Post-transcriptional regulation of cartilage development:
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MARC
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| 100 | 1 | |a Georgieva, Veronika |d 1992- |e Verfasser |0 (DE-588)1256071617 |4 aut | |
| 245 | 1 | 0 | |a Post-transcriptional regulation of cartilage development |c vorgelegt von Veronika Georgieva aus Sofia, Bulgarien |
| 264 | 1 | |a Köln |c 2021 | |
| 300 | |a iv, 123 Seiten |b Illustrationen | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Universität Köln |d 2021 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 856 | 4 | 2 | |m B:DE-101 |q application/pdf |u https://d-nb.info/1255731346/04 |3 Inhaltsverzeichnis |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=033636563&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
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| _version_ | 1804184056951209984 |
|---|---|
| adam_text | ABSTRACT
..........................................................................................................................................
III
ZUSAMMENFASSUNG
.........................................................................................................................
IV
1.
INTRODUCTION
............................................................................................................................
1
1.1.
THE
SKELETAL
SYSTEM
............................................................................................................
1
1.1.1.
ENDOCHONDRAL
OSSIFICATION
........................................................................................
1
1.1.2.
ORGANIZATION
OF
THE
GROWTH
PLATE
AND
THE
EXTRACELLULAR
MATRIX
..............................
3
1.1.3.
OSTEOCLAST-MEDIATED
ECM
REMODELING
...................................................................
5
1.2.
POST
TRANSCRIPTIONAL
GENE
REGULATION
................................................................................
9
1.2.1.
MIRNA
BIOGENESIS
...................................................................................................
9
1.2.2.
MECHANISMS
OF
MIRNA-MEDIATED
GENE
REGULATION
...............................................
11
1.2.3.
MIRNAS
IN
ENDOCHONDRAL
OSSIFICATION
..................................................................
12
1.2.4.
ROLE
OF
THE
MIRC24
CLUSTER
FOR
CARTILAGE
DEVELOPMENT
...........................................
13
1.3.
AIMS
.................................................................................................................................
14
2.
MATERIALS
AND
METHODS
........................................................................................................
15
2.1.
MATERIALS
..........................................................................................................................
15
2.1.1.
BUFFERS
......................................................................................................................
15
2.1.2.
OLIGONUCLEOTIDES
......................................................................................................
15
2.1.3.
PLASMIDS
...................................................................................................................
17
2.1.4.
ANTIBODIES
................................................................................................................
18
2.2.
CELL
BIOLOGICAL
METHODS
...................................................................................................
19
2.2.1.
EUKARYOTIC
CELL
CULTURES
...........................................................................................
19
2.2.2.
CRYOCONSERVATION
OF
CELLS
.......................................................................................
19
2.2.3.
TRANSFECTION
OF
HEK293
EBNA
CELLS
WITH
MIRNA
MIMICS
AND
PLASMIDS
......
19
2.2.4.
DUAL
LUCIFERASE
ASSAY
..............................................................................................
20
2.2.5.
CRISPR/CAS9
TARGETING
OF
COLL2AL
......................................................................
20
2.2.6.
ISOLATION
AND
CULTIVATION
OF
PRIMARY
EPIPHYSEAL
CHONDROCYTES
............................
22
2.2.7.
MIRNA
MIMIC
TRANSFECTION
OF
ATDC5
CELLS
AND
PRIMARY
EPIPHYSEAL
CHONDROCYTES
...........................................................................................................................
23
2.2.8.
STIMULATION
OF
PECS
WITH
INSULIN,
FCS,
BMP-2
AND
TGF-PI
............................
23
2.2.9.
ISOLATION
OF
SPLEEN
CELLS
..........................................................................................
24
2.2.10.
CHONDROCYTE/SPLEEN
CELL
CO-CULTURE
ASSAY
..........................................................
24
2.3.
MOLECULAR
BIOLOGICAL
METHODS
.......................................................................................
25
2.3.1.
GENOMIC
DNA
ISOLATION
FROM
BIOPSIES
.................................................................
25
2.3.2.
GENOMIC
DNA
ISOLATION
FROM
CULTURED
CELLS
.........................................................
