Investigating genetic modifiers of prion-like transmission and endo-lysosomal escape in the metazoan model system Caenorhabditis elegans:
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| 100 | 1 | |a Sandhof, Carl Alexander |d 1990- |e Verfasser |0 (DE-588)1214844146 |4 aut | |
| 245 | 1 | 0 | |a Investigating genetic modifiers of prion-like transmission and endo-lysosomal escape in the metazoan model system Caenorhabditis elegans |c presented by M. Sc. Carl Alexander Sandhof ; referees: Prof. Dr. Bernd Bukau, Prof. Dr. Aurelio Teleman |
| 264 | 1 | |a Heidelberg |c [2022] | |
| 300 | |a XVIII, 164 Seiten |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Ruprecht-Karls-University Heidelberg |d 2022 | ||
| 546 | |a Text Englisch, Zusammenfassung in englischer und deutscher Sprache | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 700 | 1 | |a Bukau, Bernd |d 1954- |0 (DE-588)143209019 |4 dgs | |
| 710 | 2 | |a Universität Heidelberg |0 (DE-588)2024349-2 |4 dgg | |
| 856 | 4 | 2 | |m B:DE-101 |q application/pdf |u https://d-nb.info/1263888291/04 |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=034164242&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
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| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-034164242 | |
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| adam_text |
TABLE
OF
CONTENTS
CONTRIBUTIONS
.
VII
ZUSAMMENFASSUNG
.
XIII
ABSTRACT
.
XVII
1.
INTRODUCTION
.
1
1.1
PROTEINOPATHIES
.
1
1.2
A-SYNUCLEIN
AND
SYNUCLEINOPATHIES
.
2
1.2.1
A-SYN
DOMAIN
ORGANIZATION
AND
FUNCTION
.
3
1.2.2
DISEASE
RELATED
A-SYN
MUTATIONS
.
4
1.2.3
.
THE
ROLE
OF
A-SYN
IN
THE
PATHOGENESIS
OF
SYNUCLEINOPATHIES
.
5
1.3
TAU
AND
TAUOPATHIES
.
7
1.3.1
THE
MICROTUBULE
BINDING
PROTEIN
TAU
.
9
1.3.2
TAU
AGGREGATION
.
10
1.3.3
POST
TRANSLATIONAL
MODIFICATIONS
OF
TAU
.
11
1.3.4
.
DISEASE
RELATED
MAPT
MUTATIONS
.
12
1.3.5
.
TOXICITY
OF
TAU
AGGREGATION
.
13
1.4
.
THE
PRION
HYPOTHESIS
IN
NEURODEGENERATIVE
DISEASES
.
15
1.4.1
CELLULAR
PATHWAYS
THAT
MEDIATE
PRION-LIKE
SPREADING
OF
INTRACELLULAR
PROTEINS
.
19
1.5
.
CAENORHABDITIS
ELEGANS
AS
METAZOAN
MODEL
SYSTEM
.
23
1.5.1
LIFE
CYCLE
.24
1.5.2
C.
ELEGANS
AS
A
SCREENING
MODEL
.
25
2.
AIM
OF
THIS
THESIS
.
29
3.
RESULTS
.
31
3.1
A-SYN
MODEL
.
31
3.1.1
A-SYNUCLEIN::RFP
EXPRESSION
IN
BWM
CELLS
IS
CYTOTOXIC
AND
IS
TRANSMITTED
TO
THE
HYPODERMIS
IN
AN
AGE
DEPENDENT
MANNER
.
31
3.1.2
A-SYN::RFP
DAMAGES
THE
ENDO-LYSOSOMAL
SYSTEM
NOT
ONLY
IN
THE
RECIPIENT
HYPODERMIS
BUT
ALSO
THE
EXPRESSING
BWM
CELLS
.
33
3.1.3
A-SYN::RFP
EXPRESSION
IN
DOPAMINERGIC
NEURONS
LEADS
TO
AXONAL
DEGENERATION
.
