Role of Hypoxia-Inducible Factor (HIF) 1α in Dendritic Cells in Immune Regulation of Atherosclerosis: = Rolle von Hypoxie induziertem Faktor (HIF) 1α in dendritischen Zellen in der Immunregulation der Atherosklerose
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Würzburg
2013
|
| Schlagworte: | |
| Online-Zugang: | Volltext http://d-nb.info/1125372370/34 Inhaltsverzeichnis |
| Beschreibung: | veröffentlicht: 2017 |
| Beschreibung: | 162 Blätter Illustrationen, Diagramme |
| Format: | Langzeitarchivierung gewährleistet, LZA |
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| 245 | 1 | 0 | |a Role of Hypoxia-Inducible Factor (HIF) 1α in Dendritic Cells in Immune Regulation of Atherosclerosis |b = Rolle von Hypoxie induziertem Faktor (HIF) 1α in dendritischen Zellen in der Immunregulation der Atherosklerose |c submitted by Sweena M. Chaudhari from Mumbai, India ; Primary supervisor: Prof. Dr. Alma Zernecke ... |
| 246 | 1 | 1 | |a Rolle von Hypoxie induziertem Faktor (HIF) 1α in dendritischen Zellen in der Immunregulation der Atherosklerose |
| 264 | 1 | |a Würzburg |c 2013 | |
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Datensatz im Suchindex
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| adam_text | ROLE OF HYPOXIA-INDUCIBLE FACTOR (HIF) 1A IN DENDRITIC CELLS IN
IMMUNE REGULATION OF ATHEROSCLEROSIS
ROLLE VON HYPOXIE INDUZIERTEM FAKTOR (HIF) 1A IN DENDRITISCHEN ZELLEN IN
DER
IMMUNREGULATION DER ATHEROSKLEROSE
DOCTORAL THESIS FOR A DOCTORAL DEGREE
AT THE GRADUATE SCHOOL OF LIFE SCIENCES
JULIUS-MAXIMILIANS-UNIVERSITAET WUERZBURG
SECTION BIOMEDICINE
SUBMITTED BY
SWEENA M. CHAUDHARI
FROM
MUMBAI, INDIA
WUERZBURG, 2013
TABLE OF CONTENTS
S U M M A R Y
.....................................................................................................................
10
ZUSAM M ENFASSUNG
...............................................................................................
12
ABBREVIATIONS..........................................................................................................14
1. IN TR O D U C TIO N
....................................................................................................
20
1.1 DENDRITIC
CELLS.........................................................................................................
20
1.1.1 ORIGIN OF DENDRITIC
CELLS...............................................................................20
1.1.2 DC MARKERS AND
SUBTYPES.........................................................................
21
1.1.3 RELATION OF DCS TO MONOCYTES AND MACROPHAGES
...............................
22
1.1.4 CD11C MARKER AND THE CD11C-ERE MOUSE M ODEL
................................
22
1.1.5 FUNCTIONS OF D CS
.......................................................................
23
1.1.6 DCS REGULATE HELPER T-CELL
BALANCE.........................................................24
1.1.7 DC
MIGRATION................................................................................................
26
1.2
ATHEROSCLEROSIS.......................................................................................................
27
1.2.1 ATHEROSCLEROSIS - A GLOBAL BURDEN
..........................................................
27
1.2.2 PATHOGENESIS OF ATHEROSCLEROSIS
.............................................................
27
1.2.3 MONOCYTES AND MACROPHAGES IN
ATHEROSCLEROSIS.................................28
1.2.4 DENDRITIC CELLS IN ATHEROSCLEROSIS
.............................................................
29
1.2.5 T CELLS IN
ATHEROSCLEROSIS...........................................................................
30
1.2.6 MOUSE MODELS TO STUDY
ATHEROGENESIS.................................................. 32
1.3 HYPOXIA-INDUCIBLE FACTOR 1 A
.................................................................................
33
1.3.1 HYPOXIA AND OXYGEN HOMEOSTASIS IN CELLS
.............................................
33
1.3.2 BIOLOGY OF THE HYPOXIA-INDUCIBLE
FACTORS.................................................34
1.3.3 NON-HYPOXIC REGULATION OF H IF IA
.............................................................
36
1.3.4 H IF IA IN IM M UNITY- LYMPHOID
CELLS.........................................................36
1.3.5 H IF IA IN IMMUNITY - MYELOID
CELLS........................................................... 37
1.3.6 H IF IA IN IMMUNITY - DENDRITIC
CELLS..........................................................38
1.3.7 H IF IA IN INNATE IMMUNITY
......
