Modulation of amyloid precursor protein trans-directed dimerization:
Saved in:
| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
Kaiserslautern
[2021]
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis Inhaltsverzeichnis |
| Physical Description: | VII, 130 Seiten Illustrationen, Diagramme 21 cm |
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MARC
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| 100 | 1 | |a August, Alexander |d 1989- |e Verfasser |0 (DE-588)1203570899 |4 aut | |
| 245 | 1 | 0 | |a Modulation of amyloid precursor protein trans-directed dimerization |c von Alexander August, M.Sc. |
| 264 | 1 | |a Kaiserslautern |c [2021] | |
| 300 | |a VII, 130 Seiten |b Illustrationen, Diagramme |c 21 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Technische Universität Kaiserslautern |d 2021 | ||
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Record in the Search Index
| _version_ | 1804184413236363264 |
|---|---|
| adam_text | TABLE
OF
CONTENTS
JV
TABLE
OF
CONTENTS
1
INTRODUCTION
1
1.1
ALZHEIMER S
DISEASE
1
1.2
THE
APP
GENE
FAMILY
3
1.2.1
STRUCTURE
AND
PROCESSING
3
1.2.2
PHYSIOLOGICAL
FUNCTIONS
OF
THE
APP
GENE
FAMILY
IN
CNS
AND
PNS
9
1.2.3
DIMERIZATION
AND
METAL
ION
INTERACTION
12
1.3
ASTROCYTE-MEDIATED
SYNAPTOGENESIS
14
1.4
THE
FUNCTION
OF
THROMBOSPONDINS
IN
CNS
AND
PNS
15
2
AIM
OF
THE
STUDY
17
3
RESULTS
18
3.1
ASTROCYTE-DERIVED
THROMBOSPONDIN-1
ACCELERATES
TRANS-DIRECTED
DIMERIZATION
OF
APP
....
18
3.1.1
THE
FACTOR
OF
INTEREST
MEDIATING
APP
DIMERIZATION
IS
AN
ASTROCYTE-SECRETED
PROTEIN
18
3.1.2
HEPARAN
SULFATE
PROTEOGLYCANS
DO
NOT
MODULATE
APP
DIMERIZATION
IN
A
BEAD
AGGREGATION
ASSAY
20
3.1.3
ESTABLISHMENT
OF
AN
ELUTION
STRATEGY
TO
ENRICH
THE
FACTOR
OF
INTEREST
22
3.1.4
IDENTIFICATION
OF
APP-INTERACTING
PROTEINS
DERIVED
FROM
CONDITIONED
MEDIUM
OF
ASTROCYTES
24
3.1.5
APP
FORMS
DIMERS
IN
CONDITIONED
MEDIUM
OF
HEK293
CELLS
THATSECRETE
TSP-1
26
3.1.6
TSP-1
IS
THE
ASTROCYTE-SECRETED
PROTEIN
IN
ACM
THAT
DRIVES
APP
DIMERIZATION
29
3.1.7
ASTROCYTE-DERIVED
TSP-1
INTERACTS
WITH
THE
ECTODOMAIN
OF
APP
AND
INDUCES
DIMERIZATION
OF
ITS
SUBDOMAINS
EL
AND
E2
WHEN
OVEREXPRESSED
32
3.1.8
TSP-1
HAS
NO
IMPACT
ON
APP
DIMERIZATION
IN
A
TRANS
CO-IMMUNOPRECIPITATION
36
3.1.9
ANALYSIS
OF
TSP-L-MEDIATED
PRESYNAPTIC
DIFFERENTIATION
IN
DEPENDENCE
ON
APP
...37
3.2
NEGATIVELY
CHARGED
CYCLIC
MOLECULES
MODULATE
APP
