Effects of nerve growth factor on TGF-beta-Smad signal transduction in PC12 cells:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
2002
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. Sprache. - Würzburg, Univ., Diss., 2003 |
| Beschreibung: | VI, 143 Bl. Ill., graph. Darst. |
Internformat
MARC
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| 100 | 1 | |a Lutz, Marion |d 1973- |e Verfasser |0 (DE-588)12451166X |4 aut | |
| 245 | 1 | 0 | |a Effects of nerve growth factor on TGF-beta-Smad signal transduction in PC12 cells |c von Marion Lutz |
| 264 | 1 | |c 2002 | |
| 300 | |a VI, 143 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a Zsfassung in dt. Sprache. - Würzburg, Univ., Diss., 2003 | ||
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Datensatz im Suchindex
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INDEX
INDEX
1 INTRODUCTION.1
1.1 TRANSFORMING GROWTH FACTOR-P (TGF-P).2
1.1.1 TGF-P SUPERFAMILY.2
1.1.2 STRUCTURE AND FUNCTIONS OF TGF-P.2
1.2 TGF-P SIGNAL TRANSDUCTION.4
1.3 TGF-P ACTIVATION.5
1.4 RECEPTORS INVOLVED IN TGF-P SIGNALING.6
1.4.1 TGF-P RECEPTOR REPERTOIRE.6
1.4.1.1 SIGNALING RECEPTORS.6
1.4.1.2 ACCESSORY RECEPTORS.7
1.4.1.3 PSEUDORECEPTORS.8
1.4.2 STRUCTURE OF TGF-P SIGNALING RECEPTORS.8
1.4.3 RECEPTOR OLIGOMERIZATION.9
1.4.4 TPRI ACTIVATION.9
1.4.5 RECEPTOR INTERACTING PROTEINS.10
1.5 SMAD PROTEINS. 13
1.5.1 CLASSIFICATION OFSMADS.13
1.5.2 STRUCTURE AND FUNCTIONS OF SMADS.14
1.5.2.1 THE MH1 DOMAIN.15
1.5.2.2 THE LINKER.15
1.5.2.3 THE MH2 DOMAIN.16
1.5.3 ACTIVATION OF SMADS.16
1.5.4 OLIGOMERIZATION OF SMADS.16
1.5.5 NUCLEOPLASMIC SHUTTLING OF SMADS.17
1.5.6 SMAD INTERACTING PROTEINS.18
1.5.7 TRANSCRIPTIONAL COMPLEXES.19
1.5.7.1 TRANSCRIPTION FACTORS.20
1.5.7.2 COACTIVATORS.21
1.5.7.3 COREPRESSORS.21
1.5.8 TERMINATION OF SMAD ACTIVITY.22
1.5.8.1 INHIBITORY SMADS.22
1.5.8.2 DEGRADATION OF SMADS.22
1.6 CROSSTALK. 23
1.6.1 CROSSTALK WITH OTHER SIGNALING PATHWAYS.23
1.6.2 PHOSPHORYLATION OF SMADS BY PROTEINS OTHER THAN TPRI.24
1.7 FUNCTION OF TGF-P IN THE NERVOUS SYSTEM.25
1.8 NERVE GROWTH FACTOR (NGF).26
1.8.1 THE FAMILY OF NEUROTROPHINS.26
1.8.2 STRUCTURE AND FUNCTIONS OF NGF.27
1.8.3 NGF RECEPTORS.28
1.8.3.1 THE LOW-AFFINITY NGF RECEPTOR P75
NTR.28
1.8.3.2 THE HIGH-AFFINITY NGF RECEPTOR TRKA.29
1.8.4 NGF SIGNAL TRANSDUCTION.30
1.9 AIM OF THE PROJECT.32
I
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/967116899
INDEX
2 MATERIAL AND SOLUTIONS.
33
2.1 CHEMICALS.33
2.2 TECHNICAL DEVICES.
33
2.3 ENZYMES.
34
2.3.1 RESTRICTION ENDONUCLEASES.34
2.3.2 DNA- AND RNA- MODIFYING ENZYMES.34
2.3.3 POLYMERASES.34
2.4 KITS.34
2.5 OLIGONUCLEOTIDES.34
2.6 PLASMIDS .
34
2.6.1 EXPRESSION VECTORS.34
2.6.2 RETROVIRAL VECTORS.35
2.7 BACTERIAL STRAINS.36
2.8 GROWTH MEDIA AND REAGENTS FOR CELL CULTURE.36
2.9 CELL LINES.37
2.10 GROWTH FACTORS.37
2.11 ANTIBODIES.37
