The role of tetraspanin Tspan8 in regulating the composition of extracellular vesicles:
Gespeichert in:
| Beteilige Person: | |
|---|---|
| Format: | Hochschulschrift/Dissertation Buch |
| Sprache: | Englisch |
| Veröffentlicht: |
Freiburg im Breisgau
April 2022
|
| Schlagwörter: | |
| Links: | https://d-nb.info/1279325674/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034328276&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| Umfang: | 200 Seiten Illustrationen, Diagramme 30 cm |
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| 245 | 1 | 0 | |a The role of tetraspanin Tspan8 in regulating the composition of extracellular vesicles |c presented by Elena Grueso Navarro |
| 264 | 1 | |a Freiburg im Breisgau |c April 2022 | |
| 300 | |a 200 Seiten |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
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| 502 | |b Dissertation |c Albert-Ludwigs-University Freiburg, Germany |d 2022 | ||
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Datensatz im Suchindex
| _version_ | 1819275972983128064 |
|---|---|
| adam_text | INDEX
ABSTRACT
..........................................................................................................................................................................
7
ZUSAMMENFASSUNG
.....................................................................................................................................................
9
INDEX
..............................................................................................................................................................................
11
ABBREVIATIONS
.............................................................................................................................................................
17
INTRODUCTION................................................................................................................................................................
5
1.
EXTRACELLULAR
VESICLES:
YOU
HAVE
A
MESSAGE
...............................................................................................
7
1.1
T
HE
ORIGINS
OF
EVS
...................................................................................................................................................
7
1.2
D
IFFERENT
TYPES
OF
EVS
.............................................................................................................................................
8
1.3
T
HE
CARGO
OF
EVS
..................................................................................................................................................
13
1.3.1
THE
PROTEIN
CARGO
OF
EVS
............................................................................................................................14
1.3.2
OVERVIEW
OF
THE
RNA
CONTENT
OF
EVS
.........................................................................................................
15
1.3.3
DNA
MOLECULES
IN
EVS
.................................................................................................................................
16
1.3.4
LIPIDS
AS
CARGO
OF
EVS.................................................................................................................................
17
1.4
T
HE
ROLE
OF
EVS
IN
CANCER
.....................................................................................................................................
18
2
THE
TETRASPANINS
AS
MOLECULAR
FACILITATORS
.............................................................................................
22
2.1
D
EFINITION
OF
TETRASPANIN
.....................................................................................................................................
22
2.2
THE
STRUCTURE
OF
TETRASPANINS
.............................................................................................................................
23
2.3
T
HE
RELEVANT
ROLE
OF
TETRASPANINS
IN
PHYSIOLOGY
AND
PATHOLOGY
........................................................................
26
2.4
T
ETRASPANINS
IN
CANCER
.........................................................................................................................................
28
3.
TETRASPANIN
TSPAN8
..............................................................................................................................................
31
3.1
TSPAN8
IN
EVS
........................................................................................................................................................36
OBJECTIVES
.....................................................................................................................................................................39
MATERIALS
&
METHODS
..............................................................................................................................................
43
1
.
MATERIALS
.............................................................................................................................................................45
1.1
I
NSTRUMENTS
................................................................................................................................................45
1.2
K
ITS
..............................................................................................................................................................
46
1.3
G
ENERAL
REAGENTS
................................................................................................................................................47
1.3.1
CHEMICAL
REAGENTS
......................................................................................................................................
47
1.3.2
BUFFERS
AND
SOLUTIONS
..................................................................................................................................
48
1.4
C
ELL
LINES
..............................................................................................................................................................
49
1.4.1
MEDIA
AND
SUPPLEMENTS
................................................................................................................
50
1.5
P
RIMERS
................................................................................................................................................................
50
1.6
A
NTIBODIES
............................................................................................................................................................50
1.7
C
ONSUMABLES
........................................................................................................................................................
51
2
.
METHODS
..............................................................................................................................................................
