Influence of glycosaminoglycan modulation on prion propagation in permanent cell cultures and organotypic cerebellar slices:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2015
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Beschreibung: | IV, 179 S. Ill., graph. Darst. 30 cm |
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| 245 | 1 | 0 | |a Influence of glycosaminoglycan modulation on prion propagation in permanent cell cultures and organotypic cerebellar slices |c vorgelegt von Hanna Marie Wolf |
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Datensatz im Suchindex
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| adam_text | TABLE OF CONTENTS
TABLE OF CONTENTS
TABLE OF CONTENTS I
SUMMARY 1
ZUSAMMENFASSUNG 3
1 INTRODUCTION 6
1.1 PRIONS 6
1.2 PRION DISEASES 7
1.3 THE CELLULAR PRION PROTEIN 11
1.3.1 THE PRION PROTEIN GENE, PROTEIN STRUCTURE AND BIOSYNTHESIS 11
1.3.2 INTRACELLULAR TRAFFICKING AND FUNCTION 14
1.3.3 CELL SURFACE RECEPTORS FOR PRP
C
15
1.4 THE PATHOGENIC FROM OF THE PRION PROTEIN 19
1.4.1 STRUCTURAL AND BIOCHEMICAL CHARACTERISTICS OF PRP
C
AND PRP
SC
19
1.4.2 MODELS OF PRION CONVERSION 20
1.4.3 THE SPECIES BARRIER AND PRION STRAINS 21
1.5 CELL CULTURE AND EX VIVO PRION MODELS 24
1.6 THE DIFFERENT STEPS OF PRION INFECTION IN VITRO AND THE INVOLVEMENT
OF GAGS.... 27
1.6.1 THE BINDING AND UPTAKE OF PRP
SC
DURING ACUTE INFECTION 28
1.6.2 INITIAL PRION FORMATION 29
1.6.3 PRP
SC
FORMATION IN PERSISTENTLY INFECTED CELLS 30
1.7 OBJECTIVES 32
2 MATERIALS AND METHODS 34
2.1 BIOLOGICAL SAFETY 34
2.2 CELL BIOLOGICAL METHODS 34
2.2.1 MEDIA AND CELL LINES 34
2.2.2 THAWING OF CELLS 36
2.2.3 CULTIVATION OF CELLS 36
2.2.4 CRYOCONSERVATION OF CELLS 36
2.2.5 DETERMINATION OF THE CELL NUMBER 37
2.2.6 TREATMENT OF CELLS 37
2.2.7 PREPARATION OF BRAIN HOMOGENATES 37
2.2.8 INFECTION OF CELLS WITH PRIONS 38
2.2.9 VIABILITY ASSAY (XTT) 38
HTTP://D-NB.INFO/1075812267
TABLE OF CONTENTS
2.2.10 BLYSCAN SULFATED GLYCOSAMINOGLYCAN ASSAY 39
2.3 PRIMARY SLICE CULTURE METHODS 40
2.3.1 MOUSE HUSBANDRY 40
2.3.2 PREPARATION OF ORGANOTYPIC CEREBELLAR BRAIN SLICES 41
2.3.3 INFECTION OF CEREBELLAR BRAIN SLICES WITH PRIONS 42
2.3.4 CULTIVATION OF CEREBELLAR BRAIN SLICES 42
2.3.5 TREATMENT OF CEREBELLAR BRAIN SLICES 43
2.3.6 TEST FOR VIABILITY (PL-STAINING) 44
2.3.7 TEST FOR MITOCHONDRIAL RESPIRATION (SEAHORSE ASSAY) 44
2.4 PROTEIN BIOCHEMICAL METHODS 46
2.4.1 PREPARATION OF LYSATES FOR SDS-PAGE 46
2.4.1.1 PREPARATION OF POST NUCLEAR CELL LYSATES 46
2.4.1.2 PREPARATION OF CEREBELLAR BRAIN SLICE LYSATES 47
2.4.2 DETERMINATION OF PROTEIN CONCENTRATION 48
2.4.2.1 DETERMINATION OF PROTEIN CONCENTRATIONS IN POST NUCLEAR CELL
LYSATES 49
2.4.2.2 DETERMINATION OF PROTEIN CONCENTRATIONS IN CEREBELLAR BRAIN
SLICE
LYSATES 49
2.4.3 PROTEINASE K (PK) DIGESTION 49
