MZB1 as a regulator of Ca_1hn2_1hn+ homeostasis, integrin activation and biosynthesis of immunoglobulins in murine B-cells:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Enth. 1 Sonderabdr. aus: Immunity ; 33. 2010 |
| Beschreibung: | VIII, 194 S. Ill., graph. Darst. 30 cm |
Internformat
MARC
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| 245 | 1 | 0 | |a MZB1 as a regulator of Ca_1hn2_1hn+ homeostasis, integrin activation and biosynthesis of immunoglobulins in murine B-cells |c vorgelegt von Marc Rosenbaum |
| 264 | 1 | |c 2013 | |
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Datensatz im Suchindex
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| adam_text | TABLE OF CONTENTS
TABLE OF CONTENTS
ABBREVIATIONS I
1. SUMMARY 1
2. INTRODUCTION 2
2.1 THE IMMUNE SYSTEM 2
2.1.1 THE IMMUNE SYSTEM - AN OVERVIEW 2
2.1.2 INNATE IMMUNE SYSTEM 2
2.1.3 NATURAL MEMORY COMPARTMENT 4
2.1.4 ADAPTIVE IMMUNITY 5
2.2 B CELL DEVELOPMENT 8
2.2.1 B-2 B CELL DEVELOPMENT 8
2.2.2 B-L B CELL DEVELOPMENT 13
2.3 PERIPHERAL B CELL FUNCTIONS 15
2.4 ENDOPLASMIC RETICULUM 18
2.4.1 THE ENDOPLASMIC RETICULUM - AN OVERVIEW 18
2.4.2 AMINKED GLYCOSYLATION 19
2.4.3 PROTEIN FOLDING 20
2.4.4 THE UNFOLDED PROTEIN RESPONSE (UPR) 22
2.4.5 THE UPR AND ITS ROLE IN PLASMA CELL DIFFERENTIATION 23
2.5 CALCIUM SIGNALING IN IMMUNE CELLS 24
2.6 INTEGRINS 25
2.7 MZB1 26
3. AIM OF STUDIES 28
4. MATERIALS 29
4.1 EQUIPMENT 29
4.2 DISPENSABLE LABORATORY MATERIAL 31
4.3 GENERAL CHEMICALS & REAGENTS 32
4.4 CHEMICALS
AND MEDIA FOR TISSUE CULTURE 36
4.5 BUFFERS AND SOLUTIONS 37
4.5.1 GENERAL BUFFERS AND SOLUTIONS 37
4.5.2 BUFFER FOR GENOTYPING 37
4.5.3 BUFFERS FOR SOUTHERN BLOTTING 37
4.5.4 BUFFERS FOR SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL
ELECTROPHORESIS 38
(SDS-PAGE) 38
4.5.5 BUFFERS AND SOLUTIONS FOR IMMUNOBLOTTING 38
4.5.6 BUFFER FOR ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) 38
4.6 ANTIBODIES 39
4.6.1 ANTIBODIES FOR IMMUNOBLOT & IMMUNOPRECIPITATION 39
4.6.2 ANTIBODIES FOR FACS 40
4.6.3 ANTIBODIES FOR ELISA AND ELISPOT 41
4.7 IMMUNIZATION REAGENTS 42
4.8 PLASMIDS 42
4.9 PRIMER SEQUENCES 44
4.9.1 PRIMERS USED FOR CLONING 44
4.9.2 PRIMERS FOR LONG RANGE PCR SCREENING OF ES CELL CLONES 45
4.9.3 SEQUENCING PRIMERS 45
4.9.4 GENOTYPING PRIMERS 46
4.9.5 PRIMERS FOR (SEMI-)QUANTITATIVE REVERSE TRANSCRIPTION PCR 46
4.9.6 PRIMERS FOR IGK REARRANGEMENT PCR 47
4.10 KITS 47
I
HTTP://D-NB.INFO/1046303090
TABLE OF CONTENTS
4.11 SOFTWARE 47
4.12 HOMEPAGES 48
5. METHODS 50
5.1 GENERAL MOLECULAR BIOLOGY TECHNIQUES SO
5.1.1 QUANTIFICATION OF NUCLEIC ACID 50
5.1.2 POLYMERASE CHAIN REACTION (PCR) 50
5.1.2.1 LONG RANGE PCR 50
5.1.2.2 GENOTYPING 50
5.1.2.3 SITE-DIRECTED MUTAGENESIS 51
5.1.2.4 SEQUENCING 52
5.1.2.5 QUANTITATIVE REVERSE TRANSCRIPTION PCR (QRT-PCR) 52
5.1.2.6 SEMI-QUANTITATIVE RT-PCR 53
