Site directed immobilization of BMP-2: two approaches for the production of osteoinductive scaffolds = Gerichtete Immobilisierung von BMP-2 : Zwei Ansätze zur Herstellung osteogener Trägerstrukturen
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Würzburg
2016
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis kostenfrei Inhaltsverzeichnis |
| Beschreibung: | veröffentlicht 2017 |
| Beschreibung: | 145 Blätter Illustrationen, Diagramme 30 cm |
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| 100 | 1 | |a Tabisz, Barbara |d 1986- |e Verfasser |0 (DE-588)114213766X |4 aut | |
| 245 | 1 | 0 | |a Site directed immobilization of BMP-2 |b two approaches for the production of osteoinductive scaffolds = Gerichtete Immobilisierung von BMP-2 : Zwei Ansätze zur Herstellung osteogener Trägerstrukturen |c submitted by Barbara Tabisz from Lezajsk |
| 246 | 1 | 1 | |a Gerichtete Immobilisierung von BMP-2 |
| 264 | 1 | |a Würzburg |c 2016 | |
| 300 | |a 145 Blätter |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a veröffentlicht 2017 | ||
| 502 | |b Dissertation |c Julius-Maximilians-Universität Würzburg |d 2017 | ||
| 546 | |a Zusammenfassung in deutscher und englischer Sprache | ||
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| 650 | 0 | 7 | |a Knochen-Morphogenese-Proteine |0 (DE-588)4305932-6 |2 gnd |9 rswk-swf |
| 653 | |a BMP-2 | ||
| 653 | |a protein immobilization | ||
| 653 | |a site-specific immobilization | ||
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| 776 | 0 | 8 | |i Erscheint auch als |n Online-Ausgabe |o urn:nbn:de:bvb:20-opus-153766 |a Tabisz, Barbara, 1986- |t Site Directed Immobilization of BMP-2: Two Approaches for the Production of Osteoinductive Scaffolds |d Würzburg : Universität Würzburg, 2017 |h Online-Ressource |
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Datensatz im Suchindex
| _version_ | 1804178408849014784 |
|---|---|
| adam_text | LIST OF
FIGURES..............................................................................................................................5
ABBREVIATIONS..............................................................................................................................7
ABSTRACT.................................................................................................................................
10
I.
INTRODUCTION..............................................................................................................
14
1. BONE MORPHOLOGY AND PHYSICAL PROPERTIES
...............................................................
14
2. REGULATION OF BONE
REMODELLING...............................................................................
14
3. CRITICAL-SIZE BONE DEFECTS
.........................................................................................
16
4. CURRENT CLINICAL TREATMENT OF NON-HEALING BONE
DEFECTS............................................16
5. ROLE OF BMP2 IN BONE FRACTURE
REPAIR......................................................................17
6. BMP2
SIGNALING........................................................................................................
19
7. CLINICAL USE OF
BMP2............................................................ 21
8. BMP2 DELIVERY
STRATEGIES.........................................................................................
23
8.1 PHYSICAL
ADSORPTION..................................................................................................
23
8.2
ENCAPSULATION...........................................................................................................26
8.3 AFFINITY INTERACTIONS
.........
.............................
26
8.4 COVALENT COUPLING: SITE-DIRECTED VS RANDOM
IMMOBILIZATION...................................28
9. AIM OF THE
STUDY......................................................................................................
30
II. MATERIAL AND
METHODS..............................................................................................
31
1.
BMP2-A2C...............................................................................................................31
1.1 EXPRESSION OF THE BMP2-A2C
VARIANT......................................................................
31
1.2 PURIFICATION OF BMP2-A2C MONOMERS FROM INCLUSION
BODIES.................................31
1.3 REFOLDING OF BMP2-A2C MONOMERS
........................................................................
32
1.4 PURIFICATION OF REFOLDED BMP2-A2C
........................................................................
32
1.5 SEPARATION OF BMP2-A2C
HOMODIMERS...................................................................
32
1.6 WESTERN BLOT ANALYSIS OF BMP2-A2C
.......................................................................
32
1.7 EVALUATION OF BIOLOGICAL ACTIVITY OF
BMP2-A2C.......................................................33
1.8 IMMOBILIZATION OF BMP2-A2C TO IODOACETYL-FUNCTIONALIZED ACRYLAMIDE
BEADS
.....
33
1.9 VISUALIZATION OF BMP2-A2C COUPLED TO IODOACETYL-FUNCTIONALIZED BEADS
...............
34
1
1.10 QUANTIFICATION OF BMP2-A2C IMMOBILIZED TO IODOACETYL-FUNCTIONALIZED
BEADS
....
