Development of CD40-targeted bifunctional scFv-TRAIL fusion proteins that induce TRAILR1- and TRAILR2-specifc cell death and dendritic cells activation:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2014
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| Schlagworte: | |
| Online-Zugang: | kostenfrei Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. und engl. Sprache |
| Beschreibung: | VI, 111 Bl. Ill., graph. Darst. |
Internformat
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| 245 | 1 | 0 | |a Development of CD40-targeted bifunctional scFv-TRAIL fusion proteins that induce TRAILR1- and TRAILR2-specifc cell death and dendritic cells activation |c von Mohamed El-Sayed Ahmed Mohamed El-Mesery |
| 246 | 1 | 3 | |a Entwicklung CD40 gerichteter bifunktioneller scFv-TRAIL Fusionsproteine die TRAILR1- und TRAILR2-spezifischen Zelltod und dendritischen Zellaktivierung induzieren |
| 264 | 1 | |c 2014 | |
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Datensatz im Suchindex
| _version_ | 1804152427218206720 |
|---|---|
| adam_text | DEVELOPMENT OF CD40-TARGETED BIFUNCTIONAL SCFV-TRAIL
FUSION PROTEINS THAT
INDUCE TRAILR1- AND
TRAILR2-SPECIFC CELL
DEATH AND DENDRITIC CELLS ACTIVATION
ENTWICKLUNG CD40 GERICHTETER BIFUNKTIONELLER SCFV-TRAIL FUSIONSPROTEINE
DIE TRAILR1- UND
TRAILR2-SPEZIFISCHEN ZELLTOD UND DENDRITISCHEN ZELLAKTIVIERUNG
INDUZIEREN
DOCTORAL THESIS FOR A DOCTORAL DEGREE
AT THE GRADUATE SCHOOL OF LIFE SCIENCES,
JULIUS-MAXIMILIANS-UNIVERSITAT WURZBURG
SECTION BIOMEDICINE
SUBMITTED BY
MOHAMED EL-SAYED AHMED
MOHAMED EL-MESERY
PHD GERMAN-EGYPTIAN RESEARCH LONG TERM SCHOLARSHIP (GERLS) HOLDER
FROM
BIOCHEMISTRY DEPARTMENT, FACULTY OF PHARMACY
MANSOURA UNIVERSITY, MANSOURA, EGYPT
WURZBURG 2014
HTTP://D-NB.INFO/1054939349
TABLE OF CONTENTS
TABLE OF CONTENTS
1. INTRODUCTION 11
1.1. RECEPTORS AND LIGANDS OF THE TNF FAMILY 11
1.1.1. TNF RECEPTOR SUPERFAMILY (TNFRSF) 11
1.1.2. TNF LIGAND FAMILY 11
1.2. TRAIL/TRAILR-SYSTEM AND ACTIVATION OF APOPTOSIS 14
1.2.1. CLASSIFICATION OF TRAILRS 14
1.2.2. MECHANISMS OF TRAIL DR-INDUCED APOPTOSIS 15
1.2.3. CLINICAL TRIALS FOR TRAIL DR TARGETING THERAPIES 17
1.2.4. MECHANISMS OF RESISTANCE AGAINST TRAIL-INDUCED APOPTOSIS 18
1.3. EXOGENOUS ACTIVATION OF TNFRS 19
1.4. RESPONSE OF TRAIL DRS TO SOLUBLE RECOMBINANT TRAIL AND STRATEGIES
TO IMPROVE ITS ACTIVITY... 19
1.5. CD40 AND ITS ROLE IN CANCER IMMUNOTHERAPY 21
1.6. THE ROLE OF DCS IN IMMUNE SYSTEM AND THE EFFECT OF TUMORS ON THEIR
FUNCTION 22
1.6.1 THE ROLE OF DCS IN IMMUNE SYSTEM 22
1.6.2. EFFECT OF TUMORS ON DCS FUNCTION 23
1.7. AIM OF THE WORK 24
2. MATERIALS 25
2.1. CHEMICALS, REAGENTS AND CELL CULTURE MEDIUMS FOR THE CELL CULTURE
25
2.2. ENZYMES 26
2.3. ANTIBODIES 26
2.4. KITS 27
2.5. INSTRUMENTS AND DISPOSABLE MATERIALS/EQUIPMENTS 28
2.6. PREPARATIONS AND BUFFERS 29
2.7. CELLS 30
2.7.1. EUKARYOTIC CELLS 30
2.7.2. DENDRITIC CELLS (DCS) 31
7
TABLE OF CONTENTS
2.7.3. PROKARYOTIC CELLS 31
2.8. PLASMIDS 31
3. METHODS 32
3.1. CELL CULTURE 32
