Mechanistic insights into the function and dysfunction of Parkin, an E3 ubiquitin ligase associated with Parkinson's disease:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | kostenfrei https://nbn-resolving.org/urn:nbn:de:bvb:19-153287 Inhaltsverzeichnis |
| Beschreibung: | 133 S. Ill., graph. Darst. |
Internformat
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| 245 | 1 | 0 | |a Mechanistic insights into the function and dysfunction of Parkin, an E3 ubiquitin ligase associated with Parkinson's disease |c Anna Theresia Gertraud Pilsl |
| 264 | 1 | |c 2012 | |
| 300 | |a 133 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a München, Univ., Diss., 2012 | ||
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| 856 | 4 | 1 | |u http://edoc.ub.uni-muenchen.de/15328/ |x Verlag |z kostenfrei |3 Volltext |
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| 912 | |a ebook | ||
| 999 | |a oai:aleph.bib-bvb.de:BVB01-025716420 | ||
Datensatz im Suchindex
| _version_ | 1804150055025770496 |
|---|---|
| adam_text | IMAGE 1
TABLE OF CONTENT
1 INTRODUCTION 8
1.1 PARKINSON S DISEASE 8
1.1.1 ETIOLOGY 8
1.1.2 PATHOLOGY 9
1.1.3 SYMPTOMS AND DIAGNOSIS 10
1.1.4 CURRENT TREATMENT OPTIONS 10
1.2 PD AND MITOCHONDRIA 11
1.2.1 IMPORTANCE OF MITOCHONDRIA FOR CELLULAR HOMEOSTASIS 11
1.2.2 PD AND MITOCHONDRIAL DYSFUNCTION 14
1.3 GENETICS OF PD 15
1.4 PINK1 17
1.5 PARKIN 20
1.5.1 PARKIN PROTECTS CELLS AGAINST A BROAD RANGE OF TOXIC INSULTS 21
1.5.2 PARKIN MEDIATES THE REMOVAL OF DAMAGED MITOCHONDRIA VIA MITOPHAGY
22
1.6 MOLECULAR INTERACTION OF PARKIN AND PINK1 25
1.7 DELLULAR QUALITY CONTROL MECHANISMS 25
1.7.1 UBIQUITIN-PROTEASOMAL SYSTEM 26
1.7.2 AUTOPHAGOSOMAL-LYSOSOMAL SYSTEM 28
2 AIMS OF THE THESIS 32
3 RESULTS 34
3.1 CHARACTERIZATION OF PARKIN-MEDIATED MITOPHAGY 34
3.1.1 DEPOLARIZATION OF MITOCHONDRIAL MEMBRANE POTENTIAL INDUCES
MITOCHONDRIAL FRAGMENTATION INDEPENDENTLY OF PARKIN 34
3.1.2 PARKIN IS RECRUITED TO DYSFUNCTIONAL MITOCHONDRIA AND INITIATES
THEIR DEGRADATION 34
3.1.3 UNCOUPLING OF MITOCHONDRIAL MEMBRANE POTENTIAL BUT NOT OTHER
CELLULAR STRESSORS MEDIATE PARKIN RECRUITMENT TO MITOCHONDRIA AND
MITOPHAGY 36
3.1.4 PATHOGENIC PARKIN MUTANTS ARE COMPROMISED IN MITOCHONDRIAL
TRANSLOCATION OR MITOPHAGY COMPLETION 40
4
HTTP://D-NB.INFO/1033482927
IMAGE 2
3.1.5 DELETION OF PARKIN FUNCTIONAL DOMAINS RESULTS IN PARKIN
INSOLUBILITY AND LOSS OF
MITOCHONDRIAL TRANSLOCATION AND MITOPHAGY 43
3.1.6 PARKIN-DEPENDENT UBIQUITINATION OF DEPOLARIZED MITOCHONDRIA 45
3.1.7 BOTH AUTOPHAGY AND PROTEASOMAL DEGRADATION ARE ESSENTIAL FOR
MITOPHAGY 48
3.1.8 PINK1 IS ESSENTIAL FOR PARKIN-MEDIATED MITOPHAGY 52
3.1.9 OVEREXPRESSION OF PARKIN AND PINK1 IS SUFFICIENT TO INDUCE
