A novel chip-based parallel transfection approach to evaluate paracrine cell interactions: = Ein neues Chip-basiertes paralleles Transfektionsverfahren für die Analyse parakriner Zellinteraktionen
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| Main Author: | |
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2012
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| Subjects: | |
| Online Access: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-33655 Inhaltsverzeichnis |
| Physical Description: | III, 106 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a A novel chip-based parallel transfection approach to evaluate paracrine cell interactions |b = Ein neues Chip-basiertes paralleles Transfektionsverfahren für die Analyse parakriner Zellinteraktionen |c von Elisabeth Kuhn |
| 246 | 1 | 1 | |a Ein neues Chip-basiertes paralleles Transfektionsverfahren für die Analyse parakriner Zellinteraktionen |
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| 300 | |a III, 106 S. |b Ill., graph. Darst. | ||
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Record in the Search Index
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| adam_text | IMAGE 1
T A B L E O F CONTENTS
TABLE O F CONTENTS
T A B L E O F C O N T E N T S I
1 S U M M A R Y 1
Z U S A M M E N F A S S U N G 2
2 I N T R O D U C T I O N 3
2.1 GENERAL PRINCIPLES O F INTERCELLULAR COMMUNICATION 3
2.1.1 CELL SURFACE RECEPTORS 4
2.1.2 INTERCELLULAR SIGNALLING 6
2.1.3 PARACRINE SIGNALLING IN PHYSIOLOGIC AND PATHOLOGIC PROCESSES 7
2.2 DEVELOPMENT O F A TUMOUR - TUMOURIGENESIS 8
2.2.1 HALLMARKS O F CANCER AND THE ROLE O F PARACRINE CELL SIGNALLING 9
2.2.2 T U M O U R MICROENVIRONMENT AS ACTIVE CONTRIBUTOR T O
TUMOURIGENESIS 13
2.3 HIGH-THROUGPUT METHODS FOR THE ANALYSIS O F G E N E FUNCTIONS 15
2.3.1 REVERSELY TRANSFECTED CELL MICROARRAY ( R T C M ) 16
2.3.2 APPLICATION AREAS O F THE R T C M METHOD 18
2.3.3 DEVELOPMENT O F THE PARACHIP - A CELL CHIP-BASED APPROACH T O
EVALUATE PARACRINE CELL INTERACTIONS 19
2.4 A I M S O F THIS STUDY 2 0
3 M A T E R I A L A N D M E T H O D S 2 2
3.1 MATERIAL 2 2
3.1.1 SPECIAL CHEMICALS 2 2
3.1.2 CONSUMABLE SUPPLIES 2 3
3.1.3 KITS 2 3
3.1.4 DEVICES 2 4
3.1.5 ENZYMES 2 4
3.1.6 PRIMARY ANTIBODIES 2 4
3.1.7 SECONDARY ANTIBODIES 2 5
3.1.8 PLASMIDS 2 5
3.1.9 BIOLOGICAL MATERIAL 2 6
3.1.10MEDIA, BUFFERS, AND SOLUTIONS 2 7
3.1.11 COMPUTER PROGRAMMES 2 8
3.2 CELL BIOLOGICAL METHODS 2 8
3.2.1 CULTIVATION O F EUKARYOTIC CELLS 2 8
3.2.2 STIMULATION O F EUKARYOTIC CELLS 2 9
3.2.3 PREPARATION O F CONDITIONED MEDIUM 3 0
3.2.4 HUMAN IFN-Y ENZYME-LINKED IMMUNOSORBENT ASSAY (EL1SA) 3 0
3.2.5 DIFFUSION RESTRICTING MATRICES 3 0
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HTTP://D-NB.INFO/1025061306
IMAGE 2
TABLE O F CONTENTS
3.2.6 DIFFERENTIAL REVERSE TRANSFECTION 31
3.3 MOLECULAR BIOLOGICAL METHODS 3 3
3.3.1 ISOLATION O F TOTAL R N A FROM EUKARYOTIC CELLS 3 3
3.3.2 C D N A SYNTHESIS 3 3
3.3.3 REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION (RT-PCR) 3 4
3.3.4 ISOLATION O F PLASMID-DNA FROM ESCHERICHIA COLI 3 5
3.3.5 MEASUREMENT O F D N A - AND RNA-CONCENTRATION 3 5
3.3.6 AGAROSE GEL ELECTROPHORESIS 3 5
3.3.7 GEL EXTRACTIONS O F D N A 3 6
3.3.8 RESTRICTION DIGEST O F D N A MOLECULES 3 6
3.3.9 DEPHOSPHORYLATION O F D N A MOLECULES 3 6
3.3.10LIGATION O F D N A FRAGMENTS 3 6
3.3.11 HEAT SHOCK TRANSFORMATION O F COMPENENT E. COLI 3 7
