Spatio-temporal investigation and quantitative analysis of the BMP signaling pathway:
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| Main Author: | |
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2013
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| Subjects: | |
| Online Access: | Volltext Inhaltsverzeichnis |
| Item Description: | Zsfassung in dt. und engl. Sprache |
| Physical Description: | 96 Bl. graph. Darst. |
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| 245 | 1 | 0 | |a Spatio-temporal investigation and quantitative analysis of the BMP signaling pathway |c von Daniela Schul |
| 246 | 1 | 3 | |a Raum-Zeitliche Untersuchung und quantitative Analyse des BMP-Signaltransduktionsweges |
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Record in the Search Index
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| adam_text | TABLE OF CONTENTS
1. SUMMARY 1
2. ZUSAMMENFASSUNG 2
3. INTRODUCTION 4
3.1 BONE MORPHOGENETIC PROTEINS 4
3.2 BMP RECEPTORS 5
3.2.1 SUBTYPES, STRUCTURE AND FUNCTION 5
3.2.2 LIGAND BINDING AND ACTIVATION OF BMP SIGNALING 6
3.2.3 DORSOMORPHIN 7
3.3 BMP SIGNAL TRANSDUCTION VIA SMAD PROTEINS 8
3.3.1 SMAD PROTEINS 8
3.3.2 SMAD NUCLEOCYTOPLASMIC SHUTTLING 10
3.3.3 SMAD TRANSCRIPTIONAL COMPLEXES 12
3.4 REGULATORY SYSTEM OF BMP SIGNALING 13
3.4.1 ANTAGONISTS 13
3.4.2 CO-RECEPTORS AND PSEUDORECEPTORS 14
3.4.3 INTRACELLULAR REGULATORY PROTEINS 14
3.5 BMPS IN EMBRYONIC DEVELOPMENT 15
3.6 DISEASES DEPENDENT ON IMPAIRED BMP SIGNALING 17
3.7 BONE MORPHOGENETIC PROTEINS IN CLINICAL APPLICATIONS 18
3.8 AIM OF THE PROJECT 18
4. MATERIALS AND METHODS 20
4.1 OLIGONUCLEOTIDE SEQUENCES 20
4.2 ANTIBODIES 21
4.3 SPECIAL TECHNICAL
DEVICES AND SOFTWARE 22
4.4 KITS . 22
4.5 FLUORESCENT DYES 22
4.6 CHEMICALS 23
4.7 C2CL2 CELL LINE 23
HTTP://D-NB.INFO/1048542513
4.8 CELL
CULTURE 23
4.8.1 CELL CULTIVATION 23
4.8.2 CRYO-CONSERVATION 23
4.8.3 TRANSFECTION 24
4.8.4 GENERATION OF STABLE C2CL2_BRE-LUC CELL LINE 24
4.8.5 CELL TREATMENT FOR THE GENE EXPRESSION EXPERIMENTS 24
4.8.6 CELL TREATMENT FOR THE TRANSIENT LUCIFERASE EXPERIMENTS 24
4.8.7 CELL TREATMENT FOR THE LUCIFERASE EXPERIMENTS 25
4.8.8 CELL TREATMENT FOR THE SMADL LIVE-SHUTTLING EXPERIMENTS AND
CONFOCAL IMAGING 26
4.8.9 CELL TREATMENT FOR THE IMMUNOFLUORESCENCE STAININGS AND
OBSERVATION OF THE SMADL
SUBCELLULAR LOCALIZATION 26
4.9 PCR 26
4.10 ENDONUCLEASE DIGESTION 27
4.11 LIGATION 27
4.12 HEAT-SHOCK TRANSFORMATION OF DNA INTO CHEMICALLY COMPETENT BACTERIA
28
4.13 CLONING 28
4.13.1 BRE-LUC REPORTER CONSTRUCT 28
4.13.2 MEGFP-SMADL EXPRESSION CONSTRUCT 29
4.14 PLASMID PREPARATION 29
4.15 TOTAL RNA ISOLATION 29
4.16 IN-VITRO CDNA TRANSCRIPTION 30
4.17 REAL-TIME PCR 30
4.18 FLUORESCENT STAINING
OF C2CL2 CELLS 31
4.18.1 IMMUNOFLUORESCENCE STAINING 31
4.18.2 CELL MEMBRANE STAINING 32
4.18.3 DNA STAINING WITH HOECHST 33342 32
4.19 MATHEMATICAL ANALYSIS 32
5. RESULTS 33
5.1 GENERATION OF THE STABLE C2CL2_BRE-LUC CELL LINE 33
5.2 GENE EXPRESSION ANALYSIS UPON SUSTAINED STIMULATION WITH BMP2 36
5.2.1 EXPRESSION ANALYSIS UTILIZING THE STABLE C2CL2_BRE-LUC CELLS 37
5.2.2 QUANTIFICATION OF BMP TARGET GENE EXPRESSION UPON SUSTAINED
STIMULATION 39
5.3 GENE EXPRESSION EXPERIMENTS AFTER SHORT TIME STIMULATION WITH BMP2
44
5.3.1 C2CL2_BRE-LUC CELLULAR RESPONSE TO SHORT-TIME RECEPTOR STIMULUS 44
5.3.2 TARGET GENE EXPRESSION ANALYSIS UPON SHORT-TIME RECEPTOR
STIMULATION 46
5.4 GENE EXPRESSION AFTER SHORT-TIME SMAD PHOSPHORYLATION 48
5.4.1 C2CL2_BRE-LUC RESPONSE TO 15 MINUTE SMAD-ACTIVATION 48
5.4.2 REAL-TIME ANALYSIS OF IDL AND SMAD6 EXPRESSION AFTER SHORT TERM
SMAD-ACTIVATION 50
5.5 FAST FOURIER TRANSFORMATION 52
5.6 SPATIO-TEMPORAL INVESTIGATION OF SMADL AFTER STIMULATION WITH BMP2
54