.
....................................................................39
1.3.8 H IF IA IN PHYSIOLOGY AND DISEASE
..................................
40
1.3.9 HYPOXIA IN
ATHEROSCLEROSIS.........................................................................41
1.3.10 ROLE OF H IF IA IN MYELOID CELLS IN ATHEROSCLEROSIS
..............................
42
1.3.11 ROLE OF H IF IA IN T CELLS IN ATHEROSCLEROSIS
.........................................
42
1.3.12 NON-HYPOXIC TRIGGERS OF H IF IA LEADING TO ATHEROSCLEROSIS
..............
43
1.4 AIM OF THIS
PROJECT................................................................................................
43
2. M A TER IA
LS..............................................................................................................45
2.1
CHEMICALS................................................................................................................
45
2.2 KITS AND
REAGENTS....................................................................................................46
2.2.1
KITS.................................................................................................................
46
2.2.2 MOLECULAR BIOLOGY
REAGENTS.......................................................................46
2.2.3 CELL CULTURE
REAGENTS..................................................................................
47
2.3
CYTOKINES.................................................................................................................
47
2.4
ANTIBODIES................................................................................................................
48
2.4.1 FLUORESCENT
ANTIBODIES...............................................................................48
2.4.2 PRIMARY
ANTIBODIES.......................................................................................48
2.4.3 SECONDARY ANTIBODIES AND DETECTION REAGENTS
......................................
49
2.5
PRIMERS.....................................................................................................................49
2.5.1 QPCR
PRIMERS...............................................................................................49
2.5.2 GENOTYPING
PRIMERS.....................................................................................50
2.5.3 CHIP
PRIMERS................................................................................................
51
2.6
PLASMIDS...................................................................................................................51
2.7
ANIMALS.....................................................................................................................51
2.8 CELL
LINES...................................................................................................................51
2.9 PLASTICWARE, GLASSWARE AND OTHER CONSUMABLES
.............................................
52
2.10 INSTRUMENTS...
........................................
53
2.11
SOFTWARES...............................................................................................................
54
2.12 BUFFERS, SOLUTIONS AND M ED IA
............................................................................
54
2.12.1
BUFFERS.........................................................................................................
54
2.12.2
SOLUTIONS......................................................................................................56
2.12.3
MEDIA...........................................................................................................
57
3. M ETHO
DS.................................................................................................................
59
3.1 ANIMAL
WORK...........................................................................................................
59
3.1.1 MICE EXPERIMENTS....................... 59
3.1.2 GENERATION OF
CD11C-CRE+ HIF1ANOX/I]* LDLR1 TRANSGENIC M IC E
..............
59
3.1.3 GENERATION OF LYSM-CRE+ H/7FAFLOX/FLOX LD/RV TRANSGENIC M ICE
...............
59
3.1.4 GENERATION OF TRANSGENIC MICE WITH STATS OVEREXPRESSION IN DCS 60
3.1.5 SACRIFICING MICE AND HARVESTING
ORGANS..................................................60
6
3.1.6 PROCESSING OF ORGANS
................................................................................
60
3.2
HISTOLOGY...................................................................................................................61
3.2.1 OIL-RED O STAINING OF AORTAS FOR PLAQUE
ANALYSIS.................................61
3.2.2 ANALYSIS OF AORTIC PLAQUE A RE A
................................................................. 62
3.2.3 PREPARATION OF AORTIC ROOTS FOR PARAFFIN SECTIONING
...............................
62
3.2.4 DE-PARAFFINIZATION OF AORTIC
ROOTS...............................................................62
3.2.5 COLLAGEN-ELASTIN STAINING OF AORTIC ROOTS FOR PLAQUE ANALYSIS
.............
63
3.2.6 ANALYSIS OF AORTIC ROOT PLAQUE A RE A
......................................................... 63
3.2.7 ANTIGEN RETRIEVAL OF AORTIC
ROOTS.................................................................64
3.2.8 MACROPHAGE-SMOOTH MUSCLE CELL STAINING OF AORTIC ROOTS
.................
64
3.2.9 T-CELL STAINING OF AORTIC ROOTS
.....................................................................
64
3.2.10 PREPARATION OF AORTIC ROOTS FOR FROZEN SECTIONING
...............................
65
3.2.11 CD11C+ CELL STAINING OF AORTIC ROOTS
......................................................
65
3.2.12 ANALYSIS OF AORTIC ROOT PLAQUE CELL
CONTENT..........................................66
3.2.13 CD11C+ CELL AND HIF1A STAINING OF AORTIC
ROOTS..................................67
3.2.14 DETECTION OF
HYPOXIA.................................................................................