TRANS
DIMERIZATION
40
3.2.1
CYCLIC
PEPTIDES
CARRYING
A
NEGATIVELY
CHARGED
PHOSPHONATE
GROUP
INDUCE
DIMERIZATION
OF
APP
40
TABLE
OF
CONTENTS
V
3.2.2
THE
CYCLIC
PEPTIDE
SK-28
DOES
NOT
MODULATE
TRANS-ORIENTED
DIMERIZATION
OF
APP
OVEREXPRESSED
IN
HEK293
CELLS
42
3.2.3
CYCLIZATION
OF
SK-28
IS
A
PREREQUISITE
TO
PROMOTE
APP
DIMERIZATION
43
3.2.4
APP
DIMERIZATION
IS
REGULATED
BY
MOLECULES
THAT
CONSIST
OF
NEGATIVELY
CHARGED
GROUPS
BOUND
TO
A
CYCLIC
BACKBONE
45
3.2.5
HEPARIN
INHIBITS
SC.4
MEDIATED
BUT
NOT
SK-28
MEDIATED
APP
DIMERIZATION
47
3.3
ZINC
IONS
BIND
TO
THE
APP
EL
DOMAIN,
THEREBY
INHIBITING
COPPER
BINDING
49
4
DISCUSSION
52
4.1
IDENTIFICATION
OF
THROMBOSPONDIN-1
AS
AN
ASTROCYTE-SECRETED
FACTOR
THAT
PROMOTES
APP
DIMERIZATION
52
4.1.1
ASTROCYTES
SECRETE
A
PROTEINACEOUS
FACTOR
OTHER
THAN
AN
HSPG
THAT
FACILITATES
TRANS
DIRECTED
DIMERIZATION
OF
APP
52
4.1.2
IDENTIFICATION
OF
ASTROCYTE-DERIVED
APP-INTERACTING
PROTEINS
54
4.1.3
THROMBOSPONDIN-1
IS
THE
ASTROCYTE-SECRETED
FACTOR
THAT
PROMOTES
TRANS-DIRECTED
DIMERIZATION
OF
APP
55
4.1.4
TSP-1
BINDS
TO
THE
EL
AND
E2
DOMAIN
OF
APP
AND
FACILITATES
THEIR
DIMERIZATION
....57
4.1.5
THE
PHYSIOLOGICAL
FUNCTION
OF
AN
INTERACTION
BETWEEN
TSP-1
AND
APP
58
4.2
CYCLIC
MOLECULES
CARRYING
NEGATIVELY
CHARGED
GROUPS
MODULATE
APP
DIMERIZATION
IN-VITRO
59
4.3
ZINC
IONS
BIND
WITH
A
NANOMOLAR
AFFLNITY
TO
THE
E
1
DOMAIN
AND
MODULATE
THE
DIMERIZATION
OFAPP
61
4.4
OUTLOOK
62
5
MATERIALS
64
5.1
LISTOFMATERIALS
USED
64
5.2
RECIPES
FOR
BUFFERS
AND
SOLUTIONS
69
5.3
CELL
LINES,
BACTERIAL
STRAINS,
YEAST
STRAINS
AND
MOUSE
STRAINS
72
5.4
CULTIVATION
MEDIA
73
5.5
ANTIBODIES
75
5.6
PLASMIDS
75
5.7
OLIGONUCLEOTIDES
AND
SIRNAS
76
6
METHODS
78
TABLE
OF
CONTENTS
____
VI
6.1
MOLECULAR
BIOLOGY
78
6.1.1
RESTRICTION
DIGESTION
OF
DNA
78
6.1.2
AGAROSE
GEL
ELECTROPHORESIS
78
6.1.3
PCR
PURIFICATION
78
6.1.4
GEL
EXTRACTION
78
6.1.5
DNALIGATION
78
6.1.6
RETRANSFORMATION
79
6.1.7
TRANSFORMATION
OF
LIGATED
DNA
79
6.1.8
SITE
DIRECTED
MUTAGENESIS
79
6.1.9
SMALL
SCALE
(MINIPREP)
DNA
PREPARATION
79
6.1.10
LARGE
SCALE
(MAXIPREP)
DNA
PREPARATION
80
6.1.11
DNA
SEQUENCING
80
6.2
PROTEINBIOCHEMISTRY
80
6.2.1
EXPRESSION
OF
APP
EL
IN
PICHIA
PASTORIS
80
6.2.2
PURIFICATION
OF
APP
EL
81
6.2.3
EXPRESSION
AND
PURIFICATION
OF
APPEX-FC
81
6.2.4
EXPRESSION
AND
PURIFICATION
OF
APLP2EX-FC
82
6.2.5
SDS-POLYACRYLAMIDE
GEL
ELECTROPHORESIS
(SDS-PAGE)
.....