34
1.11 FLUORESCENT LABELLING OF BMPRIA RECEPTOR
ECTODOMAIN.........................................34
1.12 INTERACTION OF BMP2-A2C COUPLED TO IODOACETYL-FUNCTIONALIZED BEADS
WITH
BMPRIA RECEPTOR
ECTODOMAIN................................................................................
35
1.13 EVALUATION OF OSTEOGENIC POTENTIAL OF BEADS COUPLED TO BMP2-A2C
......................
35
2.
BMP2-K3PLK................................................................................
35
2.1 CLONING OF THE BMP2-K3PLK
VARIANT........................................................................35
2.2 OPTIMIZATION OF BMP2-K3PLK PROTEIN EXPRESSION
..................................................
36
2.3 PURIFICATION OF BMP2-K3PLK FROM INCLUSION BODIES
...............................................
37
2.4 PURIFICATION OF BMP2-K3PLK ACCORDING TO WESSEL AND FLUEGGE
..............................
37
2.5 EVALUATION OF BMP2-K3PLK BY MASS SPECTROMETRY
................................................
38
2.6 VERIFICATION OF BMP2-K3PLK
REFOLDING...................................................................
38
2.7 PURIFICATION OF REFOLDED BMP2-K3PLK BY CATION-EXCHANGE
CHROMATOGRAPHY
..........
38
2.8 EVALUATION OF BMP2-K3PLK BIOLOGICAL ACTIVITY
.........
.
............................................39
2.9 SEPARATION OF BMP2-K3PLK USING A HEPARIN COLUMN
..............................................
39
2.10 SPR ANALYSIS USING BMPRIA AND ACTRIIB RECEPTOR ECTODOMAINS
.........................
39
2.11 OPTIMIZATION OF CLICK REACTION
CONDITIONS................................................................40
2.11.1 COUPLING OF BMP2-K3PLK CATALYZED BY UV IRRADIATION
..........................................
40
2.11.2 COUPLING OF BMP2-K3PLK USING COPPER(I)-CATALYZED ALKYNE-AZIDE
CYCLOADDITION .40
2.11.2.1 COUPLING OF BMP2-K3PLK TO AZYLATED POLY(2-OXAZOLINE)
...................................
40
2.11.2.2 EFFECT OF SODIUM ASCORBATE AND COPPER(II) SULFATE ON
BMP2-WT.........................40
2.11.2.3 ANALYSIS OF PROTECTIVE ROLE OF THPTA IN CUAAC REACTION
................................
41
2.11.2.4 COUPLING OF BMP2-K3PLK TO BIOTINYLATED
PEG-LINKER........................................41
2.12 EVALUATION OF CUAAC REACTION
EFFICIENCY..............................................................
41
2.13 IMMOBILIZATION OF BMP2-K3PLK TO NEUTRAVIDIN-AGAROSE
BEADS...............................42
2.14 DIRECT IMMOBILIZATION OF BMP2-K3PLK TO AZIDE-FUNCTIONALIZED BEADS
...................
42
2.15 VERIFICATION OF BMP2-K3PLK IMMOBILIZED TO FUNCTIONALIZED
BEADS........................43
2.16 QUANTIFICATION OF BMP2-K3PLK IMMOBILIZED TO NEUTRAVIDIN AGAROSE
BEADS
...........
43
2
2.17 QUANTIFICATION OF BMP2-K3PIK IMMOBILIZED TO AZIDE AGAROSE BEADS
.....................
44
2.18 INTERACTION OF IMMOBILIZED BMP2-K3PLK WITH BMPRIA ECTODOMAIN
...................
44
2.19 EVALUATION OF OSTEOGENIC POTENTIAL OF BEADS COUPLED TO BMP2-K3PLK
...................
44
III.
RESULTS.......................................................................................................................
45
1.
BMP2-A2C..............................................................................................................
45
1.1 EVALUATION OF BMP2-A2C PROTEIN EXPRESSION
.........................................................
45
1.2 PURIFICATION OF THE BMP2-A2C
VARIANT.....................................................................
46
1.3 EVALUATION OF BMP2-A2C BY WESTERN
BLOT..............................................................48
1.4 INTERACTION OF BMP2-A2C WITH BMP2 RECEPTOR ECTODOMAINS
.................................
49
1.5 EVALUATION OF BIOLOGICAL ACTIVITY OF BMP2-A2C
......................................................
50
1.6 IMMOBILIZATION OF BMP2-A2C TO IODOACETYL-FUNCTIONALIZED ACRYLAMIDE
BEADS
......