3.3.12DNA SEQUENCING 37
3.4 PROTEIN BIOCHEMISTRY METHODS 37
3.4.1 PREPARATION O F TOTAL PROTEIN EXTRACTS 3 7
3.4.2 DETERMINATION O F PROTEIN CONCENTRATION 3 8
3.4.3 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) 3 8
3.4.4 WESTERN BLOT ANALYSIS 3 8
3.5 IMMUNOFLUORESCENCE ANALYSIS 3 9
3.6 ANYLSIS O F FLUORESCENCE INTENSITY 4 0
3.7 STATISTICAL ANALYSIS 4 0
4 R E S U L T S 4 1
4.1 SELECTION O F APPROPRIATE CELL PAIRS FOR DIFFERENTIAL REVERSE
TRANSFECTION 41
4.2 DIFFERENTIAL REVERSE TRANSFECTION O F HEK 293T CELLS IN COMBINATION
WITH DIFFERENT INDICATOR CELL TYPES 4 2
4.2.1 OPTIMIZATION O F DIFFERENTIAL REVERSE TRANSFECTION O F H E K 293T
CELLS IN COMBINATION WITH PRIMARY HUMAN FIBROBLASTS 4 2
4.2.2 DIFFERENTIAL REVERSE TRANSFECTION O F H E K 2 9 3 T CELLS IN
COMBINATION WITH DIFFERENT INDICATOR CELL TYPES 4 5
4.3 ESTABLISHMENT O F AN APPROPRIATE DETECTION SYSTEM TO DEMONSTRATE
PARACRINE
INTERACTION 52
4.3.1 PARACRINE INDUCTION O F GBP-1 UPON CLASSICAL TRANSFECTION WITH AN
IFN-Y-EXPRESSION PLASMID 52
4.3.2 PARACRINE INDUCTION O F GBP-1 UPON DIFFERENTIAL REVERSE
TRANSFECTION O F AN IFN-Y EXPRESSION PLASMID 54
4.4 ESTABLISHMENT O F A METHOD TO RESTRICT DIFFUSION O F THE PARACRINE
MEDIATOR 55
4.4.1 DIFFERENT MATRICES RESTRICT DIFFUSION WITH DIFFERENT EFFICIENCIES
5 6
4.4.2 CALCULATION O F THE DIFFUSION RESTRICTION INDEX (DRI) 58
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IMAGE 3
T A B L E O F CONTENTS
4.4.3 LMP-AGAROSE MOST EFFICIENTLY RESTRICTS DIFFUSION 5 9
4.4.4 LMP-AGAROSE AT A CONCENTRATION O F 1% LIMITS DIFFUSION WITH THE
HIGHEST EFFICIENCY 6 0
4.5 DETERMINATION O F THE OPTIMAL SPOT DENSITY ON THE PARACHIP 6 2
4.5.1 MINIMAL AMOUNT O F IFN-Y PLASMID REQUIRED FOR THE PARACRINE
INDUCTION O F GBP-1 EXPRESSION 6 2
4.5.2 MAXIMAL SPOT DENSITY P E R PARACHIP 6 3
4.6 APPLICATION O F THE PARACHIP WITH DIFFERENT INDICATOR CELLS,
PARACRINE INDUCERS, A N D INDICATOR GENES 6 5
4.6.1 DETECTION O F PARACRINELY INDUCED COX-2 EXPRESSION IN FIBROBLASTS
ON THE PARACHIP 6 5
4.6.2 DETECTION O F PARACRINELY INDUCED ADHESION MOLECULES IN
ENDOTHELIAL CELLS ON T H E PARACHIP 6 7
5 D I S C U S S I O N 7 1
5.1 DIFFERENTIAL REVERSE TRANSFECTION O F H E K 2 9 3 T CELLS IN
COMBINATION WITH HFIBS 72
5.2 DIFFERENTIAL REVERSE TRANSFECTION O F H E K 2 9 3 T CELLS IN
COMBINATION WITH DIFFERENT INDICATOR CELL TYPES 7 5
5.3 RESTRICTION O F IFN-Y DIFFUSION B Y A SPECIFIC MATRIX OVERLAY 7 7
5.4 EVALUATION O F THE TESTQUALITY O F THE PARACHIP 7 8
5.5 ADVANTAGES, LIMITATIONS, AND POSSIBLE FUTURE APPLICATIONS O F THE
PARACHIP 8 0
6 R E F E R E N C E S 8 5
7 A B B R E V I A T I O N S 9 8
8 C U R R I C U L U M V I T A E 102
9 A C K N O W L E D G E M E N T S 106
- I I I -
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| spelling | Kuhn, Elisabeth 1983- Verfasser (DE-588)102466368X aut A novel chip-based parallel transfection approach to evaluate paracrine cell interactions = Ein neues Chip-basiertes paralleles Transfektionsverfahren für die Analyse parakriner Zellinteraktionen von Elisabeth Kuhn Ein neues Chip-basiertes paralleles Transfektionsverfahren für die Analyse parakriner Zellinteraktionen 2012 III, 106 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Erlangen-Nürnberg, Univ., Diss., 2012 Transfektion (DE-588)4185874-8 gnd rswk-swf Systembiologie (DE-588)4809615-5 gnd rswk-swf Zellkommunikation (DE-588)4136131-3 gnd rswk-swf Zellkontakt (DE-588)4370909-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Systembiologie (DE-588)4809615-5 s Zellkontakt (DE-588)4370909-6 s Zellkommunikation (DE-588)4136131-3 s Transfektion (DE-588)4185874-8 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:29-opus-33655 https://open.fau.de/handle/openfau/2267 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-33655 Resolvingsystem DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025194055&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Kuhn, Elisabeth 1983- A novel chip-based parallel transfection approach to evaluate paracrine cell interactions = Ein neues Chip-basiertes paralleles Transfektionsverfahren für die Analyse parakriner Zellinteraktionen Transfektion (DE-588)4185874-8 gnd Systembiologie (DE-588)4809615-5 gnd Zellkommunikation (DE-588)4136131-3 gnd Zellkontakt (DE-588)4370909-6 gnd |
| subject_GND | (DE-588)4185874-8 (DE-588)4809615-5 (DE-588)4136131-3 (DE-588)4370909-6 (DE-588)4113937-9 |
| title | A novel chip-based parallel transfection approach to evaluate paracrine cell interactions = Ein neues Chip-basiertes paralleles Transfektionsverfahren für die Analyse parakriner Zellinteraktionen |
| title_alt | Ein neues Chip-basiertes paralleles Transfektionsverfahren für die Analyse parakriner Zellinteraktionen |
| title_auth | A novel chip-based parallel transfection approach to evaluate paracrine cell interactions = Ein neues Chip-basiertes paralleles Transfektionsverfahren für die Analyse parakriner Zellinteraktionen |
| title_exact_search | A novel chip-based parallel transfection approach to evaluate paracrine cell interactions = Ein neues Chip-basiertes paralleles Transfektionsverfahren für die Analyse parakriner Zellinteraktionen |
| title_full | A novel chip-based parallel transfection approach to evaluate paracrine cell interactions = Ein neues Chip-basiertes paralleles Transfektionsverfahren für die Analyse parakriner Zellinteraktionen von Elisabeth Kuhn |
| title_fullStr | A novel chip-based parallel transfection approach to evaluate paracrine cell interactions = Ein neues Chip-basiertes paralleles Transfektionsverfahren für die Analyse parakriner Zellinteraktionen von Elisabeth Kuhn |
| title_full_unstemmed | A novel chip-based parallel transfection approach to evaluate paracrine cell interactions = Ein neues Chip-basiertes paralleles Transfektionsverfahren für die Analyse parakriner Zellinteraktionen von Elisabeth Kuhn |
| title_short | A novel chip-based parallel transfection approach to evaluate paracrine cell interactions |
| title_sort | a novel chip based parallel transfection approach to evaluate paracrine cell interactions ein neues chip basiertes paralleles transfektionsverfahren fur die analyse parakriner zellinteraktionen |
| title_sub | = Ein neues Chip-basiertes paralleles Transfektionsverfahren für die Analyse parakriner Zellinteraktionen |
| topic | Transfektion (DE-588)4185874-8 gnd Systembiologie (DE-588)4809615-5 gnd Zellkommunikation (DE-588)4136131-3 gnd Zellkontakt (DE-588)4370909-6 gnd |
| topic_facet | Transfektion Systembiologie Zellkommunikation Zellkontakt Hochschulschrift |
| url | https://open.fau.de/handle/openfau/2267 https://nbn-resolving.org/urn:nbn:de:bvb:29-opus-33655 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025194055&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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