The guanylate binding protein-1: a molecular marker of the inflammatory cytokine-activated phenotype of endothelial cells
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
2003
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | München, Univ., Diss., 2003 |
| Beschreibung: | IV, 132 S. Ill., graph. Darst. |
Internformat
MARC
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| 100 | 1 | |a Lubeseder-Martellato, Clara |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a The guanylate binding protein-1 |b a molecular marker of the inflammatory cytokine-activated phenotype of endothelial cells |c von Clara Lubeseder-Martellato |
| 264 | 1 | |c 2003 | |
| 300 | |a IV, 132 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a München, Univ., Diss., 2003 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 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=010577951&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-010577951 | |
Datensatz im Suchindex
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TABLE OF CONTENTS
TABLE OF CONTENTS
ABBREVIATIONS. 1
SUMMARY. 3
INTRODUCTION
1 FUNCTION OF THE QUIESCENT ENDOTHELIUM. 5
2 PATHOPHYSIOLOGICAL ACTIVATION OF THE ENDOTHELIUM DURING INFLAMMATION.
6
2.1 VESSEL SPROUNTIG (ANGIOGENESIS). 7
2.2 THE ROLE OF ANGIOGENIC GROWTH FACTORS IN ENDOTHELIAL CELL
ACTIVATION. 8
2.2.1 BASIC FIBROBLAST GROWTH FACTOR (BFGF). 8
2.2.2 VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF). 8
2.3 RECRUITMENT OF LEUKOCYTES. 9
2.4 THE ROLE OF INFLAMMATORY CYTOKINE IN ENDOTHELIAL CELL ACTIVATION. 10
2.4.1 INTERFERON-GAMMA (IFN-Y). 10
2.4.2 INTERLEUKIN-1 (IL-1). 10
2.4.3 TUMOR NECROSIS FACTOR-ALPHA (TNF-A). 11
2.5 THE ROLE OF INFLAMMATORY CYTOKINES IN INFLAMMATORY SKIN DISEASES. 12
2.5.1 ADVERSE DRUG REACTIONS OF THE SKIN AND DRUG ERUPTION. 13
2.5.2 PSORIASIS. 13
2.5.3 URTICARIA. 14
2.5.4 ATOPIC DERMATITIS. 15
2.5.5 ERYTHEMA EXUDATIVUM. 15
2.5.6 AIDS-ASSOCIATED KAPOSI'S SARCOMA. 16
3 MOLECULAR MARKERS OF ENDOTHELIAL CELL ACTIVATION. 18
3.1 COMPLEXITY AND REDUNDANCY OF ENDOTHELIAL CELL ACTIVATION. 19
3.2 THE GUANYLATE BINDING PROTEIN-1: A MOLECULAR MARKER OF INFLAMMATORY
CYTOKINE ACTIVATED ENDOTHELIAL CELLS. 20
4 GOALS OF THE PROJECT. 24
MATERIALS AND METHODS
1 MATERIALS. 25
1.1 CHEMICAL REAGENTS. 25
1.2 OTHER SOLUTIONS. 25
1.3 OLIGONUCLEOTIDES. 26
1.4 ENZYMES AND REAGENTS FOR MOLECULAR BIOLOGY. 26
1.5 KITS. 26
1.6 MEDI A AND SUPPLEMENTS. 26
1.7 CYTOKINE AND GROWTH FACTORS. 27
1.8 VECTORS. 27
1.9 BACTERIAL STRAINS. 27
1.10 EUKARYOTIC CELLS. 28
1.11 PARAFFIN-EMBEDDED TISSUES. 28
1.12 BLOOD SAMPLES. 28
1.13 ANTIBODIES AND LECTINE. 28
1.14 COLUMNS. 29
1.15 EQUIPMENT. 29
1.16 OTHER MATERIAL. 29
1.17 CENTRIFUGES AND ROTORS. 29
1.18 COMPUTER PROGRAMMS. 30
1.18 BUFFERS. 30
I
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/968959008
TABLE OF CONTENTS
2 METHODS. 30
2.1 CELL BIOLOGICAL METHODS. 30
2.1.1 MAMMALIAN CELL CULTURE. 30
2.1.2 CELL STIMULATION WITH DIFFERENT FACTORS. 31
2.1.3 INHIBITION OF SECRETORY PATHWAYS. 31
2.1.4 FREEZING OF CELLS. 31
2.1.5 PROLIFERATION ASSAY. 31
2.1.6 CHEMOTAXIS ASSAY. 32
2.1.7 METABOLIC LABELING OF CELLS.32
2.1.8 DETERMINATION OF CELL VIABILITY. 32
2.2 MOLECULAR BIOLOGICAL METHODS. 33
2.2.1 PREPARATION OF PLASMID DNA. 31
2.2.2 RESTRICTION DIGEST. 33
2.2.3 AGAROSE GEL ELECTROPHORESIS. 33
2.2.4 ISOLATION OF DNA FRAGMENTS FROM AGAROSE GELS. 33
2.2.5 OLIGONUCLEOTIDE PRIMERS. 34
2.2.6 POLYMERASE CHAIN REACTION (PCR). 34
2.2.7 PURIFICATION OF PCR PRODUCTS. 34
2.2.8 DNA LIGATION.34
2.2.9 SITE-DIRECTED MUTAGENESIS.35
2.2.10 CLONING OF GST-GBPL-HIS.35
2.2.11 HIS-GBP-1, HIS-GBP-2, HIS-MGBP-1.35
2.2.12 PREPARATION OF ELECTROPORATION COMPETENT CELLS.35
2.2.13 TRANSFORMATION OF ELECTROCOMPETENT CELLS. 35
2.2.14 HEAT-SHOCK TRANSFORMATION OF
E. COLI
. 36
2.2.15 SCREENING FOR POSITIVE
E. COLI
TRANSFORMANTS. 36
2.2.16
E.