35
3.1.4
DOPAMINERGIC
NEURON
A-SYN::RFP
IS
TRANSMITTED
TO
THE
HYPODERMIS
AND
INDUCES
LYSOSOMAL
RUPTURE
IN
THE
RECIPIENT
TISSUE
.36
3.1.5
REDUCING
INSULIN
SIGNALING
DELAYS
TRANSMISSION
AND
RESCUES
A-SYN
CELL
AUTONOMOUS
AND
NON-AUTONOMOUS
TOXICITY
IN
A
DAF-16
DEPENDENT
MANNER
.38
3.1.6
CHANGES
IN
RME-8/DNAJC13
FUNCTION
MODULATE
A-SYN::RFP
INTER-TISSUE
SPREADING
IN
C.
ELEGANS
.42
3.2
GENERATING
A
TAU
C.
ELEGANS
SPREADING
MODEL
TO
IDENTIFY
MODULATORS
OF
ENDO
LYSOSOMAL
ESCAPE
.
46
3.2.1
CLONING
AND
EXTRACHROMOSOMAL
EXPRESSION
OF
FLUORESCENTLY
TAGGED
TAU
FRAGMENTS
IN
THE
MECHANOSENSORY
NEURONS
.
46
3.2.2
EXPRESSION
OF
THE
F3
TAU
FRAGMENTS
IS
NEUROTOXIC
.
48
3.2.3
LIKE
A-SYN
THE
TAU
FRAGMENTS
ARE
READILY
TRANSMITTED
FROM
NEURONS
TO
THE
HYPODERMIS
.
50
3.2.4
TAU
FRAGMENT
TRANSMISSION
SENSITIZES
THE
ENDO-LYSOSOMAL
SYSTEM
IN
THE
RECIPIENT
HYPODERMIS
TO
RUPTURE
.
51
3.2.5
AN
UNBIASED
RNAI
SCREEN
FOR
SUPPRESSORS
OF
ENDO-LYSOSOMAL
RUPTURE
IDENTIFIES
KNOWN
AND
NOVEL
PATHWAYS
IMPLICATED
IN
ENDO-LYSOSOMAL
HEALTH
.54
3.2.6
A
FEW
GENES
SENSITIZE
THE
ENDO-LYSOSOMAL
SYSTEM
SPECIFICALLY
TO
F3AK280::MCH
MEDIATED
MEMBRANE
DISRUPTION
.58
3.2.7
FUNCTIONAL
SPHINGOLIPID
METABOLISM
IS
AN
ESSENTIAL
PATHWAY
TO
PROTECT
THE
ENDO-LYSOSOMAL
SYSTEM'S
INTEGRITY
.
60
3.2.8
A
DIET
HIGH
IN
MONO
(MUFA)
OR
POLYUNSATURATED
FATTY
ACIDS
(PUFA)
PROTECTS
THE
ENDO-LYSOSOMAL
MEMBRANE
FROM
RUPTURE
.
64
3.2.9
DIETS
HIGH
IN
SATURATED
FATTY
ACIDS
(SFA)
INDUCE
ENDO-LYSOSOMAL
MEMBRANE
DAMAGE
.67
3.2.10
FLUORESCENCE
RECOVERY
AFTER
PHOTOBLEACHING
(FRAP)
MEASUREMENTS
CONFIRM
AN
ENDO-LYSOSOMAL
SPECIFIC
REDUCTION
OF
MEMBRANE
FLUIDITY
UPON
KNOCKDOWN
OF
THE
GENES
OF
INTEREST
.69
4.
DISCUSSION
.
73
4.1.1
AGGREGATION
PROPENSITY
INCREASES
A-SYN
TRANSMISSION
IN
C.
ELEGANS
MORE
SO
THAN
EXPRESSION
LEVELS
74
4.1.2
ENDO-LYSOSOMAL
DAMAGE
MEDIATED
BY
A-SYN
IS
A
SHARED
PHENOTYPE
BETWEEN
DONOR
AND
RECIPIENT
CELLS
.
75
4.1.3
CHARACTERIZATION
OF
A
NOVEL
C.
ELEGANS
DOPAMINERGIC
NEURON
TO
HYPODERMIS
A-SYN
SPREADING
MODEL
.76
4.1.4
REDUCED
INSULIN
SIGNALING
PROTECTS
AGAINST
CELL
AUTONOMOUS
AND
NON-CELL
AUTONOMOUS
TOXICITY
OF
A-SYN
THROUGH
DAF-16/FOXO
ACTIVITY
.