25
2.3.3.
RNA
ISOLATION
..........................................................................................................
25
2.3.4.
REVERSE
TRANSCRIPTION
..............................................................................................
26
2.3.5.
POLYMERASE
CHAIN
REACTION
(PCR)
.........................................................................
26
2.3.6.
SEMI-QUANTITATIVE
PCR
...........................................................................................
27
2.3.7.
QUANTITATIVE
PCR
(QPCR)
......................................................................................
28
2.3.8.
AGAROSE
GEL
ELECTROPHORESIS
...................................................................................
28
2.3.9.
RESTRICTION
DIGEST
.....................................................................................................
29
2.3.10.
PHOSPHORYLATION
AND
ANNEALING
OF
OLIGONUCLEOTIDE
PAIRS
..................................
29
2.3.11.
LIGATION
................................................................................................................
29
2.3.12.
TRANSFORMATION
OF
PLASMIDS
IN
COMPETENT
E.
COLI
STRAINS
.................................
30
2.3.13.
MINI-PREPARATION
OF
PLASMID
DNA
....................................................................
30
2.3.14.
SANGER
SEQUENCING
..............................................................................................
30
2.4.
BIOCHEMICAL
METHODS
....................................................................................................
31
INTRODUCTION
2.4.1.
PROTEIN
EXTRACTION
FOR
GEL
ELECTROPHORESIS
...............................................................
31
2.4.2.
SDS-POLYACRYLAMIDE
GEL
ELECTROPHORESIS
(SDS-PAGE)
......................................
31
2.4.3.
IMMUNOBLOT
ANALYSIS
...............................................................................................
32
2.4.4.
GELATIN
ZYMOGRAPHY
................................................................................................
33
2.4.5.
PROTEIN
EXTRACTION
FOR
PROTEOME
ANALYSIS
...............................................................
33
2.4.6.
PEPTIDE
MASS
FINGER
PRINTING
...................................................................................
34
2.4.7.
ELISA
.......................................................................................................................
34
2.5.
HISTOLOGY
.........................................................................................................................
35
2.5.1.
PREPARATION
OF
FROZEN
SECTIONS
................................................................................
35
2.5.2.
PREPARATION
OF
PARAFFIN
SECTIONS
..............................................................................
35
2.5.3.
DEPARAFFINIZATION
.....................................................................................................
36
2.5.4.
IMMUNOFLUORESCENCE
ANALYSIS
................................................................................
36
2.5.5.
IMMUNOHISTOCHEMISTRY
ANALYSIS
.............................................................................
37
2.5.6.
IN
SITU
ZYMOGRAPHY
..................................................................................................
37
2.5.7.
ALP
STAINING
.............................................................................................................
38
2.5.8.
TRAP
STAINING
.........................................................................................................
38
2.6.
MICE
..................................................................................................................................
39
2.7.
STATISTICAL
ANALYSIS
............................................................................................................
39
3.
RESULTS
....................................................................................................................................
40
3.1.
PREFACE
..............................................................................................................................
40
3.2.
IN
VITRO
ANALYSIS
OF
THE
ROLE
OF
MIRC24
CLUSTER-ENCODED
RNIRNAS
FOR
CARTILAGE
ECM
PRODUCTION
....................................................................................................................................
40
3.2.1.
IN
SILICO
IDENTIFICATION
OF
PUTATIVE
MIR
TARGETS
......................................................
40
3.2.2.
TARGET
GENE
EXPRESSION
AFTER
MIR-322
TRANSFECTION
.............................................
41
3.2.3.
ECM
PROTEIN
ABUNDANCE
IN
MIR-TRANSFECTED
CHONDROCYTES
..................................
43
3.2.4.
CHARACTERIZATION
OF
MIR-322-TARGET
INTERACTIONS
...................................................
46
3.2.5.
CHARACTERIZATION
OF
THE
MIRNA-MRNA
INTERACTION
UPON
CRISPR/CAS9-MEDIATED
GENETIC
INACTIVATION
OF
THE
MIR-322-SPECIFIC
BINDING
SITE
WITHIN
THE
3
-UTR
OF
COLL2AL
GENE
47
3.3.