2.12 MARKERS.40
2.12.1 DNA-MARKERS.40
2.12.2 PROTEIN MARKERS.41
3 METHODS.42
3.1 MICROBIOLOGICAL METHODS.42
3.1.1 STERILIZATION.42
3.1.2 GROWTH MEDIA.42
3.1.3 CULTIVATION OF BACTERIA.42
3.1.4 CONSERVATION OF BACTERIA.43
3.1.4.1 SHORT-TERM CONSERVATION.43
3.1.4.2 LONG-TERM CONSERVATION.43
3.1.5 PREPARATION OF CHEMICALLY COMPETENT E.COLI.43
3.1.6 TRANSFORMATION OF COMPETENT E. COLI VIA HEAT-SHOCK.44
3.2 MOLECULAR BIOLOGICAL METHODS.44
3.2.1 STANDARD DNA PURIFICATION METHODS.44
3.2.1.1 PHENOL/CHLOROFORM EXTRACTION.44
3.2.1.2 ETHANOL PRECIPITATION.44
3.2.1.3 ISOPROPANOL PRECIPITATION.44
3.2.2 PLASMID MINIPREPARATION.45
3.2.3 PLASMID MIDIPREPARATION.45
3.2.4 DETERMINATION OF NUCLEIC ACID CONCENTRATIONS.46
3.2.5 AGAROSE GEL ELECTROPHORESIS.46
3.2.6 ELUTION OF DNA FROM AGAROSE GELS.46
3.2.7 POLYMERASE CHAIN REACTION (PCR).47
3.2.7.1 ANALYTICAL PCR.47
3.2.7.2 PREPARATIVE PCR.48
3.2.7.3 RECOMBINANT PCR.48
3.2.8 REVERSE TRANSCRIPTION.48
3.2.8.1 ISOLATION OF TOTAL RNA FROM MAMMALIAN CELLS.48
3.2.8.2 REVERSE TRANSCRIPTION.48
3.2.9 RT-PCR.49
3.2.10 DNA SEQUENCING.49
3.2.11 DIGESTION OF DNA BY RESTRICTION ENDONUCLEASES.49
3.2.12 5'-DEPHOSPHORYLATION OF DNA.49
3.2.13 LIGATION OF DNA-FRAGMENTS.49
II
INDEX
3.3 CELL BIOLOGICAL METHODS.50
3.3.1 CULTIVATION OF MAMMALIAN CELLS.50
3.3.2 DETERMINATION OF CELL NUMBERS.50
3.3.3 CRYO-CONSERVATION OF CELLS.50
3.3.4 COATING OF CELL CULTURE DISHES WITH COLLAGEN.50
3.3.5 INDUCTION OF NEURITE FORMATION IN PC12 CELLS.50
3.3.6 TRANSFECTION METHODS.
51
3.3.6.1 CALCIUM-PHOSPHATE COPRECIPITATION.
51
3.3.6.2 DEAE-DEXTRAN.
51
3.3.6.3 LIPOFECTAMINE
YY
.52
3.3.7 P-GALACTOSIDASE ASSAY.52
3.3.8 RETROVIRAL INFECTION USING REPLICATION DEFICIENT PSEUDOTYPED
MULV-VIRUSES.53
3.3.9 CHARACTERIZATION OF CELLS BY FACS ANALYSIS.54
3.3.9.1 ANTIBODY STAINING FOR FACS ANALYSIS.
54
3.3.9.2 FACS ANALYSIS OF CELLS.54
3.3.9.3 SELECTION OF GFP-POSITIVE CELLS BY FACS SORTING.55
3.4 PROTEIN CHEMICAL METHODS.55
3.4.1 CELL LYSIS.55
3.4.2 IMMUNOPRECIPITATION.56
3.4.3 SDS-POLYACRYLAMID GEL ELECTROPHORESIS.56
3.4.4 WESTERN BLOT.57
3.4.5 DETECTION OF NUCLEAR TRANSLOCATION.58
3.4.6 REPORTER GENE ASSAY.59
3.4.7 QUANTIFICATION OF TGF-PI IN THE SUPERNATANT USING MLEC CELLS.60
3.4.8 RADIOACTIVE LABELING OF TGF-PI AND NGF WITH NA[125L].61
3.4.9 BINDING AND CROSS-LINKING ASSAYS.61
3.4.9.1 BINDING AND CROSS-LINKING OF [
1 5L]-TGF-PI.61
3.4.9.2 BINDING AND CROSS-LINKING OF [125I]-NGF.62
4
RESULTS
.64
4.1 CHARACTERIZATION OF PC12 CELLS.64
4.1.1 RECEPTOR REPERTOIRE OF PC12 CELLS.64
4.1.2 INVESTIGATION OF TGF-P RESPONSIVENESS OF PC12 CELLS.66
4.2 GENERATION OF STABLE PC12 CELLS BY RETROVIRAL INFECTION.68
4.2.1 CLONING OF RETROVIRAL VECTOR CONSTRUCTS.68
4.2.1.1 THE RETROVIRAL VECTOR.68
4.2.1.2 CLONING OF A RETROVIRAL VECTOR CONTAINING THE
TFIRII-WT
GENE.69
4.2.1.3 CLONING OF A RETROVIRAL VECTOR CONTAINING THE