78
4.1.5
RME-8/DNAJC13
AS
MODULATOR
OF
TRANS
CELLULAR
A-SYN
SPREADING
.
80
4.2.1
DEVELOPING
OF
A
C.
ELEGANS
TAU
TRANSMISSION
MODEL
.81
4.2.2
PHENOTYPE
OF
THE
NEW
TRANSGENIC
C.
ELEGANS
TAU
LINES
.
81
4.2.3
WHOLE
GENOME
RNAI
SCREEN
FOR
SUPPRESSORS
OF
ENDO-LYSOSOMAL
DAMAGE.
84
4.2.4
LIPID
MEMBRANE
RIGIDIFICATION
AS
COMMON
MECHANISM
FOR
ENDO-LYSOSOMAL
DESTABILIZATION
SHARED
BETWEEN
LIPID
METABOLISM
LINKED
HITS
.88
4.2.5
DIETARY
INTERVENTIONS
AS
PREVENTIVE
MEASURE
TO
LOWER
THE
RISK
OF
PRION-LIKE
NEURODEGENERATIVE
DISEASES
.
93
5.
FUTURE
PERSPECTIVES
.
95
6.
MATERIAL
AND
METHODS
.
97
X
6.1
MATERIALS
.
97
6.1.1
SOFTWARE
AND
TECHNICAL
EQUIPMENT
.
97
6.1.2
MICROSCOPES
.
98
6.1.3
EXPENDABLE
ITEMS
.99
6.1.4
CHEMICALS
AND
ENZYMES
.99
6.1.5
FREQUENTLY
USED
BUFFERS
AND
SOLUTIONS
.
101
6.1.6
OLIGONUCLEOTIDES
.103
6.1.7
ANTIBODIES
.
104
6.2
METHODS
.105
6.2.1
MOLECULAR
BIOLOGY
METHODS
.
105
6.2.1.1
POLYMERASE
CHAIN
REACTION
.
105
6.2.1.2
ENZYMATIC
RESTRICTION
DIGEST
.
106
6.2.1.3
AGAROSE
GEL
ELECTROPHORESIS
.106
6.2.1.4
GEL
EXTRACTION
OF
DNA
FRAGMENTS
.
106
6.2.1.5
CLONING
OF
C.
ELEGANS
EXPRESSION
PLASMIDS
.
106
6.2.1.6
TRANSFORMATION
OF
ONE
SHOT
TOP10
E.
COLL
.
109
6.2.1.7
PLASMID
PREPARATION
.
109
6.2.1.8
PREPARATION
OF
E.
COLL
GLYCEROL
STOCKS
.110
6.2.2
BIOCHEMICAL
METHODS
.
110
6.2.2.1
BRADFORD
ASSAY
.
110
6.2.2.2
IMMUNOPRECIPITATION
FROM
WORM
LYSATE
.
110
6.2.2.3
SDS-PAGE
.
ILL
6.2.2.4
SEMI-DRY
BLOTTING
AND
ANTIBODY
INCUBATION
.
ILL
6.2.3
C.
ELEGANS
METHODS
.
112
6.2.3.1
C.
ELEGANS
STRAINS
AND
MAINTENANCE
.112
6.2.3.2
PREPARATION
OF
STANDARD
OP50
AND
RNAI
NGM
PLATES
.
112
6.2.3.3
SUPPLEMENTATION
OF
NGM
WITH
FATTY
ACIDS
OR
GLUCOSE
.
113
6.2.3.4
PRELOADING
OF
OP50
E.
COLI
WITH
SATURATED
FATTY
ACIDS
.
114
6.2.3.5
FREEZING
C.
ELEGANS
STRAINS
FOR
LONG
TERM
STORAGE
.114
6.2.3.6
EGG
COLLECTION
FOR
SYNCHRONIZATION
BY
BLEACHING
.
114
6.2.3.7
MICROINJECTION
OF
C.
ELEGANS
.
114
6.2.3.8
GENERATION
OF
CHIMERIC
EXTRACHROMOSOMAL
OVEREXPRESSION
LINES.
115
6.2.3.9
INTEGRATION
OF
EXTRACHROMOSOMAL
ARRAYS
BY
UV
IRRADIATION
.116
6.2.3.10
GENERATION
OF
RME-8
N861S
ENDOGENOUS
MUTANT
STRAINS
.116
6.2.3.11
WORM
LYSIS
FOR
PCR
.
