The role of RNA binding proteins in motoneuron diseases: = Die Rolle von RNA-bindenden Proteinen in Motoneuronerkrankungen
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| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
Würzburg
2016
|
| Subjects: | |
| Online Access: | Volltext Volltext Inhaltsverzeichnis |
| Physical Description: | 140 Blätter Illustrationen, Diagramme |
| Format: | Langzeitarchivierung gewährleistet, LZA |
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| 246 | 1 | 1 | |a Die Rolle von RNA-bindenden Proteinen in Motoneuronerkrankungen |
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Record in the Search Index
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| adam_text |
THE ROLE OF RNA BINDING PROTEINS IN MOTONEURON DISEASES
DIE ROLLE VON RNA-BINDENDEN PROTEINEN IN
MOTONEURONERKRANKUNGEN.
DOCTORAL THESIS FOR A DOCTORAL DEGREE
AT THE INSTITUTE FOR CLINICAL NEUROBIOLOGY,
UNIVERSITY HOSPITAL WUERZBURG
AND GRADUATE SCHOOL OF LIFE SCIENCES,
JULIUS-MAXIMILIANS-UNIVERSITAET WUERZBURG
SUBMITTED BY
RAJEEVE SIVADASAN
FROM
KADAKKAL, INDIA
WUERZBURG 2016
TABLE OF CONTENTS
1.
ABSTRACT.1
2.
ZUSAMMENFASSUNG.
3
3.
INTRODUCTION.
5
3.1 CLINICAL PHENOTYPE AND GENETIC BASIS OF SPINAL MUSCULAR ATROPHY
.
5
3.2 CELLULAR FUNCTIONS OF THE SMN PROTEIN AND MODEL SYSTEM
.
8
3.3 CLINICAL PHENOTYPE AND GENETIC BASIS OF AMYOTROPHIC LATERAL
SCLEROSIS.12
3.4 DISCOVERY OF C90RF72
MUTATIONS.
19
3.5 PATHOGENIC MECHANISM OF THE C90RF72 REPEAT
EXPANSION.20
3.6 CURRENT MODEL SYSTEMS TO STUDY C90RF72 FUNCTIONS
.
22
3.7 THE CURRENT STATE OF KNOWLEDGE ON THE FUNCTION OF C90RF72
.
25
4.
MATERIALS.
28
4.1. CELL
LINES.28
4.2 BACTERIAL
STRAINS.
28
4.3 PRIMARY MOUSE MOTONEURON
CULTURE.29
4.4 BUFFERS FOR
IMMUNOFLUORESCENCE.31
4.5 BUFFERS FOR WESTERN
BLOTTING.32
4.6 COMMERCIAL KITS AND MOLECULAR LADDERS
.
33
4.7 STOCK BUFFERS FOR PROTEIN
PURIFICATION.
33
4.7.1 BUFFERS FOR PURIFICATION O F HNRNP
R.
.
.
34
4.7.2 BUFFERS FOR PURIFICATION O F
SMN.
34
4.7.3 BUFFERS FOR PURIFICATION O F
TDP-43.
35
4.7.4 BUFFERS FOR HIS-TRAP HP COLUMN STRIPING AND CLEANING.
.
35
4.7.5 COLUMNS USED FOR PROTEIN PURIFICATION
.
36
4.8
ANTIBODIES.36
4.9 EMSA
BUFFERS.39
4.10 INSTRUMENTS
USED.
39
4.11 SOFTWARE
USED.
40
5.
METHODS.41
5.1 ANIMALS AND ETHICS
STATEMENT.
41
5.2 CELL
CULTURE.
41
5.3 CRYO STOCKING OF CELL LINES
.
41
5.4 TRANSFECTION OF HEK-293, HELA AND NSC-34
CELL.42
5.5 PRIMARY MOTONEURON
CULTURING.42
5.6 OVEREXPRESSION AND KNOCKDOWN OF C90RF72 BY SHRNA IN CULTURED MOUSE
MOTONEURONS.43
5.7 STANDARDIZATION OF LENTIVIRAL CONCENTRATION ON NSC-34 CELLS
.
44
5.8 SAMPLE PREPARATION FOR LC-MS ANALYSIS USING NSC-34 CELLS
.
44
5.9 LC-MS DATA ACQUISITION AND
ANALYSIS.
45
5.10 DATA
ANALYSIS.
45
5.11 IMMUNOFLUORESCENCE ANALYSIS OF PRIMARY MOUSE MOTONEURONS AND CELL
LINES
.
46
5.12 LENTI VIRUS
PRODUCTION.
46
5.13 PROTEIN
PURIFICATION.48
5.13.1 PREPARATION O F BACTERIAL LYSATES FOR RECOMBINANT PROTEIN
PURIFICATION.48
5.13.2 SETUP O F FPLC FOR PURIFICATION AND MAINTENANCE
.