5.1.3 RESTRICTION ENZYME (RE) DIGESTS 53
5.1.4 GENERATION OF BLUNT ENDS 54
5.1.5 REMOVAL OF 5 PHOSPHATE GROUPS FROM DNA 54
5.1.6 LIGATION OF DNA FRAGMENTS 54
5.1.7 AGAROSE GEL ELECTROPHORESIS 54
5.1.8 AGAROSE GEL EXTRACTION 54
5.1.9 PURIFICATION OF DNA FROM ENZYMATIC REACTIONS 55
5.1.10 SUBCLONING 55
5.1.10.1 PGEMT EASY VECTOR SYSTEM 55
5.1.10.2 TOPO TA CLONING 55
5.1.11 CLONING OF PLASMID CONSTRUCTS 56
5.1.11.1 PCPS MZBL-HIS(5FREP)
2
(HSS) 56
5.1.11.2 PGEX-4TL BIP 56
5.1.11.3 PGEX-4TL ERP57 56
5.1.11.4 PIRES PURO SPGFP-KDEL-HSS 56
5.1.11.5 PMYS MZBL-HSS IRES GFP 57
5.1.11.6 PMYS SPGFP-KDEL-HSS IRES TOMATO 57
5.1.12 DNA PURIFICATION 57
5.1.12.1 PHENOL-CHLOROFORM EXTRACTION OF DNA ..57
5.1.12.2 PURIFICATION OF GENOMIC DNA FROM MOUSE TISSUE 58
5.1.12.3 PURIFICATION OF GENOMIC DNA FROM CULTURED SUSPENSION CELLS 58
5.1.13 SOUTHERN BLOTTING 59
5.1.14 MRNA PREPARATION 60
5.1.15 REVERSE TRANSCRIPTION 60
5.1.16 TRANSFORMATION OF COMPETENT DH5A CELLS 60
5.1.17 PLASMID PREPARATIONS 61
5.1.17.1 MINIPREP 61
5.1.17.2 MAXIPREP 61
5.2 ANALYSIS OF PROTEINS 62
5.2.1 PREPARATION OF WHOLE CELL EXTRACTS 62
5.2.2 QUANTIFICATION OF PROTEINS 62
5.2.2.1 BRADFORD ASSAY 62
5.2.2.2 BICINCHONINIC ACID (BCA) ASSAY 63
5.2.2.3 NANODROP 63
5.2.3 TCA PRECIPITATION 63
5.2.4 PNGASE F TREATMENT 63
5.2.5 SDS-PAGE 64
5.2.6 VISUALIZATION OF PROTEINS 64
5.2.6.1 COOMASSIE STAINING 64
5.2.6.2 SILVER STAINING 64
5. 2.7 IMMUNOBLOT 65
5.3 FLUORESCENCE ACTIVATED CELL SORTING (FACS) 66
5.3.1 ISOLATION OF PRIMARY SPLENOCYTES 66
5.3.2 ISOLATION OF PRIMARY BONE MARROW CELLS 66
II
TABLE OF CONTENTS
5.3.3 STAINING OF LYMPHOCYTES 67
5.3.4 INTRACELLULAR FACS 67
5.3.5 FACS ANTIBODIES AND THEIR DILUTIONS 68
5.3.6 MAGNETIC CELL SEPARATION (MACS) 69
5.3.7 CALCIUM FLUX EXPERIMENTS 69
5.4 TISSUE CULTURE 70
5.4.1 CELL LINES 70
5.4.1.2 BL-8 70
5.4.1.3 B1-8MZB1-HSS 70
5.4.1.4 BL-8 SPGFP-KDEL-HSS 70
5.4.1.5 EMBRYONIC STEM (ES) CELLS 71
5.4.1.6 J558-IL7 71
5.4.1.7 K46 71
5.4.1.8 K46 MZB1-HSS 71
5.4.1.9 K46 SPGFP-KDEL-HSS 71
5.4.1.10 K46
SIMZBLMO
71
5.4.1.11 K46
SIMZBLMO +MZBL
CXXA
72
5.4.1.12 MOUSE EMBRYONIC FIBROBLASTS (DR-4) 72
5.4.1.13 NIH 3T3 72
5.4.1.14 NIH 3T3 FLAG-MZB1 72
5.4.1.15 OP9 72
5.4.1.16 PLATINUM E 73
5.4.1.17 PRIMARY BONE MARROW-DERIVED B CELLS 73
5.4.1.18 PRIMARY SPLENOCYTES 73
5.4.1.19 X63-IL6 73
5.4.2 STABLE TRANSFECTIONS OF SUSPENSION CELLS BY ELECTROPORATION 74
5.4.3 RETROVIRAL TRANSDUCTION 74
5.4.4 HOMOLOGOUS RECOMBINATION IN ES CELLS 75
5.4.5 IN VIVO FORMALDEHYDE CROSSLINKING OF PROTEINS 76
5.5 ANIMAL EXPERIMENTS 76
5.5.1 MOUSE STRAINS ; 76
5.5.2 GENERATION OF THE MZBL CONDITIONAL KNOCKOUT MOUSE STRAIN 77
5.5.3 IRRADIATION OF ANIMALS 77
5.5.4 IMMUNIZATION AND INJECTIONS 77
5.5.5 SERUM PREPARATION 77
5.6 ENZYMATIC ASSAYS 78
5.6.1 ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) 78
5.6.2 ELISPOT 78
5.6.3 VLA-4 REDUCTION ASSAY 79