2.4.3.1 PROTEINASE K (PK) DIGESTION OF POST NUCLEAR CELL LYSATES 49
2.4.3.2 PROTEINASE K (PK) DIGESTION OF CEREBELLAR BRAIN SLICE LYSATES 50
2.4.4 SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS
(SDS-PAGE) 50
2.4.5 WESTERN BLOT 52
2.4.6 INDIRECT IMMUNOFLUORESCENCE STAINING OF PROTEINS 56
2.4.6.1 INDIRECT IMMUNOFLUORESCENCE STAINING OF PROTEINS IN CELLS 56
2.4.6.2 INDIRECT IMMUNOFLUORESCENCE STAINING OF PROTEINS IN CEREBELLAR
SLICES 57
2.4.7 FLOW CYTOMETRY 58
2.5 DATA PROCESSING AND STATISTICS 60
2.5.1 PHOTODENSITOMETRY 60
2.5.2 MICROSCOPY 60
2.5.3 IMAGE DATA ANALYSIS 60
2.5.4 STATISTICS 61
TABLE OF CONTENTS
3 RESULTS 62
3.1 THE EFFECT OF DS-500 AND NACI03 ON PRION STRAIN INFECTIONS IN CELL
CULTURE 62
3.1.1 THE EFFECT OF DS-500 AND NACI03 ON SULFATED GLYCOSAMINOGLYCANS IN
MURINE L929 FIBROBLASTS 62
3.1.2 DS-500 AND NACICH IMPAIR PERSISTENT INFECTION INDEPENDENT OF THE
SCRAPIE STRAIN 66
3.1.3 DS-500 AND NACI0
3
AFFECT THE ESTABLISHMENT OF RML AND 22L PRION
INFECTIONS IN L929 CELLS 69
3.1.4 THE EFFECT OF DS-500 AND NACIC
3
ON THE BINDING AND/OR UPTAKE OF RML
AND 22L PRIONS IN L929 CELLS 72
3.1.4.1 DS-500 AND NACIC
3
DO NOT PREVENT THE INITIAL BINDING AND/OR
UPTAKE OF PRIONS IN L929 CELLS 73
3.1.4.2 DS-500 AND NACI03 DO NOT AFFECT THE UPTAKE OF PRIONS IN L929
CELLS, BUT CHANGE THE INTRACELLULAR DISTRIBUTION OF PRP
SC
AND
INCREASE THE SIZE OF PRP
SC
PUNCTA
78
3.1.5 GAGS, IN PARTICULAR HEPARAN SULFATE PROTEOGLYCANS, ARE NOT
REQUIRED FOR
THE UPTAKE OF RML AND 22L PRIONS IN CHO-K1 CELLS 83
3.1
.6
THE EFFECT OF DS-500 AND NACIC
3
ON PRP
C
LEVELS
86
3.2 THE ESTABLISHMENT OF THE PRION ORGANOTYPIC SLICE CULTURE ASSAY 89
3.2.1 RML AND 22L PRIONS PROPAGATE IN CEREBELLAR SLICES FROM C57BL/6JRJ
PUPS 89
3.2.2 IMMUNOFLUORESCENCE STAINING OF PRP
SC
IN CEREBELLAR SLICES 93
3.2.2.1 THE ESTABLISHMENT OF AN IMMUNOFLUORESCENCE STAINING PROTOCOL
FOR PRP
SC
93
3.2.2.2 DISTRIBUTION OF PRP
SC
IN 22L INFECTED CEREBELLAR SLICES 97
3.2.2.3 NEUROPATHOLOGICAL CHANGES IN 22L INFECTED CEREBELLAR SLICES 100
3.2.2.4 CELLULAR LOCALIZATION OF PRP
SC
IN CEREBELLAR SLICES 105
3.2.3 POSCA, A POWERFUL TOOL FOR PRION DRUG TREATMENT 107
3.2.3.1 THE EFFECT OF DS-500 AND NACICBON PERSISTENT 22L PRION
INFECTION IN CEREBELLAR SLICES 108
3.2.3.2 DS-500 AND NACIC
3
INHIBIT THE ESTABLISHMENT OF A 22L PRION
INFECTION IN CEREBELLAR SLICES 111
TABLE OF CONTENTS
4 DISCUSSION 114
4.1 THE INVOLVEMENT OF GAGS IN PRION PATHOGENESIS IN VITRO 114
4.1.1 DS-500 AND NACI03 IMPAIR PRP
SC
ACCUMULATION INDEPENDENT OF THE
CELL TYPE AND THE PRION STRAIN IN PERSISTENTLY PRION-INFECTED CELLS 115
4.1.2 THE ESTABLISHMENT OF A PERSISTENT RML AND 22L PRION INFECTION IS
IMPAIRED UPON TREATMENT WITH DS-500 AND NACI03 117
4.1.3 THE UPTAKE OF RML AND 22L PRIONS IS NOT DEPENDENT ON ENDOGENOUS
SULFATED GAGS 118
4.1.4 DIVERGENT EFFECTS OF DS-500 AND NACIC