117
6.2.3.12
WORM
LYSIS
FOR
SDS-PAGE
.118
XI
6.2.3.13
MOTILITY/THRASHING
ASSAY
.
118
6.2.3.14
MOUNTING
OF
C.
ELEGANS
FOR
MICROSCOPY
.119
6.2.3.15
QUANTIFICATION
OF
HYPODERMAL
TRANSMISSION
OF
PRION-LIKE
PROTEINS
.
119
6.2.3.16
QUANTIFICATION
OF
NEURODEGENERATION
.
119
6.2.3.17
QUANTIFICATION
OF
LYSOSOMAL
RUPTURE
(IN
VIVO
GALECTIN
PUNCTA
ASSAY)
.120
6.2.3.18
WHOLE
GENOME
IN
VIVO
GALECTIN
PUNCTA
RNAI
SCREEN
.120
6.2.4
MICROSCOPY
AND
IMAGE
PROCESSING
.
122
6.2.4.1
CONFOCAL-FLUORESCENCE
MICROSCOPY
.
122
6.2.4.2
QUANTIFICATION
OF
HYPODERMAL
A-SYN::RFP
SIGNAL
.122
6.2.4.3
WIDEFIELD-FLUORESCENCE
MICROSCOPY
.
122
6.2.4.4
CO-LOCALIZATION
MICROSCOPY
.123
6.2.4.5
FRAP
MEASUREMENTS
.
123
6.2.5
STATISTICAL
ANALYSIS
.125
6.2.5.1
HUMAN
HOMOLOGUE
ANNOTATION
.125
6.2.5.2
GO
ENRICHMENT
ANALYSIS
.
125
7
REFERENCES
.
126
8
APPENDIX
.
145
8.1
.
LIST
OF
ABBREVIATIONS
.145
8.2
C.
ELEGANS
STRAINS
.148
8.3
DNA
SEQUENCES
ORDERED
TO
CONSTRUCT
TAU
TRANSGENIC
STRAINS
.152
8.3.1
ASSEMBLED
TAU
CONSTRUCT
EXPRESSION
VECTOR
.
153
8.4
HIT
GENES
OF
THE
WHOLE
GENOME
RNAI
SCREEN
FOR
ENDO-LYSOSOMAL
RUPTURE
.
154
8.5
LIST
OF
FIGURES
.160
8.6
LIST
OF
TABLES
.162
9
ACKNOWLEDGEMENTS
.
163
XII |
| adam_txt |
TABLE
OF
CONTENTS
CONTRIBUTIONS
.
VII
ZUSAMMENFASSUNG
.
XIII
ABSTRACT
.
XVII
1.
INTRODUCTION
.
1
1.1
PROTEINOPATHIES
.
1
1.2
A-SYNUCLEIN
AND
SYNUCLEINOPATHIES
.
2
1.2.1
A-SYN
DOMAIN
ORGANIZATION
AND
FUNCTION
.
3
1.2.2
DISEASE
RELATED
A-SYN
MUTATIONS
.
4
1.2.3
.
THE
ROLE
OF
A-SYN
IN
THE
PATHOGENESIS
OF
SYNUCLEINOPATHIES
.
5
1.3
TAU
AND
TAUOPATHIES
.
7
1.3.1
THE
MICROTUBULE
BINDING
PROTEIN
TAU
.
9
1.3.2
TAU
AGGREGATION
.
10
1.3.3
POST
TRANSLATIONAL
MODIFICATIONS
OF
TAU
.
11
1.3.4
.
DISEASE
RELATED
MAPT
MUTATIONS
.
12
1.3.5
.
TOXICITY
OF
TAU
AGGREGATION
.
13
1.4
.
THE
PRION
HYPOTHESIS
IN
NEURODEGENERATIVE
DISEASES
.
15
1.4.1
CELLULAR
PATHWAYS
THAT
MEDIATE
PRION-LIKE
SPREADING
OF
INTRACELLULAR
PROTEINS
.
19
1.5
.
CAENORHABDITIS
ELEGANS
AS
METAZOAN
MODEL
SYSTEM
.
23
1.5.1
LIFE
CYCLE
.24
1.5.2
C.