IN
VIVO
ANALYSIS
OF
THE
ROLE
OF
MIRC24
CLUSTER-ENCODED
RNIRNAS
FOR
CARTILAGE
ECM
PRODUCTION
AND
STABILITY
..............................................................................................................
51
3.4.
IN
VIVO
ANALYSIS
OF
THE
ROLE
OF
MIRC24
CLUSTER-ENCODED
RNIRNAS
FOR
BONE
FORMATION
AND
REMODELING
...........................................................................................................................
71
4.
DISCUSSION
...............................................................................................................................
90
4.1.
CONSEQUENCES
OF
MIRNA-ECM
TARGET
GENE
INTERACTION
FOR
ECM
STABILITY
...............
90
4.2.
EFFECT
OF
MIRC24
CLUSTER
ABLATION
ON
EXTRACELLULAR
MATRIX
PRODUCTION
AND
DEGRADATION
92
4.3.
ROLE
OF
THE
MIRC24
CLUSTER
IN
CHONDROCYTE-MEDIATED
BONE
RESORPTION
.......................
94
4.4.
MECHANISM
OF
MIRC24
CLUSTER-MEDIATED
POSTTRANSCRIPTIONAL
GENE
REGULATION
..............
98
4.5.
OUTLOOK
...........................................................................................................................
100
SUPPLEMENTAL
...............................................................................................................................
102
REFERENCES
....................................................................................................................................
103
ABBREVIATIONS
...............................................................................................................................
117
DANKSAGUNG
..................................................................................................................................
120
LEBENSLAUF
....................................................................................................................................
121
ERKLARUNG
.....................................................................................................................................
123
|
| adam_txt |
ABSTRACT
.
III
ZUSAMMENFASSUNG
.
IV
1.
INTRODUCTION
.
1
1.1.
THE
SKELETAL
SYSTEM
.
1
1.1.1.
ENDOCHONDRAL
OSSIFICATION
.
1
1.1.2.
ORGANIZATION
OF
THE
GROWTH
PLATE
AND
THE
EXTRACELLULAR
MATRIX
.
3
1.1.3.
OSTEOCLAST-MEDIATED
ECM
REMODELING
.
5
1.2.
POST
TRANSCRIPTIONAL
GENE
REGULATION
.
9
1.2.1.
MIRNA
BIOGENESIS
.
9
1.2.2.
MECHANISMS
OF
MIRNA-MEDIATED
GENE
REGULATION
.
11
1.2.3.
MIRNAS
IN
ENDOCHONDRAL
OSSIFICATION
.
12
1.2.4.
ROLE
OF
THE
MIRC24
CLUSTER
FOR
CARTILAGE
DEVELOPMENT
.
13
1.3.
AIMS
.
14
2.
MATERIALS
AND
METHODS
.
15
2.1.
MATERIALS
.
15
2.1.1.
BUFFERS
.
15
2.1.2.
OLIGONUCLEOTIDES
.
15
2.1.3.
PLASMIDS
.
17
2.1.4.
ANTIBODIES
.
18
2.2.
CELL
BIOLOGICAL
METHODS
.
19
2.2.1.
EUKARYOTIC
CELL
CULTURES
.
19
2.2.2.
CRYOCONSERVATION
OF
CELLS
.
19
2.2.3.
TRANSFECTION
OF
HEK293
EBNA
CELLS
WITH
MIRNA
MIMICS
AND
PLASMIDS
.
19
2.2.4.
DUAL
LUCIFERASE
ASSAY
.
20
2.2.5.
CRISPR/CAS9
TARGETING
OF
COLL2AL
.
20
2.2.6.
ISOLATION
AND
CULTIVATION
OF
PRIMARY
EPIPHYSEAL
CHONDROCYTES
.
22
2.2.7.
MIRNA
MIMIC
TRANSFECTION
OF
ATDC5
CELLS
AND
PRIMARY
EPIPHYSEAL
CHONDROCYTES
.
23
2.2.8.
STIMULATION
OF
PECS
WITH
INSULIN,
FCS,
BMP-2
AND
TGF-PI
.