48
5.13.3
CHROMATOGRAPHY.
48
5.13.4 IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY (IMAC)
.
49
5.13.5 CHITIN-INTEIN
SYSTEM.
49
5.13.6 ION EXCHANGE
CHROMATOGRAPHY.49
5.13.7 SIZE-EXCLUSION
CHROMATOGRAPHY.50
5.14 CO-IMMUNOPRECIPITATIONS OF RECOMBINANT
PROTEINS.50
5.15 ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA)
.
51
5.15.1 LINEARISATION O F BETA-ACTIN 3 *UTR CDNA (VECTOR)
.
52
5.15.2. RADIOLABELING BETA-ACTIN 3 *UTR MRNA USING MEGASCRIPT
TRANSCRIPTION KIT
(AMBION).
52
5.16 CYTOSOLIC AND NUCLEAR
FRACTIONATION.53
5.17 IMMUNOPRECIPITATIONS FROM CULTURED MOUSE MOTONEURONS AND CELLS
.
54
5.18 LIVE CELL IMAGING OF ACTIN
DYNAMICS.54
5.19 G/F-ACTIN RATIO
MEASUREMENT.
55
5.20 SDS-PAGE AND WESTERN BLOTTING
.
56
5.21 COOMASSIE STAINING FOR PROTEIN
GELS.58
5.22
CLONING.
59
5.22.1 POLYMERASE CHAIN REACTION
(PCR).59
5.22.2 RESTRICTION ENZYME DIGESTION AND ANALYSIS OFDNA
FRAGMENTS.59
5.22.3 LIGATION O F RESTRICTED FRAGMENT INTO VECTOR.
.
60
5.22.4
SEQUENCING.61
5.22.5 TRANSFORMATION O F CHEMICAL COMPETENT E.
COLI.62
5.22.6 EASY PREP PLASMID
PURIFICATION.62
5.23 CROSS LINKING ANTIBODY TO BEADS FOR LC-MS
ANALYSIS.63
5.24 QUANTITATIVE
PCR.64
5.25 IMMUNOPRECIPITATION OF PROTEIN-RNA
COMPLEXES.65
5.26 STATISTICAL
ANALYSIS.
66
6.
RESULTS.
68
6.1 PROTEIN PURIFICATION OF MURINE RECOMBINANT HNRNP R AND
SMN.68
6.2 PROTEIN PURIFICATION OF MURINE RECOMBINANT
TDP-43.71
6.3 HNRNP R INTERACTION WITH SMN IN MOTONEURONS
.
73
6.4 DIRECT SMN INTERACTION WITH HNRNP R MODULATES AFFINITY OF HNRNP R
.
75
6.5 DEPLETION OF SMN ALTERS THE SUBCELLULAR DISTRIBUTION OF HNRNP R IN
MOTONEURONS. 77
6.6 TDP-43 INTERACTION WITH SMN AND HNRNP R INFLUENCED BY THE MUTATION
.
81
6.7 DIRECT INTERACTION OF SMN WITH TDP-43
.
.
83
6.8 HNRNP R SMN INTERACTION ENHANCE THE BINDING OF THE 3 *UTR OF SS-ACTIN
MRNA TO THE
HNRNP R
COMPLEX.
85
6.9 TDP-43 BINDS TO THE 3 *UTR OF
SS-ACTIN.
87
6.10 TDP-43 SMN INTERACTION ENHANCED THE BINDING OF THE 3*UTR OF SS-ACTIN
MRNA TO
TDP-43.88
6.11 TDP-43 MUTATIONS IN THE C-TERMINAL REGION INFLUENCE THE BINDING OF
MRNA
.
89
6.12 CLONING OF PLASMIDS FOR OVEREXPRESSION OF C90RF72 AND SHRNA FOR
MOUSE
C90RF72.
91
6.13 C90RF72 EXPRESSION REGULATES AXONAL GROWTH IN CULTURED MOTONEURONS
.
94
6.14 IDENTIFICATION OF THE C90RF72
INTERACTOME.
95
6.15 C90RF72 REGULATES COFILIN
PHOSPHORYLATION.99
6.16 CLONING OF A VECTOR FOR COMBINING ACTIN VISUALISATION WITH C90RF72
KNOCKDOWN
.
.
101
6.17 ACTIN DYNAMICS IS DISTURBED AFTER KNOCKDOWN OF MOUSE C90RF72
.
102
6.18 ALTERED G/F ACTIN RATIO IN MOTONEURONS AFTER OVEREXPRESSION AND
KNOCKDOWN OF
C90RF72.
104
7.
DISCUSSION.
106
7.1 SMN INTERACTS DIRECTLY WITH HNRNP R AND TDP-43 AND REGULATES THE
BINDING TO THE
3*UTR OF SS-ACTIN
MRNA.