ELEGANS
AS
A
SCREENING
MODEL
.
25
2.
AIM
OF
THIS
THESIS
.
29
3.
RESULTS
.
31
3.1
A-SYN
MODEL
.
31
3.1.1
A-SYNUCLEIN::RFP
EXPRESSION
IN
BWM
CELLS
IS
CYTOTOXIC
AND
IS
TRANSMITTED
TO
THE
HYPODERMIS
IN
AN
AGE
DEPENDENT
MANNER
.
31
3.1.2
A-SYN::RFP
DAMAGES
THE
ENDO-LYSOSOMAL
SYSTEM
NOT
ONLY
IN
THE
RECIPIENT
HYPODERMIS
BUT
ALSO
THE
EXPRESSING
BWM
CELLS
.
33
3.1.3
A-SYN::RFP
EXPRESSION
IN
DOPAMINERGIC
NEURONS
LEADS
TO
AXONAL
DEGENERATION
.
35
3.1.4
DOPAMINERGIC
NEURON
A-SYN::RFP
IS
TRANSMITTED
TO
THE
HYPODERMIS
AND
INDUCES
LYSOSOMAL
RUPTURE
IN
THE
RECIPIENT
TISSUE
.36
3.1.5
REDUCING
INSULIN
SIGNALING
DELAYS
TRANSMISSION
AND
RESCUES
A-SYN
CELL
AUTONOMOUS
AND
NON-AUTONOMOUS
TOXICITY
IN
A
DAF-16
DEPENDENT
MANNER
.38
3.1.6
CHANGES
IN
RME-8/DNAJC13
FUNCTION
MODULATE
A-SYN::RFP
INTER-TISSUE
SPREADING
IN
C.
ELEGANS
.42
3.2
GENERATING
A
TAU
C.
ELEGANS
SPREADING
MODEL
TO
IDENTIFY
MODULATORS
OF
ENDO
LYSOSOMAL
ESCAPE
.
46
3.2.1
CLONING
AND
EXTRACHROMOSOMAL
EXPRESSION
OF
FLUORESCENTLY
TAGGED
TAU
FRAGMENTS
IN
THE
MECHANOSENSORY
NEURONS
.
46
3.2.2
EXPRESSION
OF
THE
F3
TAU
FRAGMENTS
IS
NEUROTOXIC
.
48
3.2.3
LIKE
A-SYN
THE
TAU
FRAGMENTS
ARE
READILY
TRANSMITTED
FROM
NEURONS
TO
THE
HYPODERMIS
.
50
3.2.4
TAU
FRAGMENT
TRANSMISSION
SENSITIZES
THE
ENDO-LYSOSOMAL
SYSTEM
IN
THE
RECIPIENT
HYPODERMIS
TO
RUPTURE
.
51
3.2.5
AN
UNBIASED
RNAI
SCREEN
FOR
SUPPRESSORS
OF
ENDO-LYSOSOMAL
RUPTURE
IDENTIFIES
KNOWN
AND
NOVEL
PATHWAYS
IMPLICATED
IN
ENDO-LYSOSOMAL
HEALTH
.54
3.2.6
A
FEW
GENES
SENSITIZE
THE
ENDO-LYSOSOMAL
SYSTEM
SPECIFICALLY
TO
F3AK280::MCH
MEDIATED
MEMBRANE
DISRUPTION
.58
3.2.7
FUNCTIONAL
SPHINGOLIPID
METABOLISM
IS
AN
ESSENTIAL
PATHWAY
TO
PROTECT
THE
ENDO-LYSOSOMAL
SYSTEM'S
INTEGRITY
.
60
3.2.8
A
DIET
HIGH
IN
MONO
(MUFA)
OR
POLYUNSATURATED
FATTY
ACIDS
(PUFA)
PROTECTS
THE
ENDO-LYSOSOMAL
MEMBRANE
FROM
RUPTURE
.
64
3.2.9
DIETS
HIGH
IN
SATURATED
FATTY
ACIDS
(SFA)
INDUCE
ENDO-LYSOSOMAL
MEMBRANE
DAMAGE
.67
3.2.10
FLUORESCENCE
RECOVERY
AFTER
PHOTOBLEACHING
(FRAP)
MEASUREMENTS
CONFIRM
AN
ENDO-LYSOSOMAL
SPECIFIC
REDUCTION
OF
MEMBRANE
FLUIDITY
UPON
KNOCKDOWN
OF
THE
GENES
OF
INTEREST
.69
4.
