Induction and characterization of T cell responses towards uveal melanoma:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 132 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Induction and characterization of T cell responses towards uveal melanoma |c eingereicht von Sabine Britting |
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| 502 | |a Erlangen-Nürnberg, Univ., Diss., 2013 | ||
| 546 | |a Mit einer Zssfassung in dt. Sprache | ||
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Datensatz im Suchindex
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| adam_text | IMAGE 1
TABLE OF CONTENTS
1 SUMMARY 9
2 ZUSAMMENFASSUNG 11
3 INTRODUCTION 14
3.1 THE IMMUNE SYSTEM 14
3.1.1 INNATE AND ADAPTIVE IMMUNITY 14
3.1.2 ANTIGEN PROCESSING AND INTERACTION BETWEEN APC AND T LYMPHOCYTES
15
3.2 COSTIMULATORY AND INHIBITORY MOLECULES IN T CELL ACTIVATION 19
3.3 T CELL SUBSETS 21
3.4 TUMOR ANTIGENS 2
3.5 TUMOR IMMUNOLOGY 25
3.6 UVEAL MELANOMA 26
3.7 IMMUNOTHERAPY BY DENDRITIC CELL AND TUMOR CELL VACCINES IN UVEAL
MELANOMA 29
4 AIMS OF THIS THESIS 31
5 MATERIALS AND METHODS 32
5.1 MATERIALS 32
5.1.1 MEDIA, BUFFERS AND SOLUTIONS 32
5.1.2. ANTIBODIES 35
5.1.2.1. CONJUGATED ANTIBODIES 35
5.1.2.2. UNCONJUGATED ANTIBODIES 36
5.1.3. MHC PEPTIDE TETRAMER 37
5.1.4. PEPTIDES 37
5.1.5. BEADS 37
5.1.6. OLIGONUCLEOTIDES 37
5.1.7. CELL CULTURE FLASKS, PLATES AND TUBES 38
5.1.8. CYTOKINES 39
5.1.9. CELL LINES 39
5.1.9.1. PRIMARY UVEAL MELANOMA CELL LINES 39
5.1.9.2. METASTATIC UVEAL MELANOMA CELL LINES 40
5.1.9.3. CM CELL LINE 40
5.1.9.4. HYBRIDOMA CELL LINE 40
5.1.10. T CELL CLONE 40
5.1.11. SOFTWARE 41
5.2. METHODS .41
5.2.1. DETERMINATION OF CELL NUMBERS 41
HTTP://D-NB.INFO/1042312958
IMAGE 2
TABLE OF CONTENTS
5.2.2. CRYOCONSERVATION OF CELLS 41
5.2.3. THAWING OF CELLS .42
5.2.4. CULTIVATION OF CELL LINES .42
5.2.5. DENSITIY GRADIENT CENTIIFUGATION 42
5.2.6. ISOLATION OF MONOCYTES BY COUNTERCURRENT ELUTRIATION 42
5.2.7. GENERATION OF DENDRITE CELLS USING MONOCYTES 43
5.2.8. ANTIGEN-PULSING OF ANTIGEN-PRESENTING CELLS 43
5.2.9. MRNA TRANSFECTION OF MDC 43
5.2.10. CD8* T CELL CULTURE .43
5.2.11. RESTIMULATION OF CD8* T CELL CLONES 44
5.2.12. MAGNETIC CELL SEPARATION 44
5.2.13. FLOW CYTOMETRY 45
5.2.14. ANTIBODY STAINING 45
5.2.14.1. DIRECT ANTIBODY STAINING 45
5.2.14.2. INTRACELLULAR STAINING .45
5.2.14.3. PMHC-TETRAMER STAINING 46
5.2.15. PRIME-BOOST ASSAY .46
5.2.16. CYTOKINE ANALYSIS 46
5.2.16.1. DETERMINATION OF IFNY RELEASE BY ELISA TECHNIQUE 46
5.2.16.2. DETERMINATION OF IFNY RELEASING CELLS BY ELISPOT TECHNIQUE 47
5.2.16.3. CYTOKINE-BEAD-ARRAY 47
5.2.17. ISOLATION OF TOTAL TUMOR RNA 47
5.2.18. REVERSE TRANSCRIPTION 48
5.2.18.1. RT USING RANDOM PRIMERS 48
5.2.18.2. RT USING POLY T PRIMERS .48
5.2.18.3. SPECIFIC REVERSE TRANSCRIPTION 48
5.2.19. POLYMERASE CHAIN RECTION (PCR) 49
5.2.20. QUANTITATIVE POLYMERASE CHAIN REACTION 50
5.2.21. PURIFICATION OF PCR PRODUCTS 50
5.2.22. IN VITRO TRANSCRIPTION (ARCA-IVT) AND POLY A TAILING 50
5.2.23. AGAROSE GEL ELECTROPHORESIS 51
5.2.23.1. PREPARATION, STAINING AND RUNNING OF AGAROSE GELS 51
5.2.23.2. DNA LADDERS 51
5.2.23.3. RNA GELELECTROPHORESIS 52
5.2.23.4. PHOTOGRAPHY OF AGAROSE GELS 52
5.2.24. DNA GEL EXTRACTION AND PURIFICATION 53
5.2.25. SEQUENCING 53
5.2.26. WESTERN BLOTTING 54
5.2.27. IMMUNOPRECIPITATION 55
5.2.28. FLUORESCENCE MICROSCOPY 55
6 RESULTS 56
6.1 DETERMINATION OF OPTIMAL CONDITIONS FOR DC MATURATION AND T CELL
ISOLATION 56 6.1.1 GENERATION AND CHARACTERIZATION OF MDC 56
6.1.2 ISOLATION OF DIFFERENT CELL POPULATIONS FROM PBMC 59
6.1.2.1 PURIFICATION OF CD4* T LYMPHOCYTES 59
6.1.2.2 PURIFICATION OF CD8* T LYMPHOCYTES 61
IMAGE 3
TABLE OF CONTENTS
6.2 MELAN-A AND GP100 MELANOMA ANTIGEN EXPRESSION IN PRIMAIY AND