23
2.2.9.
ISOLATION
OF
SPLEEN
CELLS
.
24
2.2.10.
CHONDROCYTE/SPLEEN
CELL
CO-CULTURE
ASSAY
.
24
2.3.
MOLECULAR
BIOLOGICAL
METHODS
.
25
2.3.1.
GENOMIC
DNA
ISOLATION
FROM
BIOPSIES
.
25
2.3.2.
GENOMIC
DNA
ISOLATION
FROM
CULTURED
CELLS
.
25
2.3.3.
RNA
ISOLATION
.
25
2.3.4.
REVERSE
TRANSCRIPTION
.
26
2.3.5.
POLYMERASE
CHAIN
REACTION
(PCR)
.
26
2.3.6.
SEMI-QUANTITATIVE
PCR
.
27
2.3.7.
QUANTITATIVE
PCR
(QPCR)
.
28
2.3.8.
AGAROSE
GEL
ELECTROPHORESIS
.
28
2.3.9.
RESTRICTION
DIGEST
.
29
2.3.10.
PHOSPHORYLATION
AND
ANNEALING
OF
OLIGONUCLEOTIDE
PAIRS
.
29
2.3.11.
LIGATION
.
29
2.3.12.
TRANSFORMATION
OF
PLASMIDS
IN
COMPETENT
E.
COLI
STRAINS
.
30
2.3.13.
MINI-PREPARATION
OF
PLASMID
DNA
.
30
2.3.14.
SANGER
SEQUENCING
.
30
2.4.
BIOCHEMICAL
METHODS
.
31
INTRODUCTION
2.4.1.
PROTEIN
EXTRACTION
FOR
GEL
ELECTROPHORESIS
.
31
2.4.2.
SDS-POLYACRYLAMIDE
GEL
ELECTROPHORESIS
(SDS-PAGE)
.
31
2.4.3.
IMMUNOBLOT
ANALYSIS
.
32
2.4.4.
GELATIN
ZYMOGRAPHY
.
33
2.4.5.
PROTEIN
EXTRACTION
FOR
PROTEOME
ANALYSIS
.
33
2.4.6.
PEPTIDE
MASS
FINGER
PRINTING
.
34
2.4.7.
ELISA
.
34
2.5.
HISTOLOGY
.
35
2.5.1.
PREPARATION
OF
FROZEN
SECTIONS
.
35
2.5.2.
PREPARATION
OF
PARAFFIN
SECTIONS
.
35
2.5.3.
DEPARAFFINIZATION
.
36
2.5.4.
IMMUNOFLUORESCENCE
ANALYSIS
.
36
2.5.5.
IMMUNOHISTOCHEMISTRY
ANALYSIS
.
37
2.5.6.
IN
SITU
ZYMOGRAPHY
.
37
2.5.7.
ALP
STAINING
.
38
2.5.8.
TRAP
STAINING
.
38
2.6.
MICE
.
39
2.7.
STATISTICAL
ANALYSIS
.
39
3.
RESULTS
.
40
3.1.
PREFACE
.
40
3.2.
IN
VITRO
ANALYSIS
OF
THE
ROLE
OF
MIRC24
CLUSTER-ENCODED
RNIRNAS
FOR
CARTILAGE
ECM
PRODUCTION
.
40
3.2.1.
IN
SILICO
IDENTIFICATION
OF
PUTATIVE
MIR
TARGETS
.
40
3.2.2.
TARGET
GENE
EXPRESSION
AFTER
MIR-322
TRANSFECTION
.
41
3.2.3.
ECM
PROTEIN
ABUNDANCE
IN
MIR-TRANSFECTED
CHONDROCYTES
.
43
3.2.4.
CHARACTERIZATION
OF
MIR-322-TARGET
INTERACTIONS
.
46
3.2.5.
CHARACTERIZATION
OF
THE
MIRNA-MRNA
INTERACTION
UPON
CRISPR/CAS9-MEDIATED
GENETIC
INACTIVATION
OF
THE
MIR-322-SPECIFIC
BINDING
SITE
WITHIN
THE
3
'
-UTR
OF
COLL2AL
GENE
47
3.3.
