 26
3.9 SIRNA 26
3.10 PLASMIDS 27
3.11 ANTIBODIES 27
3.12 PRIMERS 29
3.13 CONSUMABLE ITEMS 32
3.14 LABORATORY EQUIPMENT 33
3.15 DATABASES AND SOFTWARE 34
4 METHODS 35
4.1 CELL CULTURE 35
4.1.1 CELL LINES 35
4.1.1.1 GENERAL CELL CULTURE 35
4.1.1.2 CRYOPRESERVATION OF CELLS 36
4.1.2 PRIMARY CELLS 36
4.1.2.1 PREPARATION OF L-CELL-CONDITIONED
MEDIUM (LCCM) 36
4.1.2.2 PREPARATION OF GM-CSF-CONDITIONED MEDIUM 36
HTTP://D-NB.INFO/1037033078
II CONTENTS
4.1.2.3 PREPARATION OF BONE MAIROW CELLS 36
4.1.2.4 GENERATION OF MURINE BONE MARROW-DERIVED MACROPHAGES (BMDM) 37
4.1.2.5 GENERATION OF MURINE BONE MARROW-DERIVED MACROPHAGES IN TEFLON
BAGS 37
4.1.2.6 GENERATION OF MURINE FETAL LIVER-DERIVED
MACROPHAGES (FLDM) AND DENDRITIC CELLS
(FLDC) 37
4.1.2.7 GENERATION OF MURINE BONE MARROW-DERIVED DENDRITIC CELLS
(BMDC) 38
4.1.2.8 GENERATION OF FR3L- DRIVEN DENDRITIC
CELLS FROM BONE MARROW 38
4.1.2.9 ISOLATION OF RESIDENT
PERITONEAL CELLS 38
4.1.2.10 ISOLATION OF BRONCHIAL IMMUNE CELLS 38
4.1.2.11 PREPARATION OF SINGLE CELL SUSPENSION FROM DIFFERENT ORGANS 38
4.1.2.12 ISOLATION OF HUMAN DC SUBSETS 39
4.2 IMMUNOASSAYS 39
4.2.1 ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) 39
4.2.1.1 INTERFERON^ ELISA 39
4.2.2 ROW CYTOMETRY 40
4.2.2.1 SURFACE MARKER STAINING 40
4.2.2.2 FLUORESCENCE ACSVATED CELL SORTER (FACS) 40
4.2.3 MAGNETIC ACTIVATED CELL SORTING
(MACS) 40
4.2.4 NO ASSAY 41
4.2.5 CELL PROLIFERATION ASSAY 41
4.2.6 MIXED LEUKOCYTE REACTION (MLR) 41
4.2.7 GENTAMICIN PROTECTION ASSAY 41
4.3 MOLECULAR BIOLOGY 42
4.3.1 ISOLATION OF GENOMIC DNA 42
4.3.2 ISOLATION OF PLASMID DNA 42
4.3.3 POLYMERASE CHAIN REACTION (PCR) 43
4.3.4 PURIFICATION OF DNA FROM PCR
REACTION 44
4.3.5 AGAROSE GEL ELECTROPHORESIS 44
4.3.6 PURIFICATION OF DNA FROM AGAROSE GELS 44
4.3.7 DNA DIGESTION 44
4.3.8 LIGATION 45
4.3.9 TOPO CLONING 45
4.3.10 T RANSFORMATION 45
4.3.11 SEQUENCING 46
4.3.12 TRANSFECTION OF EUKARYOTIC CELLS 46
4.3.12.1 CALCIUM-PHOSPHATE METHOD 46
4.3.12.2 LIPOFECTION 46
4.3.12.3 ELECTROPORATION 46
4.3.12.4 MRNA TRANSFECTION 47
4.3.12.4.1 PRODUCTION OF IN
VITRO TRANSCRIBED RNA 47
4.3.12.4.2 ELECTROPORATION OF DCS 47
4.3.13 T RANSDUCTION OF EUKARYOTIC CELLS 47
4.3.13.1 PRODUCTION OF VIRUS 47
4.3.13.2 INFECTION 48
4.3.14 SIRNA DELIVERY 48
4.3.14.1 PREPARATION OF SIRNA 48
4.3.14.2 TRANSFECTION OF PRIMARY CELLS 48
4.3.15 TOTAL RNA EXTRACTION 49
4.3.15.1 PHASE SEPARATION METHOD 49
4.3.15.2 RNA ISOLATION KIT 49
4.3.16 FIRST STRAND CDNA SYNTHESIS 49
4.3.17 RELATIVE QUANTIFICATION OF GENE EXPRESSION BY REAL-TIME PCR
ANALYSIS 49
4.3.18 MICROAIRAY ANALYSIS OF GENE EXPRESSION 50