68
3.3 CELL CULTURE
TECHNIQUES.........................................................................................
69
3.3.1 BONE MARROW DERIVED DENDRITIC CELL (BMDC) CULTURE
...........................
69
3.3.2 BONE MARROW DERIVED MACROPHAGE (BMM) CULTURE
..............................
69
3.3.3 STIMULATION OF CULTURED
CELLS.......................................................................70
3.3.4 ISOLATION OF SPLENIC D C S
.............................................................................
70
3.3.5 ISOLATION OF SPLENIC CD4+ T CELLS
..............................................................
70
3.3.6 ISOLATION OF NAIVE CD4+ T
CELLS..................................................................71
3.3.7 ISOLATION OF LINEAGE DEPLETED BM CELLS
....................................................
71
3.3.8 ANTIGEN SPECIFIC T-CELL
PROLIFERATION..........................................................71
3.3.9 ANTIGEN SPECIFIC T-CELL
POLARIZATION..........................................................72
3.3.10 BMDC TRANSMIGRATION
ASSAY...................................................................72
3.3.11 TRANSFECTION OF DCS WITH INHIBITION AND OVEREXPRESSION
PLASMIDS.72
3.3.12 MAINTENANCE OF HEK293F CELLS
.............................................................
72
3.4 NUCLEIC ACID DETECTION
TECHNIQUES......................................................................73
3.4.1 GENOMIC DNA ISOLATION
.............................................................................
73
3.4.2 TOTAL RNA ISOLATION
.....................................................................................
73
3.4.3 CDNA
SYNTHESIS...........................................................................................
73
3.4.4 CONVENTIONAL PCR FOR
GENOTYPING...........................................................74
3.4.5 QUANTITATIVE REAL TIME P C R
......................................................................75
3.5 PROTEIN DETECTION
TECHNIQUES...............................................................................76
3.5.1 FLOW
CYTOMETRY.............................................................................................76
3.5.2 ELISA
.............................................................................................................77
3.6 LIPID DETECTION
TECHNIQUES...................................................................................
77
3.6.1 CHOLESTEROL
ASSAY........................................................................................
77
3.6.2 TRIGLYCERIDE A S
SAY.......................................................................................78
3.7 BACTERIAL
TECHNIQUES..............................................................................................78
3.7.1 ELUTING PLASMIDS FROM FILTER PAPER
SPOTS.................................................78
3.7.2 PREPARATION OF LB PLATES
............................................................................
78
3.7.3 TRANSFORMATION OF BACTERIAL
CELLS...............................................................78
3.7.4 GROWTH OF TRANSFORMED CELLS FOR PLASMID DNA ISOLATION
.....................
78
3.7.5 PLASMID DNA
ISOLATION................................................................................79
3.7.6 LENTIVIRAL VECTOR - CRE VECTOR CO-TRANSFECTION
......................................
79
3.8 LENTIVIRUS
TECHNIQUES.............................................................................................79
3.8.1 TRANSFECTION OF HEK293F CELLS FOR LENTIVIRUS PRODUCTION
......................
79
3.8.2 HARVESTING OF
LENTIVIRUS...............................................................................79
3.8.3 VIRUS
TITRATION................................................................................................
80
3.8.4 VIRAL TRANSDUCTION OF BM CELLS FOR
IN VITRO ANALYSIS
.................................
80
3.8.5 VIRAL TRANSDUCTION OF LIN- BM CELLS FOR IN VIVO TRANSPLANTATION
...............
81
3.9 TRANSCRIPTION FACTOR BINDING SITE
ANALYSIS.......................................................... 81
3.9.1 TRANSCRIPTION FACTOR BINDING SITE PREDICTION
............................................
81
3.9.2 CHROMATIN IMMUNOPRECIPITATION A
SSAY...................................................81
3.10
STATISTICS................................................................................................................
83
4. R ESU
LTS................................................................................................................
84
4.1 PRESENCE OF HYPOXIA AND HIF1A IN ATHEROSCLEROTIC M ICE
..............................
84
4.2 GENERATION OF MICE DEFICIENT FOR HIF1A IN CD11C+ CELLS
..............................
86
4.3 ANALYSIS OF ATHEROSCLEROSIS PROGRESSION IN MICE WITH DCS DEFICIENT
FOR
HIF1A AND FED A HIGH FAT
DIET................................................................................
88
4.4 ANALYSIS OF IMMUNE CELL PHENOTYPE IN MICE WITH DCS DEFICIENT FOR
HIF1A
AND FED A HIGH FAT
DIET......................................................................
