Roles of hypoxia-inducible factor (HIF)-1a [(HIF)-1-alpha] versus HIF-2a [HIF-2-alpha] in hepatocellular tumor spheroids:
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2010
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | XI, 98 S. Ill., graph. Darst. 21 cm |
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MARC
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| 245 | 1 | 0 | |a Roles of hypoxia-inducible factor (HIF)-1a [(HIF)-1-alpha] versus HIF-2a [HIF-2-alpha] in hepatocellular tumor spheroids |c vorgelegt von Heidi Menrad |
| 264 | 1 | |c 2010 | |
| 300 | |a XI, 98 S. |b Ill., graph. Darst. |c 21 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Frankfurt (Main), Univ., Diss., 2010 (Nicht für den Austausch) | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
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| 999 | |a oai:aleph.bib-bvb.de:BVB01-021104708 | ||
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| _version_ | 1804143776084525057 |
|---|---|
| adam_text | INDEX
I
INDEX
1
SUMMARY
..............................................................................................................
1
2
ZUSA
M
MENFASS
U
NG
.............................................................................................
3
3
INTRODUCTION
................
.......................................................................................
5
3.1
TUMOR
HYPOXIA
..............................................................................................................
5
3.2
HIFS
.................................................................................................................................
7
3.2.1
OXYGEN-DEPENDENT
REGULATION
OF
THE
HIF-A-SUBUNITS
..............................................................................
8
3.2.2
ALTERNATIVE
PATHWAYS
OF
HIF
REGULATION
.....................................................................................................9
3.2.3
DIVERGENT
ROLES
OF
HIF-1
AND
HIF-2
IN
TUMOR
DEVELOPMENT
................................................................
10
3.3
VASCULARIZATION
..........................................................................................................
11
3.3.1
VASCULOGENESIS
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11
3.3.2
ANGIOGENESIS
...............................................................................................................................................
12
3.3.3
VEGF
...............................................................................................................................................................
12
3.4
CELL
DEATH
PATHWAYS
...................................................................................................
13
3.4.1
NECROSIS
...........................................................................................................................................................
13
3.4.2
APOPTOSIS
........................................................................................................................................................
14
3.4.2.1
EXTRINSIC
PATHWAY
OF
APOPTOSIS
INDUCTION
........................................................................................
14
3.4.2.2
INTRINSIC
PATHWAY
OF
APOPTOSIS
INDUCTION
..........................................................................................
16
3.4.3
AUTOPHAGY
.......................................................................................................................................................
19
3.4.3.1
THE
AUTOPHAGY-RELATED
(ATG)
PROTEINS
............................................................................................
19
3.4.3.2
AUTOPHAGY
INDUCTION
...........................................................................................................................
19
3.4.3.3
AUTOPHAGOSOME
FORMATION
.................................................................................................................20
3.4.3.4
AUTOLYSOSOME
FORMATION
....................................................................................................................
20
3.4.3.5
SELECTIVITY
OF
AUTOPHAGY
.....................................................................................................................
22
3.4.3.6
HIF-1-DEPENDENT
AUTOPHAGY
..............................................................................................................
22
3.4.3.7
HIF-1-INDEPENDENT
AUTOPHAGY
..........................................................................................................
23
3.5
HEPATOCELLULAR
CARCINOMA
AND
HIF
..........................................................................
24
3.6
AIMS
OF
THE
STUDY
......................................................................................................
25
4
MATERIAL
AND
METHODS
....................................................................................26
4.1
MATERIAL
..................................................................
26
4.1.1
CELLS
.................................................................................................................................................................
26
4.1.2
CHEMICALS
AND
REAGENTS
...............................................................................................................................
26
4.1.3
ANTIBODIES
......................................................................................................................................................
28
4.1.4
SHORT
HAIRPIN
(SH)RNA
SEQUENCES
USED
FOR
LENTIVIRAL
TRANSDUCTION
...................................................
29
4.1.5
SIRNA
SEQUENCES
USED
FOR
TRANSIENT
TRANSFECTION
...................................................................................
29
4.1.6
PRIMER
.............................................................................................................................................................
30
4.1.7
CONSUMABLE
MATERIAL
...................................................................................................................................
30
INDEX
II
4,1.8
INSTRUMENTS
AND
SOFTWARE
...........................................................................................................................
31
4.2
METHODS
.....................................................................................................................
33
4.2.1
CELL
CULTURE
..................................................................................................................................................
33
4.2.2
CULTIVATION
OF
TUMOR
SPHEROIDS
..................................................................................................................
33
4.2.3
ESTABLISHMENT
AND
CULTIVATION
OF
TUMOR
SPHEROID-EB-DERIVED
COCULTURES
........................................