4.3.19 NORTHERN BLOT 50
4.3.20 SOUTHERN BLOT 51
4.4 PROTEIN BIOCHEMISTRY 51
4.4.1 CELL LYSIS 51
4.4.2 PREPARATION OF CYTOPLASMIC AND NUCLEAR EXTRACTS 52
4.4.3 SDS-POLYACRYLAMID GEL ELECTROPHORESIS (PAGE) OF PROTEINS 52
4.4.4 WESTERN BLOT ANALYSIS 53
CONTENTS III
4.4.5 MTT ASSAY 53
4.4.6 LUCIFERASE REPORTER GENE ASSAY 54
4.4.7 STAININNG TOR IACZ-ACTIVITY 54
4.4.7.1 WHOLE-MOUNT STAINING OF EMBRYOS 54
4.4.7.2 STAINING FOR LACZ ACTIVITY IN CED CULTURE 54
4.5 ANIMAL EXPERIMENTS 55
4.5.1 MICE 55
4.5.1.1 GENERATION OF DUSP16 GENE TRAP MUTANT MICE 55
4.5.2 ANESTHESIA AND EUTHANASIA 55
4.5.3 REMOVAL OF ORGANS 55
4.5.4 GENERATION OF RADIATION CHIMERAS 56
4.5.4.1 PREPARATION OF EMBRYOS AND FETAL LIVEIS 56
4.5.4.2 IRRADIATION AND RECONSTITUTION OF RECIPIENT MICE 56
4.5.5 LPS-INDUCED SHOCK 56
4.5.6 HYDRODYNAMIC INJECTION 56
4.5.7 PATHOLOGICAL EXAMINATION OF EMBRYOS AND NEW BOM MICE 57
5 RESULTS 58
5.1 DUSP16 - OVERVIEW OF ISOFORMS AND PROTEIN FEATURES 58
5.2 DUSP16 ISOFORM MRNA EXPRESSION 60
5.2.1 REGULATION OF DUSP16 EXPRESSION IN RESPONSE TO DIFFERENT PRR 62
5.3 DETECTION OF DUSPL 6 PROTEIN 64
5.4 GENERATION OF DUSP16 GENE TRAP MICE 66
5.4.1 GENE TRAPPING OVERVIEW 66
5.4.2 VALIDATION OF DUSP16 LOCUS TARGETING 67
5.4.3 MAPPING THE INSERTION IN GENOMIC DNA (GDNA) 68
5.4.4 ANALYSIS OF DUSP16 GENE TRAP EMBRYOS 70
5.4.4.1 P-GALACTOSIDASE AS REPORTER FOR DUSP16 EXPRESSION 71
5.5 LETHALITY OF DUSP16TRAPFTRAP MICE 73
5.6 THE ROLE OF DUSP16 IN THE IMMUNE RESPONSE 74
5.6.1 OVERVIEW OF EXPERIMENTAL APPROACH 74
5.6.2 ANALYSIS OF GENE TRAP FETAL LIVER DERIVED MACROPHAGES AND
DENDRITIC CELLS 75
5.6.2.1 ACTIVATION OF MAPK 75
5.6.2.2 REGULATION OF PROTEIN EXPRESSION OF INFLAMMATORY MEDIATORS 76
5.6.3 CHARACTERIZATION OF GENE TRAP BONE MARROW MACROPHAGES AND
DENDRITIC CELLS 78
5.6.3.1 PHOSPHORYLATION OF MAPK 78
5.6.3.2 DUSP16 DEFICIENCY INFLUENCES
EXPRESSION OF INFLAMMATORY MEDIATORS 79
5.6.3.3 PRODUCTION OF INFLAMMATORY MEDIATORS BY DUSP16 GENE TRAP BMDC
AND BMDM.... 83
5.6.3.4 IN VITRO BACTERIAL INFECTION MODEL 88
5.6.3.5 IMPAIRED DIFFERENTIATION OF DENDRITIC CELLS IN ABSENCE OF DUSP16
90
5.6.3.6 USE OF SIRNA KNOCKDOWN TO ANALYSE OUSP16
FUNCTION 91
5.7 CHARACTERISATION OF DUSP16GENE TRAP BONE MARROW CHIMERAS 92
5.7.1 RECONSTITUTION OF MYELOID AND LYMPHOID
COMPARTMENT 92
5.7.2 RESPONSE OF DUSP16TRAP/TRAP CHIMERAS TO LPS CHALLENGE IN VIVO 93
5.8 DUSP9 - A PDC SPECIFIC PHOSPHATASE 96
5.8.1 PDC SPECIFIC EXPRESSION
OF DUSP9 AND FUNCTIONAL CONSEQUENCES 98
5.8.2 INDUCTION OF DUSP9 EXPRESSION DURING DIFFERENTIATION OF PDC 100
5.8.3 DUSP9 PROTEIN EXPRESSION IN PDC CORRELATES WITH ATTENUATED ERK