METASTATIC UVEAL MELANOMA CELLS 62
6.3 PRODUCTION OF MELAN-A MRNA, GP100 MRNA AND UVEAL MELANOMA MRNA FOR
TRANSFECTION OF MDC .67
6.4 CHARACTERIZATION OF MELAN-A MRNA AND GP100 MRNA TRANSFECTED MDC FOR
MELAN-A AND GP100 EXPRESSION 71
6.5 CD8* AND CD4* T CELLS ARE ACTIVATED BY MRNA TRANSFECTED MDC 79 6.5.1
EXPANSION OF MELAN-A SPECIFIC CD8* T CELLS WITH MRNA TRANSFECTED MDC
COMPARED TO PEPTIDE-PULSED MDC 79
6.5.2 ACTIVATION OF GP100 SPECIFIC CTL CLONES BY GP100 MRNA TRANSFECTED
MDC 83 6.5.3 ACTIVATION OF CD4* T CELLS BY MELAN-A MRNA TRANSFECTED MDC
85
6.6 HLA-DR*/CD80* PRIMAIY AND METASTATIC UVEAL MELANOMA VACCINES
ACTIVATE NEGATIVELY SELECTED, PURIFIED CD4* T CELLS 86
6.6.1 CD4 + T CELL ACTIVATION BY UM CELL VACCINES 86
6.6.2 CHARACTERIZATION OF UM CELL VACCINES FOR EXPRESSION OF HLA-DR AND
CD80 87 6.6.3 UM MHC II VACCINES PRIME AND BOOST NAIVE CD4* T CELLS 88
7 DISCUSSION 92
7.1 MELANOMA ANTIGEN MELAN-A AND GP100 EXPRESSION IN UM 92
7.2 DIFFERENTIATION, CHARACTERIZATION AND FUNCTION OF MRNA TRANSFECTED
MDC 93 7.2.1 GENERATION AND DIFFERENTIATION OF MDC FOR MRNA TRANSFECTION
93 7.2.2 CHARACTERIZATION OF MRNA TRANSFECTED MDC 94
7.2.3 FUNCTION OF MRNA TRANSFECTED MDC 98
7.3 T CELL ACTIVATION BY MHC II VACCINES 101
8 FUTURE PERSPECTIVES 104
9 REFERENCES 105
10 APPENDIX 124
10.1 ABBREVATIONS 124
10.2 LIST OF PUBLICATIONS 127
10.3 CURRICULUM VITAE 128
10.4 ACKNOWLEDGMENT 131
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| discipline | Biologie Medizin |
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| spelling | Britting, Sabine Verfasser aut Induction and characterization of T cell responses towards uveal melanoma eingereicht von Sabine Britting 2013 132 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Erlangen-Nürnberg, Univ., Diss., 2013 Mit einer Zssfassung in dt. Sprache Aderhautsarkom (DE-588)4299699-5 gnd rswk-swf T-Lymphozyt (DE-588)4127387-4 gnd rswk-swf Immunreaktion (DE-588)4133841-8 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Aderhautsarkom (DE-588)4299699-5 s T-Lymphozyt (DE-588)4127387-4 s Immunreaktion (DE-588)4133841-8 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026193743&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Britting, Sabine Induction and characterization of T cell responses towards uveal melanoma Aderhautsarkom (DE-588)4299699-5 gnd T-Lymphozyt (DE-588)4127387-4 gnd Immunreaktion (DE-588)4133841-8 gnd |
| subject_GND | (DE-588)4299699-5 (DE-588)4127387-4 (DE-588)4133841-8 (DE-588)4113937-9 |
| title | Induction and characterization of T cell responses towards uveal melanoma |
| title_auth | Induction and characterization of T cell responses towards uveal melanoma |
| title_exact_search | Induction and characterization of T cell responses towards uveal melanoma |
| title_full | Induction and characterization of T cell responses towards uveal melanoma eingereicht von Sabine Britting |
| title_fullStr | Induction and characterization of T cell responses towards uveal melanoma eingereicht von Sabine Britting |
| title_full_unstemmed | Induction and characterization of T cell responses towards uveal melanoma eingereicht von Sabine Britting |
| title_short | Induction and characterization of T cell responses towards uveal melanoma |
| title_sort | induction and characterization of t cell responses towards uveal melanoma |
| topic | Aderhautsarkom (DE-588)4299699-5 gnd T-Lymphozyt (DE-588)4127387-4 gnd Immunreaktion (DE-588)4133841-8 gnd |
| topic_facet | Aderhautsarkom T-Lymphozyt Immunreaktion Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026193743&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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