........................................
82
6.2.6
COOMASSIE
STAINING
83
6.2.7
COLLOIDAL
COOMASSIE
STAINING
83
6.2.8
SILVER
STAINING
83
6.2.9
WESTERN
BLOT
83
6.2.10
MASS
SPECTROMETRY
AND
DATA
ANALYSIS
84
6.2.11
ISOTHERMAL
TITRATION
CALORIMETRY
(ITC)
85
6.2.12
BEAD
AGGREGATION
ASSAY
WITH
PURIFIED
APPEX-FC
OR
APLP2EX-FC
85
6.2.13
BEAD
AGGREGATION
ASSAY
WITH
TRANSIENTLY
EXPRESSED
APPEX-FC
86
6.2.14
PULLDOWN
ASSAY
WITH
COATED
PARAMAGNETIC
PROTEIN
A
BEADS
86
6.2.15
CIS
AND
TRANS
CO-IMMUNOPRECIPITATION
(CO-IP)
87
6.2.16
IMMUNOCYTOCHEMISTRY
87
TABLE
OF
CONTENTS
VII
6.2.17
QUANTIFICATION
OF
SYNAPSES
IN
NEURONAL
CULTURES
88
6.2.18
STATISTICAL
ANALYSIS
88
6.3
CELL
BIOLOGY
88
6.3.1
CULTIVATION
OF
CELL
LINES
88
6.3.2
PREPARATION
AND
CULTIVATION
OF
ASTROCYTES
89
6.3.3
PREPARATION
AND
CULTIVATION
OF
PRIMARY
CORTICAL
NEURONS
89
6.3.4
FREEZING
OF
CELLS
90
6.3.5
THAWING
OF
CELLS
90
6.3.6
DNA
OR
SIRNA
TRANSFECTION
USING
JETPRIME
90
6.3.7
DNA
TRANSFECTION
USING
PEI
90
7
REFERENCES
91
8
APPENDIX
116
LIST
OF
ABBREVIATIONS
131
LIST
OF
FIGURES
134
LISTOFTABLES
136
DANKSAGUNG
137
LEBENSLAUF
138
ERKLAERUNG
ZUR
BETEILIGUNG
DRITTER
140
EIDESSTATTLICHE
ERKLAERUNG
141
|
| adam_txt |
TABLE
OF
CONTENTS
JV
TABLE
OF
CONTENTS
1
INTRODUCTION
1
1.1
ALZHEIMER'S
DISEASE
1
1.2
THE
APP
GENE
FAMILY
3
1.2.1
STRUCTURE
AND
PROCESSING
3
1.2.2
PHYSIOLOGICAL
FUNCTIONS
OF
THE
APP
GENE
FAMILY
IN
CNS
AND
PNS
9
1.2.3
DIMERIZATION
AND
METAL
ION
INTERACTION
12
1.3
ASTROCYTE-MEDIATED
SYNAPTOGENESIS
14
1.4
THE
FUNCTION
OF
THROMBOSPONDINS
IN
CNS
AND
PNS
15
2
AIM
OF
THE
STUDY
17
3
RESULTS
18
3.1
ASTROCYTE-DERIVED
THROMBOSPONDIN-1
ACCELERATES
TRANS-DIRECTED
DIMERIZATION
OF
APP
.