ACTIVATION 102
5.8.4 OVEREXPRESSION OF DUSP9 IN CONVENTIONAL MDC 104
5.8.5 CLONING OF THE DUSP9
PROMOTER AND LUCIFERASE REPORTER ASSAYS 106
5.8.6 EXPRESSION OF DUSP9 IN HUMAN DC SUBSETS 108
6 DISCUSSION 110
6.1 EMERGING ROLE OF DUSP
PHOSPHATASES IN THE IMMUNE SYSTEM 110
6.2 DUSP16 GENE TRAP MICE: POSSIBLE PITFALLS 110
6.2.1 CONFIRMATION OF DUSPL 6TRAP/TRAP PHENOTYPE BY SIRNA KNOCKDOWN 112
6.3 LETHALITY OF DUSP16 DEFICIENT MICE 113
6.4 REGULATION OF DUSP16 EXPRESSION 114
6.4.1 THE ROLE OF DUSPL 6 ISOFORMS 116
6.5 CONTROL OF GM-CSF INDUCED DC PROLIFERATION AND DC DEVELOPMENT 117
IV
CONTENTS
6.6 DUSP16 CONTROLS RESPONSE TO MICROBIAL STIMULI AND WHOLE BACTERIA IN
VITRO 120
6.6.1 DIFFERENTIAL IMPACT OF DUSPL
6-DEFICIENCY ON TLR-INDUCED CYTOKINE RELEASE IN
MACROPHAGES AND DC 120
6.6.2 ALTERATIONS IN MAPK ACTIVATION IN TLR-TRIGGERED DUSPL ^DEFICIENT
MACROPHAGES 121
6.6.3 POTENTIAL CROSS-TALK WITH OTHER SIGNALING PATHWAYS 121
6.6.4 POTENTIAL ROLE OF MAPK SUBSTRATES IN DUSP16-DEFICIENT MACROPHAGES
122
6.6.5 TRANSCRIPTION FACTOR REGULATION BY DUSP16 123
6.6.6 NON-MAPK TARGETS OF DUSPL
6? 124
6.6.7 OUTLOOK: MATHEMATICAL MODELING FOR BETTER
UNDERSTANDING EFFECTS OF DUSP16? 124
6.7 FN VFRO PHENOTYPE OF DUSPL 6TRAPFTRAP
CHIMERAS 125
6.7.1 DUSP16 EXPRESSION IN THE TISSUES AFTER LPS CHALLENGE 125
6.7.2 THE TISSUE AND CELLULAR SOURCE OF INCREASED IL-12 IN VIVO REMAINS
TO BE DEFINED 126
6.8 DUSP16 IN B AND T CELL BIOLOGY 128
6.8.1 B CED DEVELOPMENT AND PROLIFERATION 128
6.8.2 DUSP16 IN T
CELLS 129
6.9 DC SPECIFIC
EXPRESSION OF DUSP9. 130
6.9.1 LACK OF DUSP9 EXPRESSION IN HUMAN PDC 131
6.9.2 CLONING AND ANALYSIS OF THE MURINE DUSP9 PROMOTER 132
6.10 CONTROL OF IFN-0 AND IL-12P40 EXPRESSION BY DUSP9 AND DUSP16 133
6.10.1 MAPK SIGNALING IN IL-12P40 AND IFN-P EXPRESSION 133
6.10.2 REGULATORY MECHANISMS ACTING ON IL-12P40 TRANSCRIPTION 133
6.10.3 REGULATION OF IFN-P EXPRESSION BY MAPK, DUSP9 AND DUSP16 134
6.10.4 REDUCED ERK ACTIVATION PROMOTES IFN-P EXPRESSION 135
7 OUTLOOK 137
8 SUMMARY 138
9 ZUSAMMENFASSUNG 140
REFERENCES 142
ACKNOWLEDGEMENTS 165
|
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| indexdate | 2024-07-10T00:39:25Z |
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| spelling | Niedzielska, Magdalena Ewa Verfasser aut The role of DUSP16 and DUSP9 in the innate immune system = Die Funktion von DUSP16 und DUSP9 im angeborenen Immunsystem vorgelegt von Magdalena Ewa Niedzielska Die Funktion von DUSP16 und DUSP9 im angeborenen Immunsystem 2013 XI, 166 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Erlangen-Nürnberg, Univ., Diss., 2013 