T$RII-&CYT
MUTANT.69
4.2.1.4 CLONING OF A RETROVIRAL VECTOR CONTAINING THE
SMAD7
GENE.70
4.2.2 PRODUCTION OF REPLICATION-DEFICIENT VSV-G PSEUDOTYPED RETROVIRAL
PARTICLES.70
4.2.3 CHARACTERIZATION OF STABLE PC12 CELLS.71
4.2.3.1 CHARACTERIZATION BY FACS ANALYSIS.71
4.2.3.2 CHARACTERIZATION BY WESTERN BLOT.73
4.2.3.3 CHARACTERIZATION BY BINDING AND CROSS-LINKING.74
4.3 INVOLVEMENT OF THE TGF-P/SMAD PATHWAY IN NEURITE OUTGROWTH.75
4.4 EFFECTS OF NGF ON SMAD-DEPENDENT REPORTER CONSTRUCTS.76
4.5 VERIFICATION OF THE TGF-P INDEPENDENCE OF THE OBSERVED NGF
EFFECTS.78
4.5.1 NEUTRALIZATION OF TGF-PI IN THE MEDIUM.78
4.5.1.1 NEUTRALIZATION BY TGF-P ANTIBODIES.78
4.5.1.2 NEUTRALIZATION BY THE TPRII-B-ECD.79
4.5.2 QUANTIFICATION OF THE AMOUNT OF SECRETED TGF-PI IN THE
SUPERNATANT.80
4.6 INVESTIGATION OF THE SIGNAL TRANSDUCTION MECHANISM INDUCED BY NGF.82
4.6.1 R-SMADS INVOLVED IN NGF-INDUCED REPORTER ACTIVATION.82
4.6.1.1 EXAMINATION OF C-TERMINAL PHOSPHORYLATION OF SMAD3.83
4.6.1.2 EXAMINATION OF NUCLEAR LOCALIZATION OF SMAD3.84
4.6.2 INVESTIGATION OF SMAD4 DEPENDENCE.86
INDEX
4.6.3 EFFECTS OF THE INHIBITORY SMAD7 ON NGF-INDUCED TRANSCRIPTIONAL
ACTIVATION.87
4.7 ROLE OF TRANSMEMBRANE RECEPTORS FOR SIGNAL TRANSDUCTION.88
4.7.1 PARTICIPATION OF THE TRKA RECEPTOR.88
4.7.2 INVESTIGATION OF THE ROLE OF THE TGF-P RECEPTORS IN NGF-MEDIATED
SIGNALING.89
4.8 RECEPTOR INTERACTION STUDIES BETWEEN TGF-P AND TRKA IN COS
CELLS.91
4.8.1 EPITOPE TAGGING OF THE TRKA RECEPTOR.
91
4.8.2 VERIFICATION OF DIRECT PHYSICAL INTERACTION BETWEEN TPRII AND
TRKA.92
4.8.2.1 COIMMUNOPRECIPITATION STUDIES WITH TPRII AND TRKA.92
4.8.2.2 BINDING AND CROSS-LINKING STUDIES WITH [
125I]-NGF.93
4.8.2.3 COMPETITION ASSAY WITH [125L]-TGF-PI AND COLD NGF.
94
4.9 OTHER PATHWAYS AND MOLECULES THAT ARE POTENTIALLY INVOLVED.95
4.9.1 CONTRIBUTION OF THE MAPK/ERK PATHWAY.96
4.9.2 CONTRIBUTION OF THE TAK1-P38 KINASE PATHWAY.97
4.10 INVESTIGATION OF OTHER CELL LINES.99
4.10.1 RECEPTOR REPERTOIRE OF DIFFERENT CELL LINES.99
4.10.2 NGF-INDUCED SMAD-DEPENDENT TRANSCRIPTION IN OTHER CELL LINES.101
5 DISCUSSION.103
5.1 PC12 CELLS AS CELL SYSTEM TO INVESTIGATE THE CROSSTALK .103
5.2 GENERATION AND CHARACTERIZATION OF STABLE PC12 CELLS.104
5.3 TGF-P SIGNALING IN NGF-INDUCED NEURITE OUTGROWTH OF PC12 CELLS.104
5.4 MECHANISM OF NGF-MEDIATED SMAD-DEPENDENT REPORTER ACTIVATION.105
5.4.1 NGF EFFECTS ARE INDEPENDENT OF TGF-PI.106
5.4.2 THE MECHANISM OF SMAD ACTIVATION AND DOWNSTREAM SIGNAL
TRANSDUCTION.107
5.4.3 INHIBITORY FUNCTIONS OF SMAD7.109
5.5 ROLE OF TRANSMEMBRANE RECEPTORS IN NGF-INDUCED SMAD ACTIVATION.110
5.6 PARTICIPATION OF OTHER SIGNAL TRANSDUCTION PATHWAYS.112
5.7 EVALUATION OF OTHER CELL SYSTEMS.114
5.8 SUMMARIZING DISCUSSION AND CURRENT MODEL.114
6 SUMMARY.117
6 ZUSAMMENFASSUNG.118
7 REFERENCES.119
APPENDIX
A.1 ABBREVIATIONS.134