106
7.2 DEPLETION OF SMN ALTERS THE SUBCELLULAR DISTRIBUTION OF HNRNP R IN
MOTONEURONS. 107
7.3 MUTATIONS IN THE C-TERMINAL REGION OF TDP-43 REGULATE THE BINDING OF
PROTEIN AND
RNA.
109
7.4 C90RF72 INTERACTION WITH COFILIN MODULATES ACTIN DYNAMICS IN
MOTONEURONS
.
110
8. REFERENCES FOR THIS THESIS
.
.
.114
9.
APPENDIX.126
9.1 LIST OF
FIGURES:.
126
9.2 LIST OF
TABLES:.
129
9.3
ABBREVIATIONS.131
10.
AFFIDAVIT.
134
11.
ACKNOWLEDGMENTS.135
12. CURRICULUM
VITAE.138 |
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| spelling | Sivadasan, Rajeeve 1980- Verfasser (DE-588)1123578737 aut The role of RNA binding proteins in motoneuron diseases = Die Rolle von RNA-bindenden Proteinen in Motoneuronerkrankungen submitted by Rajeeve Sivadasan from Kadakkal, India ; Primary Supervisor: Prof. Dr. Michael Sendtner aus dem Lebenslauf der Dissertation Die Rolle von RNA-bindenden Proteinen in Motoneuronerkrankungen Würzburg 2016 140 Blätter Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier Dissertation Julius-Maximilians-Universität Würzburg 2016 Langzeitarchivierung gewährleistet, LZA Zusammenfassung in deutscher und englischer Sprache RNS-Bindungsproteine (DE-588)4178253-7 gnd rswk-swf Krankheit (DE-588)4032844-2 gnd rswk-swf Motoneuron (DE-588)4135747-4 gnd rswk-swf RNA binding proteins (DE-588)4113937-9 Hochschulschrift gnd-content Motoneuron (DE-588)4135747-4 s Krankheit (DE-588)4032844-2 s RNS-Bindungsproteine (DE-588)4178253-7 s DE-604 Sendtner, Michael dgs https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-141907 Resolving-System kostenfrei Volltext http://d-nb.info/1122021054/34 Langzeitarchivierung Nationalbibliothek kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029408699&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Sivadasan, Rajeeve 1980- The role of RNA binding proteins in motoneuron diseases = Die Rolle von RNA-bindenden Proteinen in Motoneuronerkrankungen RNS-Bindungsproteine (DE-588)4178253-7 gnd Krankheit (DE-588)4032844-2 gnd Motoneuron (DE-588)4135747-4 gnd |
| subject_GND | (DE-588)4178253-7 (DE-588)4032844-2 (DE-588)4135747-4 (DE-588)4113937-9 |
| title | The role of RNA binding proteins in motoneuron diseases = Die Rolle von RNA-bindenden Proteinen in Motoneuronerkrankungen |
| title_alt | aus dem Lebenslauf der Dissertation Die Rolle von RNA-bindenden Proteinen in Motoneuronerkrankungen |
| title_auth | The role of RNA binding proteins in motoneuron diseases = Die Rolle von RNA-bindenden Proteinen in Motoneuronerkrankungen |
| title_exact_search | The role of RNA binding proteins in motoneuron diseases = Die Rolle von RNA-bindenden Proteinen in Motoneuronerkrankungen |
| title_full | The role of RNA binding proteins in motoneuron diseases = Die Rolle von RNA-bindenden Proteinen in Motoneuronerkrankungen submitted by Rajeeve Sivadasan from Kadakkal, India ; Primary Supervisor: Prof. Dr. Michael Sendtner |
| title_fullStr | The role of RNA binding proteins in motoneuron diseases = Die Rolle von RNA-bindenden Proteinen in Motoneuronerkrankungen submitted by Rajeeve Sivadasan from Kadakkal, India ; Primary Supervisor: Prof. Dr. Michael Sendtner |
| title_full_unstemmed | The role of RNA binding proteins in motoneuron diseases = Die Rolle von RNA-bindenden Proteinen in Motoneuronerkrankungen submitted by Rajeeve Sivadasan from Kadakkal, India ; Primary Supervisor: Prof. Dr. Michael Sendtner |
| title_short | The role of RNA binding proteins in motoneuron diseases |
| title_sort | the role of rna binding proteins in motoneuron diseases die rolle von rna bindenden proteinen in motoneuronerkrankungen |
| title_sub | = Die Rolle von RNA-bindenden Proteinen in Motoneuronerkrankungen |
| topic | RNS-Bindungsproteine (DE-588)4178253-7 gnd Krankheit (DE-588)4032844-2 gnd Motoneuron (DE-588)4135747-4 gnd |
| topic_facet | RNS-Bindungsproteine Krankheit Motoneuron Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-141907 http://d-nb.info/1122021054/34 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029408699&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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