52
1.7 QUANTIFICATION OF BMP2-A2C IMMOBILIZED TO IODOACETYL ACRYLAMIDE
BEADS...........54
1.8 INTERACTION OF IMMOBILIZED BMP2-A2C WITH BMPRIA RECEPTOR ECTODOMAIN
........
56
1.9 DIFFERENTIATION OF C2C12 CELLS UPON EXPOSURE TO BMP2-A2C-COUPLED
BEADS
........
57
2.
BMP2-K3PLK............................................................................................................60
2.1 CLONING OF
BMP2-K3PLK..........................................................................................60
2.2 OPTIMIZATION OF BMP2-K3PLK PROTEIN
EXPRESSION...................................................61
2.3 MASS SPECTROMETRY OF BMP2-K3PLK MONOMERS FROM SMALL SCALE
PREPARATION
.......
64
2.4 REFOLDING AND PURIFICATION OF BMP2-K3PLK (SMALL SCALE)
......................................
65
2.5 MASS SPECTROMETRY OF REFOLDED BMP2-K3PLK (SMALL SCALE)
....................................
66
2.6 LARGE SCALE PREPARATION OF
BMP2-K3PLK.................................................................
68
2.7 WESTERN BLOT ANALYSIS OF REFOLDED BMP2-K3PLK HOMODIMERS (LARGE
SCALE)
............
69
2.8 ANALYSIS OF BMP2-K3PLK DIMERS BY MASS SPECTROMETRY (LARGE SCALE)
...................
70
2.9 SPR ANALYSIS USING BMPRIA AND ACTRIIB RECEPTOR ECTODOMAINS
.........................
71
2.10 EVALUATION OF BIOLOGICAL ACTIVITY OF REFOLDED
BMP2-K3PLK....................................73
2.11 OPTIMIZATION OF COUPLING
CONDITIONS.......................................................................
75
2.11.1 CLICK REACTION CATALYZED BY UV
IRRADIATION..............................................................75
2.11.2 COPPER CATALYZED AZIDE-ALKYNE CYCLOADDITION
.........................................................
78
2.12 SITE-SPECIFIC BIOTINYLATION OF
BMP2-K3PLK.............................................................
85
2.13 IMMOBILIZATION OF BMP2-K3PLK TO NEUTRAVIDIN-AGAROSE BEADS
...............................
86
2.13.1 QUANTIFICATION OF BMP2-K3PLK IMMOBILIZED TO NEUTRAVIDIN
BEADS.........................88
2.13.2 INTERACTION OF BEAD-IMMOBILIZED BMP2-K3PLK WITH THE BMPRIA
RECEPTOR
ECTODOMAIN...................................................... 89
2.13.3 OSTEOGENIC POTENTIAL OF BMP2-K3PLK COUPLED TO NEUTRAVIDIN-AGAROSE
BEADS
.........
90
2.14 DIRECT IMMOBILIZATION OF BMP2-K3PLK TO AZIDE-FUNCTIONALIZED AGAROSE
BEADS
......
92
2.15 QUANTIFICATION OF BMP2-K3PLK IMMOBILIZED TO AZIDE-FUNCTIONALIZED
AGAROSE BEADS
..................................................................................................................................93
2.16 INTERACTION OF AGAROSE BEAD-IMMOBILIZED BMP2-K3PLK WITH BMPRIA
RECEPTOR
ECTODOMAIN...............................................................................................................
95
2.17 OSTEOGENIC POTENTIAL OF BMP2-K3PLK-COUPLED TO AZIDE-FUNCTIONALIZED
BEADS
.......
96
IV.
DISCUSSION..............................................................................................................
100
V.
CONCLUSIONS............................................................................................................
113
VI.
OUTLOOK...................................................................................................................114
VII.
APPENDIX................................................................................................................
115
ACKNOWLEDGMENTS..............................................................................................................141
AFFIDAVIT.............................................................................................................................