MITOPHAGY IN THE ABSENCE OF MITOCHONDRIAL DEPOLARIZATION 54
3.1.10 OVEREXPRESSION OF PINK1 INDUCES INSOLUBILITY OF PARKIN 56
3.1.11 MITOCHONDRIAL DEPOLARIZATION INDUCES INSOLUBILITY OF PARKIN 57
3.1.12 IN THE ABSENCE OF PINK1 CCCP DOES NOT RENDER PARKIN INSOLUBLE 58
3.1.13 PATHOGENIC PINK1 MUTANTS HAVE DIFFERENTIAL EFFECTS ON PARKIN
SOLUBILITY AND MITOPHAGY 59
3.1.14 MITOCHONDRIAL^ TARGETED PARKIN INDUCES MITOPHAGY UPON DISSIPATION
OF MITOCHONDRIAL MEMBRANE POTENTIAL 60
3.1.15 MITOCHONDRIAL TARGETING OF PARKIN IS NOT SUFFICIENT TO INDUCE
MITOPHAGY IN THE ABSENCE OF PINK1 63
3.2 PATHOGENIC PARKIN SPECIES ARE DEGRADED VIA THE PROTEASOME IN A
P62-DEPENDENT MANNER 64
3.2.1 PATHOGENIC PARKIN MUTANTS ARE PRONE TO MISFOLDING AND FORM
CYTOSOLIC AGGREGATES 64
3.2.2 P62 COLOCALIZES WITH MUTANT PARKIN AGGREGATES 66
3.2.3 P62 LEVELS INFLUENCE THE ABUNDANCE OF PARKIN AGGREGATES 71
3.2.4 PROTEASOMAL INHIBITION ENHANCES MUTANT PARKIN AGGREGATE FORMATION
...73
3.2.5 THE AUTOPHAGOSOMAL-LYSOSOMAL PATHWAY IS DISPENSABLE FOR
DEGRADATION OF MUTANT PARKIN AGGREGATES 75
3.2.6 A CELL CULTURE MODEL TO MONITOR THE DEGRADATION OF PATHOGENIC
PARKIN MUTANTS.77
3.2.7 ANALYSIS OF MUTANT PARKIN W453X IN A TRANSGENIC MOUSE MODEL OF
PARKIN AGGREGATION 80
4 DISCUSSION 83
4.1 PARKIN AND MITOPHAGY 83
4.2 DEGRADATION OF PATHOGENIC PARKIN MUTANTS 92
5 MATERIAL 97
5.1 DRUGS AND INHIBITORS 97
5.2 ENZYMES 98
5
IMAGE 3
5.3 PRIMERS AND OLIGONUCLEOTIDES 98
5.4 BUFFERS .....98
5.5 MAMMALIAN CELL LINES AND PRIMARY CORTICAL NEURONS -101
5.6 CELL CULTURE REAGENTS AND MEDIA 101
5.6.1 REAGENTS AND MEDIA FOR MAMMALIAN CELL CULTURE 101
5.6.2 MEDIA FOR BACTERIA 102
5.7 ANTIBODIES 102
5.7.1 PRIMARY ANTIBODIES 102
5.7.2 SECONDARY ANTIBODIES 103
5.8 PLASMIDS 104
5.8.1 COMMERCIAL VECTORS 104
5.8.2 MAMMALIAN EXPRESSION VECTORS 104
5.8.3 LENTIVIRAL EXPRESSION VECTORS 105
6 METHODS 106
6.1 NUCLEIC ACID TECHNIQUES 106
6.1.1 POLYMERASE CHAIN REACTION 106
6.1.2 RESTRICTION DIGEST 106
6.1.3 AGAROSE GEL ELECTROPHORESIS 106
6.. 1.4 PURIFICATION OF DNA FROM AGAROSE AND CLEAN-UP OF PCR REACTIONS
106
6.1.5 DEPHOSPHORYLATION OF LINEARIZED VECTORS 107
6.1.6 LIGATION OF DNA FRAGMENTS 107
6.1.7 PREPARATION OF COMPETENT BACTERIA 107
6.1.8 TRANSFORMATION OF BACTERIA AND SCREENING 107
6.1.9 DNA SEQUENCING 107
6.1.10 PREPARATIVE AND ANALYTICAL SCALE PLASMID DNA PREPARATION 108
6.2 CELL CULTURE METHODS 108
6.2.1 CULTURE AND MAINTENANCE OF MAMMALIAN CELL LINES 108
6.2.2 PREPARATION AND CULTIVATION OF PRIMARY CORTICAL NEURONS 108
6.2.3 LIPOSOME-MEDIATED TRANSIENT TRANSFECTION 109
6.2.4 ESTABLISHMENT OF STABLE CELL LINES VIA LENTIVIRAL TRANSDUCTION 109
6.2.5 TRANSIENT SILENCING OF TARGET GENES USING SIRNA 109
6.2.6 CELL HARVEST AND LYSIS 109
6
IMAGE 4