3.2. CLONING AND PRODUCTION OF THE EXPRESSION PLASMIDS 32
3.3. PROTEIN PRODUCTION 32
3.4. PROTEIN PURIFICATION 33
3.5. FACS ANALYSIS 33
3.5.1. DETECTION OF CELL SURFACE MARKERS 33
3.5.2. SCFV:G28-TRAIL BINDING TO CD40-TRANSFECTANT CELLS 34
3.6. EQUILIBRIUM BINDING STUDIES 34
3.6.1. BINDING STUDIES IN HEK293 CELLS TRANSIENTLY TRANSFECTED WITH THE
VARIOUS TRAILRS 34
3.6.2. BINDING STUDIES IN JURKAT AND HT1080 CELLS 35
3.7. IN VITRO BINDING STUDIES 35
3.8. IMMUNOPRECIPITATION (IP) ANALYSIS 35
3.9. WESTERN BLOT 36
3.9.1. SDS-PAGE 36
3.9.2. BLOTTING ON NITROCELLULOSE MEMBRANES 37
3.9.3. MEMBRANE DETECTION 37
3.10. CELL VIABILITY ASSAYS 37
3.11. TOTAL CELL LYSATES 38
3.12. SILVER STAINING 38
3.13. IL8 ELISA 38
3.14. ISOLATION, CULTIVATION AND STIMULATION OF MONOCYTE-DERIVED DCS 39
3.14.1. PREPARATIONS OF DCS 39
3.14.2. FACS ANALYSIS OF MONOCYTES, IDCS AND MDCS 39
3.14.3. CELL VIABILITY ASSAYS 40
TABLE OF CONTENTS
3.14.4. IL12 ELISA 40
3.15. STATISTICAL ANALYSIS 41
4. RESULTS 42
4.1. CHARACTERIZATION OF TRAILR1- AND TRAILR2-SPECIFIC TRAIL MUTANTS 42
4.1.1. BINDING STUDIES WITH TRAIL MUTANTS AND TRAIL WILD TYPE 42
4.1.2. IMMUNOPRECIPITATION (IP) ANALYSIS 46
4.1.3. CELL VIABILITY ASSAYS 47
4.2. TARGETING AND ACTIVATION OF CD40 WITH SCFV-TRAIL FUSION PROTEINS 51
4.2.1. DESIGN AND PRODUCTION OF SCFV-TRAIL FUSION PROTEIN TARGETING CD40
51
4.2.2. EVALUATION OF INHERENT FUNCTIONALITY OF SCFV AND TRAIL DOMAIN OF
SCFV:G28-TRAIL FUSION
PROTEIN 52
4.2.3. ANALYSIS OF CD40-DEPENDENT ENHANCEMENT OF APOPTOSIS INDUCTION BY
CD40-TARGETED TRAIL
FUSION PROTEINS 54
4.2.4. CD40-BOUND SCFV:G28-TRAIL INDUCES CELL DEATH IN CD40-NEGATIVE
BYSTANDER CELLS 57
4.2.5. ANALYSIS OF CD40-DEPENDENT ACTIVITY OF TRAILR1- AND
TRAILR2-SPECIFIC SCFV:G28-TRAIL
FUSION PROTEINS 60
4.2.6. ANALYSIS OF EFFECTS OF SCFV:G28-TRAIL ON CD40 SIGNALING 63
5. DISCUSSION 69
5.1. CHARACTERIZATION OF TRAILR1- AND TRAILR2-SPECIFIC TRAIL MUTANTS 69
5.2. TARGETING AND ACTIVATION OF CD40 WITH SCFV-TRAIL FUSION PROTEINS 71
6. SUMMARY 77
7. ZUSAMMENFASSUNG 79
8. REFERENCES 81
9. ANNEX 94
9.1 DNA SEQUENCES 94
9.1.1. FLAG-TNC-TRAIL 94
9.1.2. FLAG-TNC-TRAILMUTRL 94
9.1.3. FLAG-TNC-TRAILMUTR2 95
9
TABLE OF CONTENTS
9.1.4. GPL-FLAG-TNC-TRAIL 96
9.1.5. GPL-FLAG-TNC-TRAILMUTRL 97
9.1.6 GPL-FLAG-TNC-TRAILMUTR2 98
9.1.7 FC-FLAG-TRAIL 99
9.1.8. FC-FLAG-TRAILMUTRL 100
9.1.9. FC-FLAG-TRAILMUTR2 101
9.1.10. SCFV:G28-FLAG-TNC-TRAIL 102
9.1.11. SCFV:G28-FLAG-TNC-TRAILMUTRL 103
9.1.12. SCFV:G28-FLAG-TNC-TRAILMUTR2 105
9.2. LIST OF ABBREVIATIONS 107
9.3. LIST OF PUBLICATIONS ORIGINATING FROM THIS THESIS 110
9.4. CURRICULUM VITAE ILL
10
|