34
4.2.4
GROWTH
DETERMINATION
OF
MONOLAYER
CELL
CLONES
....................................................................................34
4.2.5
ATP
MEASUREMENTS
.......................................................................................................................................
34
4.2.6
LACTATE
PRODUCTION
........................................................................................................................................34
4.2.7
LENTIVIRAL
TRANSDUCTION
.................................................................................................................................
35
4.2.8
TRANSIENT
TRANSFECTION
...................................................................................................................................
35
4.2.9
DETERMINATION
OF
PROTEIN
EXPRESSION
BY
WESTERN
ANALYSIS
...................................................................35
4.2.9.1
PROTEIN
ISOLATION
FROM
TUMOR
SPHEROIDS
............................................................................................
35
4.2.9.2
PROTEIN
ISOLATION
FROM
MONOLAYER
CELLS
............................................................................................
35
4.2.9.3
INVESTIGATION
OF
PROTEIN
CONCENTRATIONS
(LOWRY)
.............................................................................
36
4.2.9.4
SODIUM
DODECYL
SULFATE
(SDS)-PAGE/WESTEM
ANALYSIS
..............................................................
36
4.2.10
ANALYSIS
OF
MRNA
EXPRESSION
...................................................................................................................
36
4.2.10.1
RNA
ISOLATION
.....................................................................................................................................
36
4.2.10.2
REVERSE
TRANSCRIPTION-POLYMERASE
CHAIN
REACTION
(RT-PCR)
.......................................................
37
4.2.10.3
QUANTITATIVE
PCR
(QPCR)
.................................................................................................................
37
4.2.1
1
IMMUNO-CYTO-/HISTO-CHEMISTRY
........................................................
38
4.2.11.1
TRYPAN
BLUE
STAINING
..........................................................................................................................
38
4.2.11.2
PROPIDIUM
IODIDE
(PI)
STAINING
..........................................................................................................
38
4.2.11.3
MITO
AND
LYSOTRACKER
COSTAINING
....................................................................................................
39
4.2.11.4
CMFDA
STAINING
.................................................................................................................................
39
4.2.11.5
CD3I
STAINING
.....................................................................................................................................
39
4.2.11.6
IMMUNOHISTOCHEMICAL
ANALYSIS
USING
THE
CSA
KIT
.........................................................................
39
4.2.11.7
IMMUNOHISTOCHEMICAL
ANALYSIS
USING
FLURESCCNCC
LABELING
...........................................................40
4.2.11.8
QUANTIFICATION
OF
VIABLE
AND
CLEAVED
CASPASE-3
POSITIVE
CELLS
.......................................................40
4.2.12
TRANSMISSION
ELECTRON
MICROSCOPY
(TEM)
...............................................................................................
41
4.2.13
STATISTICAL
ANALYSIS
..........................................................................................................................................
41
5
RESULTS
...............................................................................................................
42
5.1
TUMOR
DEVELOPMENT
PRIOR
TO
ANGIOGENESIS
INDUCTION
-
THE
TUMOR
SPHEROIDS
....
42
5.1.1
CELL
LINE
SELECTION
.........................................................................................................................................
42
5.1.2
KNOCKDOWN
OF
HIF-1
A
OR
HIF-2A
INCREASES
THE
EXPRESSION
OF
THE
OTHER
SUBUNIT
...........................
43
5.1.3
GROWTH
AND
CELL
VIABILITY
WERE
ENHANCED
IN
SH
L
A
AND
SH2A
TUMOR
SPHEROIDS
.................................
46
5.1.4
REDUCED
CASPASE-3
ACTIVITY,
DYSREGULATED
BCL-XI
AND
BAX
EXPRESSION
IN
SHLU
AND
SH2A
TUMOR
SPHEROIDS
........................................................................................................................................................
51
5.1.5
INCREASED
BNIP3
EXPRESSION
IN
SH2A
CELLS
..............................................................................................
52
5.1.6
INCREASED
AUTOPHAGY
IN
SH2A
TUMOR
SPHEROIDS
.......................................................................................
53
5.1.7
KNOCKDOWN
OF
HIF-1
A
REVERSED
SURVIVAL
ADVANTAGES
IN
SH2A
TUMOR
SPHEROIDS
..............................
56
5.2
INDUCTION
OF
THE
ANGIOGENIC
SWITCH
-
THE
TUMOR
SPHEROID-EB
COCULTURE
MODEL..
58
5.2.1
HIF
EXPRESSION
IN
THE
COCULTURE
MODEL
....................................................................................................
59
INDEX
III
5.2.2
INCREASED
GROWTH
AND
DECREASED
CELL
DEATH
IN
TUMOR
SPHEROID-EB
COCULTURES
................................
59
5.2.3
CD3I
EXPRESSION
IS
ASSOCIATED
WITH
HLF-2A
EXPRESSION
IN
COCULTURES
.............................................