6.3 PROTEIN BIOCHEMISTRY 110
6.3.1 MEASUREMENT OF TOTAL PROTEIN CONTENT 110
6.3.2 SDS-PAGE AND IMMUNOBLOT ANALYSIS 110
6.3.3 DETERGENT-SOLUBILITY ASSAY 110
6.4 MICROSCOPY 110
6.4.1 INDIRECT IMMUNOFLUORESCENCE 110
6.4.2 MITOPHAGY ASSAY 111
6.4.3 ACTIVATED CASPASE-3 ASSAY 111
6.5 STATISTICAL ANALYSIS 111
6.6 SOFTWARE AND DATABASES 111
7 REFERENCES 113
8 ACKNOWLEDGEMENTS 129
9 PUBLICATIONS 131
10 SUMMARY 132
7
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| spelling | Pilsl, Anna Theresia Gertraud 1983- Verfasser (DE-588)103111629X aut Mechanistic insights into the function and dysfunction of Parkin, an E3 ubiquitin ligase associated with Parkinson's disease Anna Theresia Gertraud Pilsl 2012 133 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:19-153287 http://edoc.ub.uni-muenchen.de/15328/ Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:19-153287 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025716420&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Pilsl, Anna Theresia Gertraud 1983- Mechanistic insights into the function and dysfunction of Parkin, an E3 ubiquitin ligase associated with Parkinson's disease |
| subject_GND | (DE-588)4113937-9 |
| title | Mechanistic insights into the function and dysfunction of Parkin, an E3 ubiquitin ligase associated with Parkinson's disease |
| title_auth | Mechanistic insights into the function and dysfunction of Parkin, an E3 ubiquitin ligase associated with Parkinson's disease |
| title_exact_search | Mechanistic insights into the function and dysfunction of Parkin, an E3 ubiquitin ligase associated with Parkinson's disease |
| title_full | Mechanistic insights into the function and dysfunction of Parkin, an E3 ubiquitin ligase associated with Parkinson's disease Anna Theresia Gertraud Pilsl |
| title_fullStr | Mechanistic insights into the function and dysfunction of Parkin, an E3 ubiquitin ligase associated with Parkinson's disease Anna Theresia Gertraud Pilsl |
| title_full_unstemmed | Mechanistic insights into the function and dysfunction of Parkin, an E3 ubiquitin ligase associated with Parkinson's disease Anna Theresia Gertraud Pilsl |
| title_short | Mechanistic insights into the function and dysfunction of Parkin, an E3 ubiquitin ligase associated with Parkinson's disease |
| title_sort | mechanistic insights into the function and dysfunction of parkin an e3 ubiquitin ligase associated with parkinson s disease |
| topic_facet | Hochschulschrift |
| url | http://edoc.ub.uni-muenchen.de/15328/ https://nbn-resolving.org/urn:nbn:de:bvb:19-153287 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025716420&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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