5.6.4 INSULIN TURBIDITY ASSAY 79
5.7 FLUORESCENCE LIFETIME IMAGING (FLIM) 80
5.8 AFFINITY PURIFICATIONS 81
5.8.1 COUPLING OF ANTIBODIES TO BEADS 81
5.8.2 IMMUNO-AFFINITY PURIFICATION 81
5.8.3 SFREP-TACTIN PULL DOWN 82
5.8.4 CO-IMMUNOPRECIPITATION 82
5.8.5 LECTIN MICROPURIFICATION 83
5.8.6 PROTEIN EXPRESSION AND PURIFICATION 84
5.8.7 GST PULL DOWN ASSAY 85
5.9 MASS SPECTROMETRY (MS) ANALYSIS .....85
5.9.1 IN-GEL DIGESTION 85
5.9.2 STAGE TIP PURIFICATION 87
5.9.3 NANO-LC COLUMN PACKING 87
5.9.4 LC-MS ANALYSIS 88
5.10 DATA AND STATISTICAL ANALYSIS 89
5.10.1 MS DATA ANALYSIS :.89
III
TABLE OF CONTENTS
5.10.1.2 MS DATA ANALYSIS OF THE IMMUNO-AFFINITY APPROACH 89
5.10.1.2 MS DATA ANALYSIS OF
STREP-
TACTIN PULL DOWN APPROACH 90
5.10.2 STATISTICAL ANALYSIS 91
6. RESULTS 93
6.1 MZB1 PROTEIN REGULATES CALCIUM HOMEOSTASIS, ANTIBODY SECRETION, AND
INTEGRIN
ACTIVATION IN INNATE-LIKE B CELLS 93
6.1.1 ARTICLE 95
6.1.2 SUPPLEMENTAL FIGURES AND FIGURE LEGENDS 109
6.2 MZB1 AND ITS ROLE IN B CELL DIFFERENTIATION 119
CONDITIONAL ABLATION OF THE MZBL GENE DURING EARLY B CELL DEVELOPMENT
119
ABLATION OF MZBL DURING THE EARLY B CELL STAGE RESULTS IN A BLOCK OF
PRO-B CELL
DIFFERENTIATION WITH A DRASTIC LOSS OF PERIPHERAL B CELLS 121
THE BLOCK IN B CELL DIFFERENTIATION CANNOT BE RESCUED BY A PRODUCTIVELY
REARRANGED NHC
124
MZBL^MBL
CRE
PRO/PRE-B CELLS SHOW A DEFECT IN THE SURFACE EXPRESSION OF THE NHC 126
MZBL IS REQUIRED FOR THE JV-LINKED GLYCOSYLATION OF |IHC IN LATE PRO-B
CELLS 128
6.3 MZB1 AND ITS IMPLICATION IN THE HUMORAL IMMUNE RESPONSE 133
CONDITIONAL ABLATION OF MZBL DURING THE TRANSITIONAL B CELL STAGES DOES
NOT AFFECT THE
MATURE SPLENIC B CELL COMPARTMENT 133
MZ B CELLS OF MZBL^CD^L-CRE MICE SHOW NORMAL BCR-MEDIATED CA
2+
FLUX AND A
NORMAL ER CA
2+
STORE CONTENT RELATIVE TO MZ B CELLS OF MZBL
+/+
CD21-CRE MICE 135
LOSS OF MZBL RESULTS IN SIGNIFICANTLY REDUCED T CELL-DEPENDENT AND T
CELL-INDEPENDENT
TYPE 2 IMMUNE RESPONSES 136
6.4 GERMLINE DELETION OF MZBL AND ITS DIFFERENTIAL PHENOTYPE 139
MZBR ~
MICE SHOW NORMAL NUMBERS OF BONE MARROW PRE-B CELLS AND NORMAL
FREQUENCIES
OF SPLENIC B CELLS 139
MZ B CELLS OF MZBL MICE DISPLAY NORMAL SURFACE LEVELS OF VLA-4 AND LFA-1
AS WELL
AS MEMBRANE-BOUND IGM 143
MZBR ~
MICE SHOW AN IMPAIRED TI-2 IMMUNE RESPONSE 145
THE MBL
CRE
ALLELE SYNERGIZES WITH MZBL DEFICIENCY TO GENERATE A B CELL
DEVELOPMENTAL
BLOCK AT THE TRANSITION OF PRO-B TO PRE-B CELLS 146
PHARMACOLOGICAL INDUCTION OF ER STRESS IN AN MZBL-DEFICIENT BACKGROUND
RESULTS IN A
PERTURBATION OF B CELL DEVELOPMENT AT THE PRO-B TO PRE-B CELL TRANSITION
148
6.5 MZB1 AND ITS INTERACTION PARTNERS 150
MZB1 ASSOCIATES WITH COMPONENTS OF THE ER PROTEIN FOLDING AND
//-GLYCOSYLATION
MACHINERIES 150
MZB1 SHOWS HOMOLOGY TO CNPY2, A MEMBER OF THE CANOPY PROTEIN FAMILY 155
7. DISCUSSION 156
MZB1 AND ITS ROLE IN CA
2+
-REGULATING PROCESSES AND PROLIFERATION 156