52
2.1
C
ULTURE
OF
MAMMALIAN
CELLS
...............................................................................................................................
52
2.1.1
CULTURE
AND
STORAGE
OF
CELLS
.......................................................................................................................
52
2.1.2
MYCOPLASMA
TEST
........................................................................................................................................
52
2.1.3
CULTURE
OF
CELLS
IN
3D
...................................................................................................................................
53
2.2
E
VALUATION
OF
CELL
VIABILITY
.................................................................................................................................
54
2.2.1
TRYPAN
BLUE
.................................................................................................................................................
54
2.2.2
ANNEXIN
V/PI
FLOW
CYTOMETRY
ASSAY
.........................................................................................................
54
2.3
C
HARACTERIZATION
OF
CELL
-
SURFACE
MARKERS
WITH
FLOW
CYTOMETRY
........................................................................
55
2.4
C
ELL
PROLIFERATION
ASSAYS
......................................................................................................................................
55
2.4.1
CELL
CYCLE
ANALYSIS
........................................................................................................................................
55
2.4.2
WST-1
ASSAY
.................................................................................................................................................
55
2.5
EV
ISOLATION
FROM
CELL
CULTURE
SUPERNATANT
........................................................................................................
56
2.5.1
PRODUCTION
OF
EVS
IN
VITRO:
2D
CELL
CULTURE
................................................................................................
56
2.5.2
PRODUCTION
OF
EVS
IN
VITRO:
3D
CELL
CULTURE
................................................................................................
56
2.5.3
EV
ISOLATION
FROM
SERIAL
ULTRACENTRIFUGATION
(UC)
.....................................................................................
56
2.5.4
ISOLATION
OF
EVS
USING
SIZE
EXCLUSION
CHROMATOGRAPHY
(SEC)
.................................................................
57
2.5.5
ISOLATION
OF
EVS
USING
SUCROSE
DENSITY
GRADIENT
......................................................................................
58
2.6
C
HARACTERIZATION
OF
EVS
......................................................................................................................................
59
2.6.1
NANOPARTICLE
TRACKING
ANALYSIS
(NTA)
.......................................................................................................
59
2.6.2
TRANSMISSION
ELECTRON
MICROSCOPY
(TEM)
.................................................................................................
59
2.6.3
DETECTION
OFGFP
+
EVS
BY
NANO-FLOW
CYTOMETRY
(NANOFCM)
..................................................................
59
2.6.4
CHARACTERIZATION
OF
TETRASPANIN
DISTRIBUTION
AND
CO-LOCALIZATION
IN
EVS:
SP-IRIS
TECHNOLOGY
(NANOVIEW
TECHNOLOGY)
........................................................................................................................................
60
2.7
F
ULLY
QUANTITATIVE
ANALYSIS
OF
EVS
..............................................................................................................
60
2.7.1
LABELLING
OF
EVS
...........................................................................................................................................
60
2.7.2
QUANTITATIVE
ANALYSIS
WITH
HIGH-RESOLUTION
FLOW
CYTOMETRY
...................................................................
60
2.8
P
ROTEIN
ANALYSIS
.....................................................................................................................................................
61
2.8.1
PREPARATION
OF
PROTEIN
EXTRACTS
..................................................................................................................
61
2.8.2
PROTEOMICS
....................................................................................................................................................
61
2.8.3
WESTERN
BLOT
.................................................................................................................................................63
2.8.4
DOT
BLOT
........................................................................................................................................................63
2.9
RNA
ANALYSIS
................................................................................................................................................64
2.9.1
RNA
ISOLATION
................................................................................................................................................64
2.9.2
RNA
PROFILING
AND
FRAGMENT
ANALYSIS
........................................................................................................
65
2.9.3
TRANSCRIPTOMICS
...........................................................................................................................................65
2.9.4
QUANTITATIVE
REAL
TIME-PCR
(QRT-PCR)
ANALYSIS
......................................................................................
67
2.10
DNA
ANALYSIS
.......................................................................................................................................................