143
|
| any_adam_object | 1 |
| author | Tabisz, Barbara 1986- |
| author_GND | (DE-588)114213766X |
| author_facet | Tabisz, Barbara 1986- |
| author_role | aut |
| author_sort | Tabisz, Barbara 1986- |
| author_variant | b t bt |
| building | Verbundindex |
| bvnumber | BV044875026 |
| collection | ebook |
| ctrlnum | (OCoLC)1017028529 (DE-599)DNB1142137899 |
| discipline | Biologie Medizin |
| format | Thesis Book |
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| publishDateSort | 2016 |
| record_format | marc |
| spelling | Tabisz, Barbara 1986- Verfasser (DE-588)114213766X aut Site directed immobilization of BMP-2 two approaches for the production of osteoinductive scaffolds = Gerichtete Immobilisierung von BMP-2 : Zwei Ansätze zur Herstellung osteogener Trägerstrukturen submitted by Barbara Tabisz from Lezajsk Gerichtete Immobilisierung von BMP-2 Würzburg 2016 145 Blätter Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier veröffentlicht 2017 Dissertation Julius-Maximilians-Universität Würzburg 2017 Zusammenfassung in deutscher und englischer Sprache Trägersubstanz (DE-588)4131813-4 gnd rswk-swf Knochenbruch (DE-588)4031318-9 gnd rswk-swf Knochen-Morphogenese-Proteine (DE-588)4305932-6 gnd rswk-swf BMP-2 protein immobilization site-specific immobilization (DE-588)4113937-9 Hochschulschrift gnd-content Knochen-Morphogenese-Proteine (DE-588)4305932-6 s Trägersubstanz (DE-588)4131813-4 s Knochenbruch (DE-588)4031318-9 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-153766 Tabisz, Barbara, 1986- Site Directed Immobilization of BMP-2: Two Approaches for the Production of Osteoinductive Scaffolds Würzburg : Universität Würzburg, 2017 Online-Ressource B:DE-101 application/pdf http://d-nb.info/1142137899/04 Inhaltsverzeichnis https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-153766 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=030269378&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Tabisz, Barbara 1986- Site directed immobilization of BMP-2 two approaches for the production of osteoinductive scaffolds = Gerichtete Immobilisierung von BMP-2 : Zwei Ansätze zur Herstellung osteogener Trägerstrukturen Trägersubstanz (DE-588)4131813-4 gnd Knochenbruch (DE-588)4031318-9 gnd Knochen-Morphogenese-Proteine (DE-588)4305932-6 gnd |
| subject_GND | (DE-588)4131813-4 (DE-588)4031318-9 (DE-588)4305932-6 (DE-588)4113937-9 |
| title | Site directed immobilization of BMP-2 two approaches for the production of osteoinductive scaffolds = Gerichtete Immobilisierung von BMP-2 : Zwei Ansätze zur Herstellung osteogener Trägerstrukturen |
| title_alt | Gerichtete Immobilisierung von BMP-2 |
| title_auth | Site directed immobilization of BMP-2 two approaches for the production of osteoinductive scaffolds = Gerichtete Immobilisierung von BMP-2 : Zwei Ansätze zur Herstellung osteogener Trägerstrukturen |
| title_exact_search | Site directed immobilization of BMP-2 two approaches for the production of osteoinductive scaffolds = Gerichtete Immobilisierung von BMP-2 : Zwei Ansätze zur Herstellung osteogener Trägerstrukturen |
| title_full | Site directed immobilization of BMP-2 two approaches for the production of osteoinductive scaffolds = Gerichtete Immobilisierung von BMP-2 : Zwei Ansätze zur Herstellung osteogener Trägerstrukturen submitted by Barbara Tabisz from Lezajsk |
| title_fullStr | Site directed immobilization of BMP-2 two approaches for the production of osteoinductive scaffolds = Gerichtete Immobilisierung von BMP-2 : Zwei Ansätze zur Herstellung osteogener Trägerstrukturen submitted by Barbara Tabisz from Lezajsk |
| title_full_unstemmed | Site directed immobilization of BMP-2 two approaches for the production of osteoinductive scaffolds = Gerichtete Immobilisierung von BMP-2 : Zwei Ansätze zur Herstellung osteogener Trägerstrukturen submitted by Barbara Tabisz from Lezajsk |
| title_short | Site directed immobilization of BMP-2 |
| title_sort | site directed immobilization of bmp 2 two approaches for the production of osteoinductive scaffolds gerichtete immobilisierung von bmp 2 zwei ansatze zur herstellung osteogener tragerstrukturen |
| title_sub | two approaches for the production of osteoinductive scaffolds = Gerichtete Immobilisierung von BMP-2 : Zwei Ansätze zur Herstellung osteogener Trägerstrukturen |
| topic | Trägersubstanz (DE-588)4131813-4 gnd Knochenbruch (DE-588)4031318-9 gnd Knochen-Morphogenese-Proteine (DE-588)4305932-6 gnd |
| topic_facet | Trägersubstanz Knochenbruch Knochen-Morphogenese-Proteine Hochschulschrift |
| url | http://d-nb.info/1142137899/04 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-153766 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030269378&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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