MZB1 AND ITS IMPACT ON INTEGRIN-MEDIATED ADHESION 158
THE ROLE OF MZB1 IN B CELL DIFFERENTIATION 160
THE LINK BETWEEN MZB1, ER STRESS AND THE BIOSYNTHESIS OF IGM 163
8. ACKNOWLEDGEMENTS 169
9. REFERENCES .......170
10. APPENDIX 192
10.1 LONG RANGE PCR DATA 192
IV
|
| any_adam_object | 1 |
| author | Rosenbaum, Marc |
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| author_facet | Rosenbaum, Marc |
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| dewey-ones | 571 - Physiology & related subjects |
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| dewey-search | 571.967193535 |
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| discipline | Biologie Medizin |
| format | Thesis Book |
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| spelling | Rosenbaum, Marc Verfasser (DE-588)1045456063 aut MZB1 as a regulator of Ca_1hn2_1hn+ homeostasis, integrin activation and biosynthesis of immunoglobulins in murine B-cells vorgelegt von Marc Rosenbaum 2013 VIII, 194 S. Ill., graph. Darst. 30 cm txt rdacontent n rdamedia nc rdacarrier Enth. 1 Sonderabdr. aus: Immunity ; 33. 2010 Freiburg (Breisgau), Univ., Diss., 2013 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027250433&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Rosenbaum, Marc MZB1 as a regulator of Ca_1hn2_1hn+ homeostasis, integrin activation and biosynthesis of immunoglobulins in murine B-cells |
| subject_GND | (DE-588)4113937-9 |
| title | MZB1 as a regulator of Ca_1hn2_1hn+ homeostasis, integrin activation and biosynthesis of immunoglobulins in murine B-cells |
| title_auth | MZB1 as a regulator of Ca_1hn2_1hn+ homeostasis, integrin activation and biosynthesis of immunoglobulins in murine B-cells |
| title_exact_search | MZB1 as a regulator of Ca_1hn2_1hn+ homeostasis, integrin activation and biosynthesis of immunoglobulins in murine B-cells |
| title_full | MZB1 as a regulator of Ca_1hn2_1hn+ homeostasis, integrin activation and biosynthesis of immunoglobulins in murine B-cells vorgelegt von Marc Rosenbaum |
| title_fullStr | MZB1 as a regulator of Ca_1hn2_1hn+ homeostasis, integrin activation and biosynthesis of immunoglobulins in murine B-cells vorgelegt von Marc Rosenbaum |
| title_full_unstemmed | MZB1 as a regulator of Ca_1hn2_1hn+ homeostasis, integrin activation and biosynthesis of immunoglobulins in murine B-cells vorgelegt von Marc Rosenbaum |
| title_short | MZB1 as a regulator of Ca_1hn2_1hn+ homeostasis, integrin activation and biosynthesis of immunoglobulins in murine B-cells |
| title_sort | mzb1 as a regulator of ca 1hn2 1hn homeostasis integrin activation and biosynthesis of immunoglobulins in murine b cells |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027250433&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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