DISCUSSION
.
73
4.1.1
AGGREGATION
PROPENSITY
INCREASES
A-SYN
TRANSMISSION
IN
C.
ELEGANS
MORE
SO
THAN
EXPRESSION
LEVELS
74
4.1.2
ENDO-LYSOSOMAL
DAMAGE
MEDIATED
BY
A-SYN
IS
A
SHARED
PHENOTYPE
BETWEEN
DONOR
AND
RECIPIENT
CELLS
.
75
4.1.3
CHARACTERIZATION
OF
A
NOVEL
C.
ELEGANS
DOPAMINERGIC
NEURON
TO
HYPODERMIS
A-SYN
SPREADING
MODEL
.76
4.1.4
REDUCED
INSULIN
SIGNALING
PROTECTS
AGAINST
CELL
AUTONOMOUS
AND
NON-CELL
AUTONOMOUS
TOXICITY
OF
A-SYN
THROUGH
DAF-16/FOXO
ACTIVITY
.
78
4.1.5
RME-8/DNAJC13
AS
MODULATOR
OF
TRANS
CELLULAR
A-SYN
SPREADING
.
80
4.2.1
DEVELOPING
OF
A
C.
ELEGANS
TAU
TRANSMISSION
MODEL
.81
4.2.2
PHENOTYPE
OF
THE
NEW
TRANSGENIC
C.
ELEGANS
TAU
LINES
.
81
4.2.3
WHOLE
GENOME
RNAI
SCREEN
FOR
SUPPRESSORS
OF
ENDO-LYSOSOMAL
DAMAGE.
84
4.2.4
LIPID
MEMBRANE
RIGIDIFICATION
AS
COMMON
MECHANISM
FOR
ENDO-LYSOSOMAL
DESTABILIZATION
SHARED
BETWEEN
LIPID
METABOLISM
LINKED
HITS
.88
4.2.5
DIETARY
INTERVENTIONS
AS
PREVENTIVE
MEASURE
TO
LOWER
THE
RISK
OF
PRION-LIKE
NEURODEGENERATIVE
DISEASES
.
93
5.
FUTURE
PERSPECTIVES
.
95
6.
MATERIAL
AND
METHODS
.
97
X
6.1
MATERIALS
.
97
6.1.1
SOFTWARE
AND
TECHNICAL
EQUIPMENT
.
97
6.1.2
MICROSCOPES
.
98
6.1.3
EXPENDABLE
ITEMS
.99
6.1.4
CHEMICALS
AND
ENZYMES
.99
6.1.5
FREQUENTLY
USED
BUFFERS
AND
SOLUTIONS
.
101
6.1.6
OLIGONUCLEOTIDES
.103
6.1.7
ANTIBODIES
.
104
6.2
METHODS
.105
6.2.1
MOLECULAR
BIOLOGY
METHODS
.
105
6.2.1.1
POLYMERASE
CHAIN
REACTION
.
105
6.2.1.2
ENZYMATIC
RESTRICTION
DIGEST
.
106
6.2.1.3
AGAROSE
GEL
ELECTROPHORESIS
.106
6.2.1.4
GEL
EXTRACTION
OF
DNA
FRAGMENTS
.
106
6.2.1.5
CLONING
OF
C.
ELEGANS
EXPRESSION
PLASMIDS
.
106
6.2.1.6
TRANSFORMATION
OF
ONE
SHOT
TOP10
E.
COLL
.
109
6.2.1.7
PLASMID
PREPARATION
.
109
6.2.1.8
PREPARATION
OF
E.
COLL
GLYCEROL
STOCKS
.110
6.2.2
BIOCHEMICAL
METHODS
.
110
6.2.2.1
BRADFORD
ASSAY
.
110
6.2.2.2
IMMUNOPRECIPITATION
FROM
WORM
LYSATE
.
110
6.2.2.3
SDS-PAGE
.
ILL
6.2.2.4
SEMI-DRY
BLOTTING
AND
ANTIBODY
INCUBATION
.
ILL
6.2.3
C.
ELEGANS
METHODS
.