COLI PERMANENT CULTURES. 36
2.2.17 DETERMINATION OF INDUCTION KINETICS. 36
2.2.18 DETERMINATION OF PROTEIN SOLUBILITY.36
2.3 BIOCHEMICAL METHODS.37
2.3.1 INHIBITION OF PROTEASE ACTIVITY.37
2.3.2 PREPARATION OF CELLULAR EXTRACTS USING RIPA BUFFER.\.37
2.3.3 THAW-LYSIS OF CELLS. 37
2.3.4 TRITON EXTRACTION OF CELLULAR PROTEINS. 37
2.3.5 PRECIPITATION OF PROTEINS BY TCA. 38
2.3.6 DETERMINATION OF PROTEIN CONCENTRATION.38
2.3.7 WESTERN BLOTTING. 38
2.3.8 COOMASSIE BLUE STAINING OF PROTEINS. 39
2.3.9 SYPRO ORANGE STAINING OF PROTEINS. 39
2.3.10 SILVER STAINING OF PROTEINS. 39
2.3.11 PROTEIN PURIFICATION THROUGH NITA AFFINITY CHROMATOGRAPHY. 39
2.3.12 PROTEIN PURIFICATION OF GST-GBP-L-HIS THROUGH NITA AFFINITY
CHROMATOGRAPHY FOLLOWED BY GLUTATHIONE AFFINITY CHROMATOGRAPH. 40
2.3.13 DIALYSIS. 40
2.3.14 DESALTING. 40
2.3.15 IMMUNOPRECIPITATION. 40
2.4 PRODUCTION OF ANTI GBP-1 ANTIBODIES. 41
2.4.1 MONOCLONAL ANTIBODIES. 41
2.4.2 POLYCLONAL ANTIBODIES. 41
2.5 IMMUNCYTO- AND IMMUNOHISTO-CHEMISTRY. 41
2.5.1 IMMUNOHISTOCHEMISTRY ON PARAFFIN EMBEDDED SECTIONS. 41
TABLE OF CONTENTS
2.5.2 INDIRECT IMMUNOFLUORESCENCE ON PARAFFIN EMBEDDED SECTIONS. 42
2.5.3 IMMUNOCYTOCHEMISTRY. 42
2.5.4 INDIRECT IMMUNOFLUORESCENCE ON FIXED CELLS.43
2.6 ENZYME-LINKED IMMUNOASSAY (ELISA). 43
2.7 STATISTICAL ANALYSIS. 43
RESULTS
1 GENERATION OF MONO- AND POLY-CLONAL ANTIBODIES AGAINST GBP-1. 45
1.1 EXPRESSION CLONING AND PURIFICATION OF RECOMBINANT GBP-1 PROTEINS.
45
1.2 PRODUCTION OF ANTI-GBP-1 ANTIBODIES. 48
1.3 CHARACTERIZATION OF ANTI-GBP-1 ANTIBODIES. 49
2 CHARACTERIZATION OF GBP-1 EXPRESSION IN INFLAMMATORY
CYTOKINE-ACTIVATED