67
2.10.1
ISOLATION
OF
DNA
........................................................................................................................................
67
2.10.2
DNA
SIZING
ANALYSIS
....................................................................................................................................
68
2.11
CO-IMMUNOPRECIPITATION
.....................................................................................................................................
68
2.12
I
MMUNOFLUORESCENCE
AND
SUPER
-
RESOLUTION
MICROSCOPY
(SRM)
.......................................................................
69
2.13
S
TATISTICAL
ANALYSIS
.............................................................................................................................................
69
CONTRIBUTIONS
............................................................................................................................................................
71
RESULTS
...........................................................................................................................................................................
73
1.
A
FIBROSARCOMA
CELL
MODEL
TO
STUDY
THE
IMPACT
OF
TETRASPANINS
IN
CELL-DERIVED
EVS
............
75
1.1
C
HARACTERIZATION
OF
THE
EXOGENOUS
EXPRESSION
OF
T
SPAN
8
AND
CD9
TETRASPANINS
..............................................76
1.2
A
LOOK
INTO
THE
POPULATION
OF
EVS
RELEASED
BY
TETRASPANIN-TRANSFECTED
HT1080
DERIVATES
..............................78
1.2.1
TSPAN8
AND
CD9
TETRASPANINS
ARE
MOSTLY
RECRUITED
TO
THE
SMALL
EVS
.....................................................
78
1.2.2
ESTABLISHMENT
OF
A
PROTOCOL
FOR
ISOLATION
OF
SMALL
EVS............................................................................
80
1.2.3
TSPAN8,
AND
NOT
CD9,
ENHANCES
THE
PRODUCTION
OF
EVS
IN
HT1080
CELLS
IN
VITRO
....................................
86
1.2.4
EXPLORING
TETRASPANIN
DISTRIBUTION
WITHIN
EVS
FROM
HT1080
CELL
DERIVATES
...........................................
90
1.3
C
HARACTERIZATION
OF
THE
T
SPAN
8
EXPRESSION
IN
A
GFP
TAGGED
CELL
SYSTEM
...........................................................
92
1.3.1
EVALUATION
OF
THE
TSPAN8GFP
+
POPULATION
OF
EVS
....................................................................................
95
2.
HOW
DO
TETRASPANINS
IMPACT
EV
CARGO?
A
MULTI-OMIC
APPROACH
......................................................
98
2.1
C
OMPREHENSIVE
ANALYSIS
OF
THE
EV
PROTEOME
.......................................................................................................98
2.1.1
THE
EXPRESSION
OF
TSPAN8
IN
HT1080
CELLS
CHANGES
THE
EV
PROTEOME
..................................................
98
2.1.2
TSPAN8
IMPACTS
MORE
STRONGLY
THE
PROTEOMIC
CONTENT
OF
EVS
IN
COMPARISON
TO
CD9
........................
101
2.1.3
VALIDATION
OF
PROTEOMIC
DATA
WITH
IMMUNOBLOT
....................................................................................
106
2.1.4
EVALUATION
OF
THE
PROTEIN
TOPOLOGY
ON
EVS
.............................................................................................
107
2.1.5
SPECIFIC
ELUTION
OF
HISTONES
WITH
EVS
......................................................................................................
ILL
2.2
T
ETRASPANIN
EXPRESSION
DOES
NOT
AFFECT
PROLIFERATION
IN
HT1080
CELLS
............................................................
112
2.3
C
OMPREHENSIVE
ANALYSIS
OF
THE
NUCLEIC
ACID
CARGO
OF
EVS
.................................................................................
115
2.3.1
QUANTITATIVE
AND
QUALITATIVE
DIFFERENCES
IN
EV-RNA
UPON
EXPRESSION
OFTSPAN8
...............................
115
2.3.2
ANALYSIS
OF
SMALL
RNAS
WITH
RNASEQ
.....................................................................................................
116
2.3.3
ANALYSIS
OF
LONG
RNAS
WITH
MACE-SEQ
...................................................................................................