62
5.2.4
VEGF
)2
I
EXPRESSION
IS
ASSOCIATED
WITH
HLF-2U
ACCUMULATION
IN
COCULTURES
...................................
63
6
DISCUSSION
.........................................................................................................
65
6.1
TUMOR
DEVELOPMENT
PRIOR
TO
ANGIOGENESIS
INDUCTION
-
THE
TUMOR
SPHEROIDS
....
65
6.1.1
RECIPROCAL
REGULATION
OF
LLLF-A-SUBUNIT
EXPRESSION
AND
HIF
ACTIVITY
...............................................65
6.1.2
LOSS
OF
ONE
HIF-SUBUNITS
DOES
NOT
ALTER
PROLIFERATION,
GLUCOSE
METABOLISM,
OR
ATP
CONTENT.
...69
6.1.3
LOSS
OF
ONE
LLLF-A-SUBUNIT
RESULTS
IN
A
SURVIVAL
ADVANTAGE
.................................................................
70
6.2
INDUCTION
OF
THE
ANGIOGENIC
SWITCH
-
THE
TUMOR
SPHEROID-EB
COCULTURE
MODEL..
73
6.2.1
HIF-2A
EXPRESSION
POSITIVELY
CORRELATES
WITH
VEGF121
AND
CD31
EXPRESSION
...............................
74
6.2.2
COCULTURE
WITH
EBS
SUPPORTS
GROWTH
AND
SURVIVAL
OF
TUMOR
SPHEROIDS
.............................................
74
6.3
CONCLUDING
REMARKS
..................................................................................................
77
7
REFERENCES
...................................................
78
8
APPENDIX
..........................................................................................................
91
9
PUBLICATIONS
.....................................................................................................
94
10
DANKSAGUNG
.....................................................................................................
96
11
CURRICULUM
VITAE
.............................................................................................
97
12
ERKLARUNG
.........................................................................................................
98
|
| any_adam_object | 1 |
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| id | DE-604.BV037190284 |
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| indexdate | 2024-07-09T22:53:02Z |
| institution | BVB |
| language | English |
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| physical | XI, 98 S. Ill., graph. Darst. 21 cm |
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| spelling | Menrad, Heidi 1982- Verfasser (DE-588)143039903 aut Roles of hypoxia-inducible factor (HIF)-1a [(HIF)-1-alpha] versus HIF-2a [HIF-2-alpha] in hepatocellular tumor spheroids vorgelegt von Heidi Menrad 2010 XI, 98 S. Ill., graph. Darst. 21 cm txt rdacontent n rdamedia nc rdacarrier Frankfurt (Main), Univ., Diss., 2010 (Nicht für den Austausch) (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=021104708&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Menrad, Heidi 1982- Roles of hypoxia-inducible factor (HIF)-1a [(HIF)-1-alpha] versus HIF-2a [HIF-2-alpha] in hepatocellular tumor spheroids |
| subject_GND | (DE-588)4113937-9 |
| title | Roles of hypoxia-inducible factor (HIF)-1a [(HIF)-1-alpha] versus HIF-2a [HIF-2-alpha] in hepatocellular tumor spheroids |
| title_auth | Roles of hypoxia-inducible factor (HIF)-1a [(HIF)-1-alpha] versus HIF-2a [HIF-2-alpha] in hepatocellular tumor spheroids |
| title_exact_search | Roles of hypoxia-inducible factor (HIF)-1a [(HIF)-1-alpha] versus HIF-2a [HIF-2-alpha] in hepatocellular tumor spheroids |
| title_full | Roles of hypoxia-inducible factor (HIF)-1a [(HIF)-1-alpha] versus HIF-2a [HIF-2-alpha] in hepatocellular tumor spheroids vorgelegt von Heidi Menrad |
| title_fullStr | Roles of hypoxia-inducible factor (HIF)-1a [(HIF)-1-alpha] versus HIF-2a [HIF-2-alpha] in hepatocellular tumor spheroids vorgelegt von Heidi Menrad |
| title_full_unstemmed | Roles of hypoxia-inducible factor (HIF)-1a [(HIF)-1-alpha] versus HIF-2a [HIF-2-alpha] in hepatocellular tumor spheroids vorgelegt von Heidi Menrad |
| title_short | Roles of hypoxia-inducible factor (HIF)-1a [(HIF)-1-alpha] versus HIF-2a [HIF-2-alpha] in hepatocellular tumor spheroids |
| title_sort | roles of hypoxia inducible factor hif 1a hif 1 alpha versus hif 2a hif 2 alpha in hepatocellular tumor spheroids |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=021104708&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT menradheidi rolesofhypoxiainduciblefactorhif1ahif1alphaversushif2ahif2alphainhepatocellulartumorspheroids |