3
ON CELLULAR PRP
C
LEVELS 124
4.1.5 DS-500 AND NACI0
3
IMPAIR PRION INFECTION DOWNSTREAM OF THE INITIAL
PRP
SC
UPTAKE 125
4.2 THE ESTABLISHMENT OF THE PRION ORGANOTYPIC SLICE CULTURE ASSAY 127
4.2.1 PROPAGATION OF RML AND 22L PRIONS IN CEREBELLAR SLICES OF
C57BL/6JRJ
MICE 128
4.2.2 SUCCESSFUL ESTABLISHMENT OF AN IMMUNOFLUORESCENCE STAINING
PROTOCOL
FOR PRP
SC
133
4.2.3 DISTRIBUTION AND CELLULAR LOCALIZATION OF PRP
SC
IN 22L INFECTED
ORGANOTYPIC CEREBELLAR SLICES 134
4.2.4 NEUROPATHOLOGICAL CHANGES ARE FAITHFULLY REPRODUCED IN 22L
INFECTED
ORGANOTYPIC CEREBELLAR SLICES 137
4.2.5 POSCA, A POWERFUL TOOL FOR PRION DRUG TREATMENT AND FUTURE
PERSPECTIVES 139
BIBLIOGRAPHY 142
ABBREVIATIONS 173
PUBLICATIONS AND CONGRESS CONTRIBUTIONS 178
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| spelling | Wolf, Hanna Verfasser (DE-588)1074372395 aut Influence of glycosaminoglycan modulation on prion propagation in permanent cell cultures and organotypic cerebellar slices vorgelegt von Hanna Marie Wolf 2015 IV, 179 S. Ill., graph. Darst. 30 cm txt rdacontent n rdamedia nc rdacarrier Bonn, Univ., Diss., 2015 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1075812267/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028352811&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Wolf, Hanna Influence of glycosaminoglycan modulation on prion propagation in permanent cell cultures and organotypic cerebellar slices |
| subject_GND | (DE-588)4113937-9 |
| title | Influence of glycosaminoglycan modulation on prion propagation in permanent cell cultures and organotypic cerebellar slices |
| title_auth | Influence of glycosaminoglycan modulation on prion propagation in permanent cell cultures and organotypic cerebellar slices |
| title_exact_search | Influence of glycosaminoglycan modulation on prion propagation in permanent cell cultures and organotypic cerebellar slices |
| title_full | Influence of glycosaminoglycan modulation on prion propagation in permanent cell cultures and organotypic cerebellar slices vorgelegt von Hanna Marie Wolf |
| title_fullStr | Influence of glycosaminoglycan modulation on prion propagation in permanent cell cultures and organotypic cerebellar slices vorgelegt von Hanna Marie Wolf |
| title_full_unstemmed | Influence of glycosaminoglycan modulation on prion propagation in permanent cell cultures and organotypic cerebellar slices vorgelegt von Hanna Marie Wolf |
| title_short | Influence of glycosaminoglycan modulation on prion propagation in permanent cell cultures and organotypic cerebellar slices |
| title_sort | influence of glycosaminoglycan modulation on prion propagation in permanent cell cultures and organotypic cerebellar slices |
| topic_facet | Hochschulschrift |
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