124
2.3.4
QUALITATIVE
AND
QUANTITATIVE
CHANGES
IN
EV-DNA
UPON
TSPAN8
EXPRESSION
........................................
129
3.
DOES
TSPAN8
LOCALIZATION
IN
THE
CELL
EXPLAIN
THE
NUCLEAR
ORIGIN
OF
EV
CARGO?
.........................131
3.1
C
ELLULAR
T
SPAN
8
CO
-
PRECIPITATES
WITH
NUCLEAR
PROTEINS
....................................................................................
131
3.2
S
UPER
-
RESOLUTION
MICROSCOPY
REVEALS
T
SPAN
8
LOCALIZATION
IN
THE
NUCLEAR
RIM
................................................
137
DISCUSSION
...................................................................................
141
E
FFECTS
OF
T
SPAN
8
EXPRESSION
IN
THE
POPULATION
OF
EVS:
ENHANCED
YIELD
PRODUCTION
..............................................
143
E
FFECTS
OF
TETRASPANIN
T
SPAN
8
IN
EXTRACELLULAR
VESICLE
S
CARGO
:
ENHANCED
RECRUITMENT
OF
NUCLEAR
MOLECULES
...
146
T
SPAN
8
EXHIBITS
AN
ALTERNATIVE
NUCLEAR
LOCALIZATION
...............................................................................................150
CONCLUSIONS
..............................................................................................................................................................
155
BIBLIOGRAPHY
.............................................................................................................................................................
159
ANNEX
..........................................................................................................................................................................187
P
UBLICATIONS
RELATED
TO
THIS
D
OCTORAL
THESIS
..........................................................................................................
189
S
UPPLEMENTARY
MATERIAL
..........................................................................................................................................
191
ACKNOWLEDGEMENTS..............................................................................................................................................195
|
| any_adam_object | 1 |
| author | Grueso Navarro, Elena |
| author_GND | (DE-588)1277087601 |
| author_facet | Grueso Navarro, Elena |
| author_role | aut |
| author_sort | Grueso Navarro, Elena |
| author_variant | n e g ne neg |
| building | Verbundindex |
| bvnumber | BV049066223 |
| ctrlnum | (OCoLC)1356724457 (DE-599)KXP1830237314 |
| discipline | Medizin |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV049066223 |
| illustrated | Illustrated |
| indexdate | 2024-12-20T19:59:47Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-034328276 |
| oclc_num | 1356724457 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR |
| owner_facet | DE-355 DE-BY-UBR |
| physical | 200 Seiten Illustrationen, Diagramme 30 cm |
| publishDate | 2022 |
| publishDateSearch | 2022 |
| publishDateSort | 2022 |
| record_format | marc |
| spellingShingle | Grueso Navarro, Elena The role of tetraspanin Tspan8 in regulating the composition of extracellular vesicles |
| subject_GND | (DE-588)4113937-9 |
| title | The role of tetraspanin Tspan8 in regulating the composition of extracellular vesicles |
| title_auth | The role of tetraspanin Tspan8 in regulating the composition of extracellular vesicles |
| title_exact_search | The role of tetraspanin Tspan8 in regulating the composition of extracellular vesicles |
| title_full | The role of tetraspanin Tspan8 in regulating the composition of extracellular vesicles presented by Elena Grueso Navarro |
| title_fullStr | The role of tetraspanin Tspan8 in regulating the composition of extracellular vesicles presented by Elena Grueso Navarro |
| title_full_unstemmed | The role of tetraspanin Tspan8 in regulating the composition of extracellular vesicles presented by Elena Grueso Navarro |
| title_short | The role of tetraspanin Tspan8 in regulating the composition of extracellular vesicles |
| title_sort | the role of tetraspanin tspan8 in regulating the composition of extracellular vesicles |
| topic_facet | Hochschulschrift |
| url | https://d-nb.info/1279325674/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034328276&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT gruesonavarroelena theroleoftetraspanintspan8inregulatingthecompositionofextracellularvesicles |