92
4.5 ANALYSIS OF IMMUNE CELL PHENOTYPE IN MICE WITH DCS DEFICIENT FOR
HIF1A AT
THE BASELINE
LEVEL......................................................................................................97
4.6 CHARACTERIZATION OF MATURE BMDCS DEFICIENT FOR H IF 1 A
................................
99
4.7 CHARACTERIZATION OF SPLENIC DCS ISOLATED FROM ATHEROSCLEROTIC W T
LDLRA AND
CKO LDLF -
MICE......................................................................................................
104
4.8 EXPRESSION OF STAT PROTEINS IN DCS DEFICIENT IN H IF 1 A
.............................
108
4.9 A HIF1 A-STATS-IL-12-IFNY PATHWAY IN ATHEROSCLEROSIS
.............................
110
4.10 DETERMINATION OF HIF1A BINDING SITES ON GENE PROMOTERS AND EVIDENCE
OF
DIRECT TRANSCRIPTION FACTOR ACTIVITY BY
CHIP......................................................... 113
5. D IS C U S S IO N
..........................................................................................................116
6. R E FE R E N C E S
.......................................................................................................
131
ACKNOW LEDG EM ENTS
.........................................................................................
156
CURRICULUM V IT A E
...............................................................................................
158
LIST OF PUBLICATIONS
.........................................................................................
159
LIST OF ORAL AND POSTER PRESENTATIO NS
............................................
160
A FFID A V
IT...................................................................................................................161
|
| any_adam_object | 1 |
| author | Chaudhari, Sweena M. 1985- |
| author_GND | (DE-588)1127356283 (DE-588)129581607 |
| author_facet | Chaudhari, Sweena M. 1985- |
| author_role | aut |
| author_sort | Chaudhari, Sweena M. 1985- |
| author_variant | s m c sm smc |
| building | Verbundindex |
| bvnumber | BV044219650 |
| collection | ebook |
| ctrlnum | (OCoLC)981906073 (DE-599)DNB1125372370 |
| dewey-full | 610 |
| dewey-hundreds | 600 - Technology (Applied sciences) |
| dewey-ones | 610 - Medicine and health |
| dewey-raw | 610 |
| dewey-search | 610 |
| dewey-sort | 3610 |
| dewey-tens | 610 - Medicine and health |
| discipline | Medizin |
| format | Thesis Book |
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| genre | (DE-588)4113937-9 Hochschulschrift gnd-content |
| genre_facet | Hochschulschrift |
| id | DE-604.BV044219650 |
| illustrated | Illustrated |
| indexdate | 2024-07-10T07:46:56Z |
| institution | BVB |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-029625650 |
| oclc_num | 981906073 |
| open_access_boolean | 1 |
| owner | DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-12 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 DE-860 DE-2174 |
| owner_facet | DE-384 DE-473 DE-BY-UBG DE-703 DE-1051 DE-824 DE-29 DE-12 DE-91 DE-BY-TUM DE-19 DE-BY-UBM DE-1049 DE-92 DE-739 DE-898 DE-BY-UBR DE-355 DE-BY-UBR DE-706 DE-20 DE-1102 DE-860 DE-2174 |
| physical | 162 Blätter Illustrationen, Diagramme |
| psigel | ebook |
| publishDate | 2013 |
| publishDateSearch | 2013 |
| publishDateSort | 2013 |
| record_format | marc |
| spelling | Chaudhari, Sweena M. 1985- Verfasser (DE-588)1127356283 aut Role of Hypoxia-Inducible Factor (HIF) 1α in Dendritic Cells in Immune Regulation of Atherosclerosis = Rolle von Hypoxie induziertem Faktor (HIF) 1α in dendritischen Zellen in der Immunregulation der Atherosklerose submitted by Sweena M. Chaudhari from Mumbai, India ; Primary supervisor: Prof. Dr. Alma Zernecke ... Rolle von Hypoxie induziertem Faktor (HIF) 1α in dendritischen Zellen in der Immunregulation der Atherosklerose Würzburg 2013 162 Blätter Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier veröffentlicht: 2017 Dissertation Julius-Maximilians-Universität Würzburg 2014 Langzeitarchivierung gewährleistet, LZA Zusammenfassung in deutscher und englischer Sprache Hypoxie (DE-588)4133549-1 gnd rswk-swf