Mit einer Zssfassung in dt. Sprache MAP-Kinase (DE-588)4576144-9 gnd rswk-swf Aktivierung Chemie (DE-588)4141761-6 gnd rswk-swf Immunsystem (DE-588)4026643-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Immunsystem (DE-588)4026643-6 s MAP-Kinase (DE-588)4576144-9 s Aktivierung Chemie (DE-588)4141761-6 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026069168&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Niedzielska, Magdalena Ewa The role of DUSP16 and DUSP9 in the innate immune system = Die Funktion von DUSP16 und DUSP9 im angeborenen Immunsystem MAP-Kinase (DE-588)4576144-9 gnd Aktivierung Chemie (DE-588)4141761-6 gnd Immunsystem (DE-588)4026643-6 gnd |
| subject_GND | (DE-588)4576144-9 (DE-588)4141761-6 (DE-588)4026643-6 (DE-588)4113937-9 |
| title | The role of DUSP16 and DUSP9 in the innate immune system = Die Funktion von DUSP16 und DUSP9 im angeborenen Immunsystem |
| title_alt | Die Funktion von DUSP16 und DUSP9 im angeborenen Immunsystem |
| title_auth | The role of DUSP16 and DUSP9 in the innate immune system = Die Funktion von DUSP16 und DUSP9 im angeborenen Immunsystem |
| title_exact_search | The role of DUSP16 and DUSP9 in the innate immune system = Die Funktion von DUSP16 und DUSP9 im angeborenen Immunsystem |
| title_full | The role of DUSP16 and DUSP9 in the innate immune system = Die Funktion von DUSP16 und DUSP9 im angeborenen Immunsystem vorgelegt von Magdalena Ewa Niedzielska |
| title_fullStr | The role of DUSP16 and DUSP9 in the innate immune system = Die Funktion von DUSP16 und DUSP9 im angeborenen Immunsystem vorgelegt von Magdalena Ewa Niedzielska |
| title_full_unstemmed | The role of DUSP16 and DUSP9 in the innate immune system = Die Funktion von DUSP16 und DUSP9 im angeborenen Immunsystem vorgelegt von Magdalena Ewa Niedzielska |
| title_short | The role of DUSP16 and DUSP9 in the innate immune system |
| title_sort | the role of dusp16 and dusp9 in the innate immune system die funktion von dusp16 und dusp9 im angeborenen immunsystem |
| title_sub | = Die Funktion von DUSP16 und DUSP9 im angeborenen Immunsystem |
| topic | MAP-Kinase (DE-588)4576144-9 gnd Aktivierung Chemie (DE-588)4141761-6 gnd Immunsystem (DE-588)4026643-6 gnd |
| topic_facet | MAP-Kinase Aktivierung Chemie Immunsystem Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026069168&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT niedzielskamagdalenaewa theroleofdusp16anddusp9intheinnateimmunesystemdiefunktionvondusp16unddusp9imangeborenenimmunsystem AT niedzielskamagdalenaewa diefunktionvondusp16unddusp9imangeborenenimmunsystem |