A.2 SEQUENCES OF OLIGONUCLEOTIDES.136
A.3 REPORTERGEN CONSTRUCTS.138
PUBLIKATIONEN.139
LEBENSLAUF.141
DANKSAGUNG.142
ERKLARUNG.143
IV |
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| id | DE-604.BV016451838 |
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| indexdate | 2024-11-22T17:35:31Z |
| institution | BVB |
| language | English |
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| physical | VI, 143 Bl. Ill., graph. Darst. |
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| spelling | Lutz, Marion 1973- Verfasser (DE-588)12451166X aut Effects of nerve growth factor on TGF-beta-Smad signal transduction in PC12 cells von Marion Lutz 2002 VI, 143 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. Sprache. - Würzburg, Univ., Diss., 2003 Transforming Growth Factor beta (DE-588)4370120-6 gnd rswk-swf Nervenwachstumsfaktor (DE-588)4171485-4 gnd rswk-swf Signaltransduktion (DE-588)4318717-1 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Transforming Growth Factor beta (DE-588)4370120-6 s Nervenwachstumsfaktor (DE-588)4171485-4 s Signaltransduktion (DE-588)4318717-1 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010172454&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Lutz, Marion 1973- Effects of nerve growth factor on TGF-beta-Smad signal transduction in PC12 cells Transforming Growth Factor beta (DE-588)4370120-6 gnd Nervenwachstumsfaktor (DE-588)4171485-4 gnd Signaltransduktion (DE-588)4318717-1 gnd |
| subject_GND | (DE-588)4370120-6 (DE-588)4171485-4 (DE-588)4318717-1 (DE-588)4113937-9 |
| title | Effects of nerve growth factor on TGF-beta-Smad signal transduction in PC12 cells |
| title_auth | Effects of nerve growth factor on TGF-beta-Smad signal transduction in PC12 cells |
| title_exact_search | Effects of nerve growth factor on TGF-beta-Smad signal transduction in PC12 cells |
| title_full | Effects of nerve growth factor on TGF-beta-Smad signal transduction in PC12 cells von Marion Lutz |
| title_fullStr | Effects of nerve growth factor on TGF-beta-Smad signal transduction in PC12 cells von Marion Lutz |
| title_full_unstemmed | Effects of nerve growth factor on TGF-beta-Smad signal transduction in PC12 cells von Marion Lutz |
| title_short | Effects of nerve growth factor on TGF-beta-Smad signal transduction in PC12 cells |
| title_sort | effects of nerve growth factor on tgf beta smad signal transduction in pc12 cells |
| topic | Transforming Growth Factor beta (DE-588)4370120-6 gnd Nervenwachstumsfaktor (DE-588)4171485-4 gnd Signaltransduktion (DE-588)4318717-1 gnd |
| topic_facet | Transforming Growth Factor beta Nervenwachstumsfaktor Signaltransduktion Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=010172454&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT lutzmarion effectsofnervegrowthfactorontgfbetasmadsignaltransductioninpc12cells |