5.6.1 ANAYLSIS OF SMADL SUBCELLULAR DISTRIBUTION USING
IMMUNOFLUORESCENCE 54
5.6.2 LIVE OBSERVATION OF THE SMADL SUBCELLULAR DISTRIBUTION 56
5.6.3 PHOSPHO-SMADL AMOUNT AS A FUNCTION OF THE LIGAND CONCENTRATION 58
6. DISCUSSION 61
6.1 BMP TARGET GENE EXPRESSION LEVEL
IS DOSE-DEPENDENT 62
6.2 SHORT-TIME RECEPTOR STIMULI ARE SUFFICIENT TO DRIVE LONG-TERM TARGET
GENE TRANSCRIPTION
65
6.3 OSCILLATING
TARGET GENE EXPRESSION IS INDEPENDENT OF THE BMP CONCENTRATION 67
6.4 TARGET GENE EXPRESSION OSCILLATION
IS DIRECTLY DEPENDENT ON BMP RECEPTOR KINASE 69
6.5 CRUCIAL DIFFERENCES REGARDING THE SPATIO-TEMPORAL INTRACELLULAR
LOCALIZATION OF THE R-
SMAD FAMILY MEMBERS UPON STIMULATION 72
CURRICULUM VITAE 76
AFFIDAVIT 78
BIBLIOGRAPHY 79
ACKNOWLEDGEMENTS 95
PUBLICATIONS 96
|
| any_adam_object | 1 |
| author | Schul, Daniela 1984- |
| author_GND | (DE-588)1047353164 |
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| author_role | aut |
| author_sort | Schul, Daniela 1984- |
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| bvnumber | BV041642227 |
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| discipline | Biologie |
| format | Thesis Book |
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| spelling | Schul, Daniela 1984- Verfasser (DE-588)1047353164 aut Spatio-temporal investigation and quantitative analysis of the BMP signaling pathway von Daniela Schul Raum-Zeitliche Untersuchung und quantitative Analyse des BMP-Signaltransduktionsweges 2013 96 Bl. graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. und engl. Sprache Würzburg, Univ., Diss., 2013 Mit einer Zssfassung in dt. Sprache Knochen-Morphogenese-Proteine (DE-588)4305932-6 gnd rswk-swf Signaltransduktion (DE-588)4318717-1 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Knochen-Morphogenese-Proteine (DE-588)4305932-6 s Signaltransduktion (DE-588)4318717-1 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-84224 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-84224 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=027082999&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Schul, Daniela 1984- Spatio-temporal investigation and quantitative analysis of the BMP signaling pathway Knochen-Morphogenese-Proteine (DE-588)4305932-6 gnd Signaltransduktion (DE-588)4318717-1 gnd |
| subject_GND | (DE-588)4305932-6 (DE-588)4318717-1 (DE-588)4113937-9 |
| title | Spatio-temporal investigation and quantitative analysis of the BMP signaling pathway |
| title_alt | Raum-Zeitliche Untersuchung und quantitative Analyse des BMP-Signaltransduktionsweges |
| title_auth | Spatio-temporal investigation and quantitative analysis of the BMP signaling pathway |
| title_exact_search | Spatio-temporal investigation and quantitative analysis of the BMP signaling pathway |
| title_full | Spatio-temporal investigation and quantitative analysis of the BMP signaling pathway von Daniela Schul |
| title_fullStr | Spatio-temporal investigation and quantitative analysis of the BMP signaling pathway von Daniela Schul |
| title_full_unstemmed | Spatio-temporal investigation and quantitative analysis of the BMP signaling pathway von Daniela Schul |
| title_short | Spatio-temporal investigation and quantitative analysis of the BMP signaling pathway |
| title_sort | spatio temporal investigation and quantitative analysis of the bmp signaling pathway |
| topic | Knochen-Morphogenese-Proteine (DE-588)4305932-6 gnd Signaltransduktion (DE-588)4318717-1 gnd |
| topic_facet | Knochen-Morphogenese-Proteine Signaltransduktion Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-84224 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027082999&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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