112
6.2.3.1
C.
ELEGANS
STRAINS
AND
MAINTENANCE
.112
6.2.3.2
PREPARATION
OF
STANDARD
OP50
AND
RNAI
NGM
PLATES
.
112
6.2.3.3
SUPPLEMENTATION
OF
NGM
WITH
FATTY
ACIDS
OR
GLUCOSE
.
113
6.2.3.4
PRELOADING
OF
OP50
E.
COLI
WITH
SATURATED
FATTY
ACIDS
.
114
6.2.3.5
FREEZING
C.
ELEGANS
STRAINS
FOR
LONG
TERM
STORAGE
.114
6.2.3.6
EGG
COLLECTION
FOR
SYNCHRONIZATION
BY
BLEACHING
.
114
6.2.3.7
MICROINJECTION
OF
C.
ELEGANS
.
114
6.2.3.8
GENERATION
OF
CHIMERIC
EXTRACHROMOSOMAL
OVEREXPRESSION
LINES.
115
6.2.3.9
INTEGRATION
OF
EXTRACHROMOSOMAL
ARRAYS
BY
UV
IRRADIATION
.116
6.2.3.10
GENERATION
OF
RME-8
N861S
ENDOGENOUS
MUTANT
STRAINS
.116
6.2.3.11
WORM
LYSIS
FOR
PCR
.
117
6.2.3.12
WORM
LYSIS
FOR
SDS-PAGE
.118
XI
6.2.3.13
MOTILITY/THRASHING
ASSAY
.
118
6.2.3.14
MOUNTING
OF
C.
ELEGANS
FOR
MICROSCOPY
.119
6.2.3.15
QUANTIFICATION
OF
HYPODERMAL
TRANSMISSION
OF
PRION-LIKE
PROTEINS
.
119
6.2.3.16
QUANTIFICATION
OF
NEURODEGENERATION
.
119
6.2.3.17
QUANTIFICATION
OF
LYSOSOMAL
RUPTURE
(IN
VIVO
GALECTIN
PUNCTA
ASSAY)
.120
6.2.3.18
WHOLE
GENOME
IN
VIVO
GALECTIN
PUNCTA
RNAI
SCREEN
.120
6.2.4
MICROSCOPY
AND
IMAGE
PROCESSING
.
122
6.2.4.1
CONFOCAL-FLUORESCENCE
MICROSCOPY
.
122
6.2.4.2
QUANTIFICATION
OF
HYPODERMAL
A-SYN::RFP
SIGNAL
.122
6.2.4.3
WIDEFIELD-FLUORESCENCE
MICROSCOPY
.
122
6.2.4.4
CO-LOCALIZATION
MICROSCOPY
.123
6.2.4.5
FRAP
MEASUREMENTS
.
123
6.2.5
STATISTICAL
ANALYSIS
.125
6.2.5.1
HUMAN
HOMOLOGUE
ANNOTATION
.125
6.2.5.2
GO
ENRICHMENT
ANALYSIS
.
125
7
REFERENCES
.
126
8
APPENDIX
.
145
8.1
.
LIST
OF
ABBREVIATIONS
.145
8.2
C.
ELEGANS
STRAINS
.148
8.3
DNA
SEQUENCES
ORDERED
TO
CONSTRUCT
TAU
TRANSGENIC
STRAINS
.152
8.3.1
ASSEMBLED
TAU
CONSTRUCT
EXPRESSION
VECTOR
.
153
8.4
HIT
GENES
OF
THE
WHOLE
GENOME
RNAI
SCREEN
FOR
ENDO-LYSOSOMAL
RUPTURE
.
154
8.5
LIST
OF
FIGURES
.160
8.6
LIST
OF
TABLES
.162
9
ACKNOWLEDGEMENTS
.