Entzündung (DE-588)4014975-4 gnd rswk-swf Arteriosklerose (DE-588)4068866-5 gnd rswk-swf Dendritische Zelle (DE-588)4313846-9 gnd rswk-swf Hypoxia Atherosclerosis Dendritic cells (DE-588)4113937-9 Hochschulschrift gnd-content Arteriosklerose (DE-588)4068866-5 s Dendritische Zelle (DE-588)4313846-9 s Entzündung (DE-588)4014975-4 s Hypoxie (DE-588)4133549-1 s DE-604 Zernecke-Madsen, Alma 1976- (DE-588)129581607 dgs Erscheint auch als Online-Ausgabe, PDF urn:nbn:de:bvb:20-opus-91853 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-91853 Resolving-System kostenfrei Volltext http://d-nb.info/1125372370/34 Langzeitarchivierung Nationalbibliothek DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029625650&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Chaudhari, Sweena M. 1985- Role of Hypoxia-Inducible Factor (HIF) 1α in Dendritic Cells in Immune Regulation of Atherosclerosis = Rolle von Hypoxie induziertem Faktor (HIF) 1α in dendritischen Zellen in der Immunregulation der Atherosklerose Hypoxie (DE-588)4133549-1 gnd Entzündung (DE-588)4014975-4 gnd Arteriosklerose (DE-588)4068866-5 gnd Dendritische Zelle (DE-588)4313846-9 gnd |
| subject_GND | (DE-588)4133549-1 (DE-588)4014975-4 (DE-588)4068866-5 (DE-588)4313846-9 (DE-588)4113937-9 |
| title | Role of Hypoxia-Inducible Factor (HIF) 1α in Dendritic Cells in Immune Regulation of Atherosclerosis = Rolle von Hypoxie induziertem Faktor (HIF) 1α in dendritischen Zellen in der Immunregulation der Atherosklerose |
| title_alt | Rolle von Hypoxie induziertem Faktor (HIF) 1α in dendritischen Zellen in der Immunregulation der Atherosklerose |
| title_auth | Role of Hypoxia-Inducible Factor (HIF) 1α in Dendritic Cells in Immune Regulation of Atherosclerosis = Rolle von Hypoxie induziertem Faktor (HIF) 1α in dendritischen Zellen in der Immunregulation der Atherosklerose |
| title_exact_search | Role of Hypoxia-Inducible Factor (HIF) 1α in Dendritic Cells in Immune Regulation of Atherosclerosis = Rolle von Hypoxie induziertem Faktor (HIF) 1α in dendritischen Zellen in der Immunregulation der Atherosklerose |
| title_full | Role of Hypoxia-Inducible Factor (HIF) 1α in Dendritic Cells in Immune Regulation of Atherosclerosis = Rolle von Hypoxie induziertem Faktor (HIF) 1α in dendritischen Zellen in der Immunregulation der Atherosklerose submitted by Sweena M. Chaudhari from Mumbai, India ; Primary supervisor: Prof. Dr. Alma Zernecke ... |
| title_fullStr | Role of Hypoxia-Inducible Factor (HIF) 1α in Dendritic Cells in Immune Regulation of Atherosclerosis = Rolle von Hypoxie induziertem Faktor (HIF) 1α in dendritischen Zellen in der Immunregulation der Atherosklerose submitted by Sweena M. Chaudhari from Mumbai, India ; Primary supervisor: Prof. Dr. Alma Zernecke ... |
| title_full_unstemmed | Role of Hypoxia-Inducible Factor (HIF) 1α in Dendritic Cells in Immune Regulation of Atherosclerosis = Rolle von Hypoxie induziertem Faktor (HIF) 1α in dendritischen Zellen in der Immunregulation der Atherosklerose submitted by Sweena M. Chaudhari from Mumbai, India ; Primary supervisor: Prof. Dr. Alma Zernecke ... |
| title_short | Role of Hypoxia-Inducible Factor (HIF) 1α in Dendritic Cells in Immune Regulation of Atherosclerosis |
| title_sort | role of hypoxia inducible factor hif 1α in dendritic cells in immune regulation of atherosclerosis rolle von hypoxie induziertem faktor hif 1α in dendritischen zellen in der immunregulation der atherosklerose |
| title_sub | = Rolle von Hypoxie induziertem Faktor (HIF) 1α in dendritischen Zellen in der Immunregulation der Atherosklerose |
| topic | Hypoxie (DE-588)4133549-1 gnd Entzündung (DE-588)4014975-4 gnd Arteriosklerose (DE-588)4068866-5 gnd Dendritische Zelle (DE-588)4313846-9 gnd |
| topic_facet | Hypoxie Entzündung Arteriosklerose Dendritische Zelle Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-91853 http://d-nb.info/1125372370/34 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029625650&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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