IN
VIVO
ANALYSIS
OF
THE
ROLE
OF
MIRC24
CLUSTER-ENCODED
RNIRNAS
FOR
CARTILAGE
ECM
PRODUCTION
AND
STABILITY
.
51
3.4.
IN
VIVO
ANALYSIS
OF
THE
ROLE
OF
MIRC24
CLUSTER-ENCODED
RNIRNAS
FOR
BONE
FORMATION
AND
REMODELING
.
71
4.
DISCUSSION
.
90
4.1.
CONSEQUENCES
OF
MIRNA-ECM
TARGET
GENE
INTERACTION
FOR
ECM
STABILITY
.
90
4.2.
EFFECT
OF
MIRC24
CLUSTER
ABLATION
ON
EXTRACELLULAR
MATRIX
PRODUCTION
AND
DEGRADATION
92
4.3.
ROLE
OF
THE
MIRC24
CLUSTER
IN
CHONDROCYTE-MEDIATED
BONE
RESORPTION
.
94
4.4.
MECHANISM
OF
MIRC24
CLUSTER-MEDIATED
POSTTRANSCRIPTIONAL
GENE
REGULATION
.
98
4.5.
OUTLOOK
.
100
SUPPLEMENTAL
.
102
REFERENCES
.
103
ABBREVIATIONS
.
117
DANKSAGUNG
.
120
LEBENSLAUF
.
121
ERKLARUNG
.
123 |
| any_adam_object | 1 |
| any_adam_object_boolean | 1 |
| author | Georgieva, Veronika 1992- |
| author_GND | (DE-588)1256071617 |
| author_facet | Georgieva, Veronika 1992- |
| author_role | aut |
| author_sort | Georgieva, Veronika 1992- |
| author_variant | v g vg |
| building | Verbundindex |
| bvnumber | BV048256332 |
| ctrlnum | (OCoLC)1312274647 (DE-599)DNB1255731346 |
| discipline | Allgemeine Naturwissenschaft Medizin |
| discipline_str_mv | Allgemeine Naturwissenschaft Medizin |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV048256332 |
| illustrated | Illustrated |
| index_date | 2024-07-03T19:58:33Z |
| indexdate | 2024-07-10T09:33:17Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-033636563 |
| oclc_num | 1312274647 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR DE-83 |
| owner_facet | DE-355 DE-BY-UBR DE-83 |
| physical | iv, 123 Seiten Illustrationen |
| publishDate | 2021 |
| publishDateSearch | 2021 |
| publishDateSort | 2021 |
| record_format | marc |
| spelling | Georgieva, Veronika 1992- Verfasser (DE-588)1256071617 aut Post-transcriptional regulation of cartilage development vorgelegt von Veronika Georgieva aus Sofia, Bulgarien Köln 2021 iv, 123 Seiten Illustrationen txt rdacontent n rdamedia nc rdacarrier Dissertation Universität Köln 2021 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf https://d-nb.info/1255731346/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=033636563&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Georgieva, Veronika 1992- Post-transcriptional regulation of cartilage development |
| subject_GND | (DE-588)4113937-9 |
| title | Post-transcriptional regulation of cartilage development |
| title_auth | Post-transcriptional regulation of cartilage development |
| title_exact_search | Post-transcriptional regulation of cartilage development |
| title_exact_search_txtP | Post-transcriptional regulation of cartilage development |
| title_full | Post-transcriptional regulation of cartilage development vorgelegt von Veronika Georgieva aus Sofia, Bulgarien |
| title_fullStr | Post-transcriptional regulation of cartilage development vorgelegt von Veronika Georgieva aus Sofia, Bulgarien |
| title_full_unstemmed | Post-transcriptional regulation of cartilage development vorgelegt von Veronika Georgieva aus Sofia, Bulgarien |
| title_short | Post-transcriptional regulation of cartilage development |
| title_sort | post transcriptional regulation of cartilage development |
| topic_facet | Hochschulschrift |
| url | https://d-nb.info/1255731346/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=033636563&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT georgievaveronika posttranscriptionalregulationofcartilagedevelopment |
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