163
XII |
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| author | Sandhof, Carl Alexander 1990- |
| author_GND | (DE-588)1214844146 (DE-588)143209019 |
| author_facet | Sandhof, Carl Alexander 1990- |
| author_role | aut |
| author_sort | Sandhof, Carl Alexander 1990- |
| author_variant | c a s ca cas |
| building | Verbundindex |
| bvnumber | BV048899812 |
| ctrlnum | (OCoLC)1335520390 (DE-599)KXP1810098378 |
| format | Thesis Book |
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| genre_facet | Hochschulschrift |
| id | DE-604.BV048899812 |
| illustrated | Illustrated |
| index_date | 2024-07-03T21:50:37Z |
| indexdate | 2025-01-24T11:02:21Z |
| institution | BVB |
| institution_GND | (DE-588)2024349-2 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-034164242 |
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| owner_facet | DE-355 DE-BY-UBR |
| physical | XVIII, 164 Seiten Illustrationen, Diagramme 30 cm |
| publishDate | 2022 |
| publishDateSearch | 2022 |
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| spelling | Sandhof, Carl Alexander 1990- Verfasser (DE-588)1214844146 aut Investigating genetic modifiers of prion-like transmission and endo-lysosomal escape in the metazoan model system Caenorhabditis elegans presented by M. Sc. Carl Alexander Sandhof ; referees: Prof. Dr. Bernd Bukau, Prof. Dr. Aurelio Teleman Heidelberg [2022] XVIII, 164 Seiten Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Ruprecht-Karls-University Heidelberg 2022 Text Englisch, Zusammenfassung in englischer und deutscher Sprache (DE-588)4113937-9 Hochschulschrift gnd-content Bukau, Bernd 1954- (DE-588)143209019 dgs Universität Heidelberg (DE-588)2024349-2 dgg B:DE-101 application/pdf https://d-nb.info/1263888291/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034164242&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p emakn 0,05093 20220804 DE-101 https://d-nb.info/provenance/plan#emakn 2\p dnb 20220909 DE-101 https://d-nb.info/provenance/plan#dnb 3\p dnb 20220909 DE-101 https://d-nb.info/provenance/plan#dnb |
| spellingShingle | Sandhof, Carl Alexander 1990- Investigating genetic modifiers of prion-like transmission and endo-lysosomal escape in the metazoan model system Caenorhabditis elegans |
| subject_GND | (DE-588)4113937-9 |
| title | Investigating genetic modifiers of prion-like transmission and endo-lysosomal escape in the metazoan model system Caenorhabditis elegans |
| title_auth | Investigating genetic modifiers of prion-like transmission and endo-lysosomal escape in the metazoan model system Caenorhabditis elegans |
| title_exact_search | Investigating genetic modifiers of prion-like transmission and endo-lysosomal escape in the metazoan model system Caenorhabditis elegans |
| title_exact_search_txtP | Investigating genetic modifiers of prion-like transmission and endo-lysosomal escape in the metazoan model system Caenorhabditis elegans |
| title_full | Investigating genetic modifiers of prion-like transmission and endo-lysosomal escape in the metazoan model system Caenorhabditis elegans presented by M. Sc. Carl Alexander Sandhof ; referees: Prof. Dr. Bernd Bukau, Prof. Dr. Aurelio Teleman |
| title_fullStr | Investigating genetic modifiers of prion-like transmission and endo-lysosomal escape in the metazoan model system Caenorhabditis elegans presented by M. Sc. Carl Alexander Sandhof ; referees: Prof. Dr. Bernd Bukau, Prof. Dr. Aurelio Teleman |
| title_full_unstemmed | Investigating genetic modifiers of prion-like transmission and endo-lysosomal escape in the metazoan model system Caenorhabditis elegans presented by M. Sc. Carl Alexander Sandhof ; referees: Prof. Dr. Bernd Bukau, Prof. Dr. Aurelio Teleman |
| title_short | Investigating genetic modifiers of prion-like transmission and endo-lysosomal escape in the metazoan model system Caenorhabditis elegans |
| title_sort | investigating genetic modifiers of prion like transmission and endo lysosomal escape in the metazoan model system caenorhabditis elegans |
| topic_facet | Hochschulschrift |
| url | https://d-nb.info/1263888291/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034164242&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT sandhofcarlalexander investigatinggeneticmodifiersofprionliketransmissionandendolysosomalescapeinthemetazoanmodelsystemcaenorhabditiselegans AT bukaubernd investigatinggeneticmodifiersofprionliketransmissionandendolysosomalescapeinthemetazoanmodelsystemcaenorhabditiselegans AT universitatheidelberg investigatinggeneticmodifiersofprionliketransmissionandendolysosomalescapeinthemetazoanmodelsystemcaenorhabditiselegans |
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