18
3.1.1
THE
FACTOR
OF
INTEREST
MEDIATING
APP
DIMERIZATION
IS
AN
ASTROCYTE-SECRETED
PROTEIN
18
3.1.2
HEPARAN
SULFATE
PROTEOGLYCANS
DO
NOT
MODULATE
APP
DIMERIZATION
IN
A
BEAD
AGGREGATION
ASSAY
20
3.1.3
ESTABLISHMENT
OF
AN
ELUTION
STRATEGY
TO
ENRICH
THE
FACTOR
OF
INTEREST
22
3.1.4
IDENTIFICATION
OF
APP-INTERACTING
PROTEINS
DERIVED
FROM
CONDITIONED
MEDIUM
OF
ASTROCYTES
24
3.1.5
APP
FORMS
DIMERS
IN
CONDITIONED
MEDIUM
OF
HEK293
CELLS
THATSECRETE
TSP-1
26
3.1.6
TSP-1
IS
THE
ASTROCYTE-SECRETED
PROTEIN
IN
ACM
THAT
DRIVES
APP
DIMERIZATION
29
3.1.7
ASTROCYTE-DERIVED
TSP-1
INTERACTS
WITH
THE
ECTODOMAIN
OF
APP
AND
INDUCES
DIMERIZATION
OF
ITS
SUBDOMAINS
EL
AND
E2
WHEN
OVEREXPRESSED
32
3.1.8
TSP-1
HAS
NO
IMPACT
ON
APP
DIMERIZATION
IN
A
TRANS
CO-IMMUNOPRECIPITATION
36
3.1.9
ANALYSIS
OF
TSP-L-MEDIATED
PRESYNAPTIC
DIFFERENTIATION
IN
DEPENDENCE
ON
APP
.37
3.2
NEGATIVELY
CHARGED
CYCLIC
MOLECULES
MODULATE
APP
TRANS
DIMERIZATION
40
3.2.1
CYCLIC
PEPTIDES
CARRYING
A
NEGATIVELY
CHARGED
PHOSPHONATE
GROUP
INDUCE
DIMERIZATION
OF
APP
40
TABLE
OF
CONTENTS
V
3.2.2
THE
CYCLIC
PEPTIDE
SK-28
DOES
NOT
MODULATE
TRANS-ORIENTED
DIMERIZATION
OF
APP
OVEREXPRESSED
IN
HEK293
CELLS
42
3.2.3
CYCLIZATION
OF
SK-28
IS
A
PREREQUISITE
TO
PROMOTE
APP
DIMERIZATION
43
3.2.4
APP
DIMERIZATION
IS
REGULATED
BY
MOLECULES
THAT
CONSIST
OF
NEGATIVELY
CHARGED
GROUPS
BOUND
TO
A
CYCLIC
BACKBONE
45
3.2.5
HEPARIN
INHIBITS
SC.4
MEDIATED
BUT
NOT
SK-28
MEDIATED
APP
DIMERIZATION
47
3.3
ZINC
IONS
BIND
TO
THE
APP
EL
DOMAIN,
THEREBY
INHIBITING
COPPER
BINDING
49
4
DISCUSSION
52
4.1
IDENTIFICATION
OF
THROMBOSPONDIN-1
AS
AN
ASTROCYTE-SECRETED
FACTOR
THAT
PROMOTES
APP
DIMERIZATION
52
4.1.1
ASTROCYTES
SECRETE
A
PROTEINACEOUS
FACTOR
OTHER
THAN
AN
HSPG
THAT
FACILITATES
TRANS
DIRECTED
DIMERIZATION
OF
APP
52
4.1.2
IDENTIFICATION
OF
ASTROCYTE-DERIVED
APP-INTERACTING
PROTEINS
54
4.1.3
THROMBOSPONDIN-1
IS
THE
ASTROCYTE-SECRETED
FACTOR
THAT
PROMOTES
TRANS-DIRECTED
DIMERIZATION
OF
APP
55
4.1.4
TSP-1
BINDS
TO
THE
EL
AND
E2
DOMAIN
OF
APP
AND
FACILITATES
THEIR
DIMERIZATION
.57
4.1.5
THE
PHYSIOLOGICAL
FUNCTION
OF
AN
INTERACTION
BETWEEN
TSP-1
AND
APP
58
4.2
CYCLIC
MOLECULES
CARRYING
NEGATIVELY
CHARGED
GROUPS
MODULATE
APP
DIMERIZATION
IN-VITRO
59
4.3
ZINC
IONS
BIND
WITH
A
NANOMOLAR
AFFLNITY
TO
THE
E
1
DOMAIN
AND
MODULATE
THE
DIMERIZATION
OFAPP
61
4.4
OUTLOOK
62
5
MATERIALS
64
5.1
LISTOFMATERIALS
USED
64
5.2
RECIPES
FOR
BUFFERS
AND
SOLUTIONS
69
5.3
CELL
LINES,
BACTERIAL
STRAINS,
YEAST
STRAINS
AND
MOUSE
STRAINS
72
5.4
CULTIVATION
MEDIA
73
5.5
ANTIBODIES
75
5.6
PLASMIDS
75
5.7
OLIGONUCLEOTIDES