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| spelling | El-Mesery, Mohamed 1983- Verfasser (DE-588)1054736197 aut Development of CD40-targeted bifunctional scFv-TRAIL fusion proteins that induce TRAILR1- and TRAILR2-specifc cell death and dendritic cells activation von Mohamed El-Sayed Ahmed Mohamed El-Mesery Entwicklung CD40 gerichteter bifunktioneller scFv-TRAIL Fusionsproteine die TRAILR1- und TRAILR2-spezifischen Zelltod und dendritischen Zellaktivierung induzieren 2014 VI, 111 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. und engl. Sprache Würzburg, Univ., Diss., 2014 Antigen CD40 (DE-588)7544036-2 gnd rswk-swf Tumor-Nekrose-Faktor (DE-588)4202932-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Tumor-Nekrose-Faktor (DE-588)4202932-6 s Antigen CD40 (DE-588)7544036-2 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-100114 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-100114 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027455436&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | El-Mesery, Mohamed 1983- Development of CD40-targeted bifunctional scFv-TRAIL fusion proteins that induce TRAILR1- and TRAILR2-specifc cell death and dendritic cells activation Antigen CD40 (DE-588)7544036-2 gnd Tumor-Nekrose-Faktor (DE-588)4202932-6 gnd |
| subject_GND | (DE-588)7544036-2 (DE-588)4202932-6 (DE-588)4113937-9 |
| title | Development of CD40-targeted bifunctional scFv-TRAIL fusion proteins that induce TRAILR1- and TRAILR2-specifc cell death and dendritic cells activation |
| title_alt | Entwicklung CD40 gerichteter bifunktioneller scFv-TRAIL Fusionsproteine die TRAILR1- und TRAILR2-spezifischen Zelltod und dendritischen Zellaktivierung induzieren |
| title_auth | Development of CD40-targeted bifunctional scFv-TRAIL fusion proteins that induce TRAILR1- and TRAILR2-specifc cell death and dendritic cells activation |
| title_exact_search | Development of CD40-targeted bifunctional scFv-TRAIL fusion proteins that induce TRAILR1- and TRAILR2-specifc cell death and dendritic cells activation |
| title_full | Development of CD40-targeted bifunctional scFv-TRAIL fusion proteins that induce TRAILR1- and TRAILR2-specifc cell death and dendritic cells activation von Mohamed El-Sayed Ahmed Mohamed El-Mesery |
| title_fullStr | Development of CD40-targeted bifunctional scFv-TRAIL fusion proteins that induce TRAILR1- and TRAILR2-specifc cell death and dendritic cells activation von Mohamed El-Sayed Ahmed Mohamed El-Mesery |
| title_full_unstemmed | Development of CD40-targeted bifunctional scFv-TRAIL fusion proteins that induce TRAILR1- and TRAILR2-specifc cell death and dendritic cells activation von Mohamed El-Sayed Ahmed Mohamed El-Mesery |
| title_short | Development of CD40-targeted bifunctional scFv-TRAIL fusion proteins that induce TRAILR1- and TRAILR2-specifc cell death and dendritic cells activation |
| title_sort | development of cd40 targeted bifunctional scfv trail fusion proteins that induce trailr1 and trailr2 specifc cell death and dendritic cells activation |
| topic | Antigen CD40 (DE-588)7544036-2 gnd Tumor-Nekrose-Faktor (DE-588)4202932-6 gnd |
| topic_facet | Antigen CD40 Tumor-Nekrose-Faktor Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-100114 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027455436&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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