AND
SIRNAS
76
6
METHODS
78
TABLE
OF
CONTENTS
_
VI
6.1
MOLECULAR
BIOLOGY
78
6.1.1
RESTRICTION
DIGESTION
OF
DNA
78
6.1.2
AGAROSE
GEL
ELECTROPHORESIS
78
6.1.3
PCR
PURIFICATION
78
6.1.4
GEL
EXTRACTION
78
6.1.5
DNALIGATION
78
6.1.6
RETRANSFORMATION
79
6.1.7
TRANSFORMATION
OF
LIGATED
DNA
79
6.1.8
SITE
DIRECTED
MUTAGENESIS
79
6.1.9
SMALL
SCALE
(MINIPREP)
DNA
PREPARATION
79
6.1.10
LARGE
SCALE
(MAXIPREP)
DNA
PREPARATION
80
6.1.11
DNA
SEQUENCING
80
6.2
PROTEINBIOCHEMISTRY
80
6.2.1
EXPRESSION
OF
APP
EL
IN
PICHIA
PASTORIS
80
6.2.2
PURIFICATION
OF
APP
EL
81
6.2.3
EXPRESSION
AND
PURIFICATION
OF
APPEX-FC
81
6.2.4
EXPRESSION
AND
PURIFICATION
OF
APLP2EX-FC
82
6.2.5
SDS-POLYACRYLAMIDE
GEL
ELECTROPHORESIS
(SDS-PAGE)
.
.
82
6.2.6
COOMASSIE
STAINING
83
6.2.7
COLLOIDAL
COOMASSIE
STAINING
83
6.2.8
SILVER
STAINING
83
6.2.9
WESTERN
BLOT
83
6.2.10
MASS
SPECTROMETRY
AND
DATA
ANALYSIS
84
6.2.11
ISOTHERMAL
TITRATION
CALORIMETRY
(ITC)
85
6.2.12
BEAD
AGGREGATION
ASSAY
WITH
PURIFIED
APPEX-FC
OR
APLP2EX-FC
85
6.2.13
BEAD
AGGREGATION
ASSAY
WITH
TRANSIENTLY
EXPRESSED
APPEX-FC
86
6.2.14
PULLDOWN
ASSAY
WITH
COATED
PARAMAGNETIC
PROTEIN
A
BEADS
86
6.2.15
CIS
AND
TRANS
CO-IMMUNOPRECIPITATION
(CO-IP)
87
6.2.16
IMMUNOCYTOCHEMISTRY
87
TABLE
OF
CONTENTS
VII
6.2.17
QUANTIFICATION
OF
SYNAPSES
IN
NEURONAL
CULTURES
88
6.2.18
STATISTICAL
ANALYSIS
88
6.3
CELL
BIOLOGY
88
6.3.1
CULTIVATION
OF
CELL
LINES
88
6.3.2
PREPARATION
AND
CULTIVATION
OF
ASTROCYTES
89
6.3.3
PREPARATION
AND
CULTIVATION
OF
PRIMARY
CORTICAL
NEURONS
89
6.3.4
FREEZING
OF
CELLS
90
6.3.5
THAWING
OF
CELLS
90
6.3.6
DNA
OR
SIRNA
TRANSFECTION
USING
JETPRIME
90
6.3.7
DNA
TRANSFECTION
USING
PEI
90
7
REFERENCES
91
8
APPENDIX
116
LIST
OF
ABBREVIATIONS
131
LIST
OF
FIGURES
134
LISTOFTABLES
136
DANKSAGUNG
137
LEBENSLAUF
138
ERKLAERUNG
ZUR
BETEILIGUNG
DRITTER
140
EIDESSTATTLICHE
ERKLAERUNG
141 |
| any_adam_object | 1 |
| any_adam_object_boolean | 1 |
| author | August, Alexander 1989- |
| author_GND | (DE-588)1203570899 |
| author_facet | August, Alexander 1989- |
| author_role | aut |
| author_sort | August, Alexander 1989- |
| author_variant | a a aa |
| building | Verbundindex |
| bvnumber | BV048467160 |
| ctrlnum | (OCoLC)1313915668 (DE-599)DNB1254623787 |
| discipline | Biologie |
| discipline_str_mv | Biologie |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV048467160 |
| illustrated | Illustrated |
| index_date | 2024-07-03T20:35:41Z |
| indexdate | 2024-07-10T09:38:56Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-033845039 |
| oclc_num | 1313915668 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR DE-29T |
| owner_facet | DE-355 DE-BY-UBR DE-29T |
| physical | VII, 130 Seiten Illustrationen, Diagramme 21 cm |
| publishDate | 2021 |
| publishDateSearch | 2021 |
| publishDateSort | 2021 |
| record_format | marc |
| spelling | August, Alexander 1989- Verfasser (DE-588)1203570899 aut Modulation of amyloid precursor protein trans-directed dimerization von Alexander August, M.Sc. Kaiserslautern [2021] VII, 130 Seiten Illustrationen, Diagramme 21 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Technische Universität Kaiserslautern 2021 4\p Elution (DE-588)4274495-7 gnd (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf https://d-nb.info/1254623787/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=033845039&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p emakn 0,28713 20220407 DE-101 https://d-nb.info/provenance/plan#emakn 2\p dnb 20220419 DE-101 https://d-nb.info/provenance/plan#dnb 3\p dnb 20220419 DE-101 https://d-nb.info/provenance/plan#dnb 4\p emagnd 0,08510 20220407 DE-101 https://d-nb.info/provenance/plan#emagnd |
| spellingShingle | August, Alexander 1989- Modulation of amyloid precursor protein trans-directed dimerization 4\p Elution (DE-588)4274495-7 gnd |
| subject_GND | (DE-588)4274495-7 (DE-588)4113937-9 |
| title | Modulation of amyloid precursor protein trans-directed dimerization |
| title_auth | Modulation of amyloid precursor protein trans-directed dimerization |
| title_exact_search | Modulation of amyloid precursor protein trans-directed dimerization |
| title_exact_search_txtP | Modulation of amyloid precursor protein trans-directed dimerization |
| title_full | Modulation of amyloid precursor protein trans-directed dimerization von Alexander August, M.Sc. |
| title_fullStr | Modulation of amyloid precursor protein trans-directed dimerization von Alexander August, M.Sc. |
| title_full_unstemmed | Modulation of amyloid precursor protein trans-directed dimerization von Alexander August, M.Sc. |
| title_short | Modulation of amyloid precursor protein trans-directed dimerization |
| title_sort | modulation of amyloid precursor protein trans directed dimerization |
| topic | 4\p Elution (DE-588)4274495-7 gnd |
| topic_facet | Elution Hochschulschrift |
| url | https://d-nb.info/1254623787/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=033845039&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT augustalexander modulationofamyloidprecursorproteintransdirecteddimerization |
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