ENDOTHELIAL CELLS
IN VITRO. 52
2.1.1 EFFECTS OF INFLAMMATORY CYTOKINES ON GBP-1 EXPRESSION. 52
2.1.2 EFFECTS OF ANGIOGENIOC GROWTH FACTORS ON INFLAMMATORY CYTOKINE-
INDUCED GBP-1 EXPRESSION. 55
2.1.3 EFFECTS OF DIFFERENT FACTORS ON GBP-1 EXPRESSION. 57
2.2. STUDIES OF GBP-1 SUBCELLULAR LOCALIZATION IN HUVEC.60
2.3 COLOCALIZATION STUDIES OF GBP-1 WITH MARKERS FOR DIFFERENT
ORGANELLES.61
2.4 STUDIES OF GBP-1 ASSOCIATION WITH DETERGENT-RESISTANT MEMBRANES. 63
2.5 STUDIES OF IFN-Y-INDUCED GBP-1 EXPRESSION IN DIFFERENT
CELL TYPES. 64
SUMMARY OF CHAPTERS 1 TO 2 : IC-INDUCED GBP-1 EXPRESSION
IN VITRO. 65
3 CHARACTERIZATION OF INFLAMMATORY CYTOKINE ACTIVATION OF ENDOTHELIAL
CELLS
IN VIVO. 66
3.1 EXPRESSION OF GBP-1 IN NORMAL HUMAN TISSUE. 66
3.2 GBP-1 EXPRESSION IN ENDOTHELIAL CELLS IN SKIN DISEASES WITH
A HIGH-INFLAMMATORY COMPONENT. 67
3.3 GBP-1 EXPRESSION IN KAPOSI'S SARCOMA. 71
3.4 GBP-1 EXPRESSION IN NON-PROLIFERATING VESSEL ENDOTHELIAL CELLS.72
SUMMARY OF CHAPTER 3: GBP-1 IS A MARKER OF INFLAMMATORY
CYTOKINE-ACTIVATED
ENDOTHELIAL CELLS IN VIVO.75
4 STUDIES OF GBP-1 SECRETION.75
4.1 STUDIES OF GBP-1 SECRETED BY HUVEC IN THE ABSENCE OF CELL DEATH. 78
4.2 DEVELOPMENT OF AN ANTI-GBP-1 ENZYME-LINKED
IMMUNOADSORBENT ASSAY (ELISA). 80
4.3 MODULATION OF GBP-1 SECRETION.82
4.4 STUDIES OF CELL SPECIFIC GBP-1 SECRETION. 87
4.5 DETECTION OF GBP-1 IN BLOOD SAMPLES OF PATIENTS. 87
4.5.1 DETECTION OF GBP-1 IN THE PLASMA OF PATIENTS UNDER IFN-A
TREATMENT.89
4.5.2 DETECTION OF GBP-1 IN THE PLASMA OF AIDS PATIENTS.90
4.5.3 DETECTION OF GBP-1 IN THE SERUM OF PATIENTS WITH
INFLAMMATORY SKIN DISEASES.92
III
TABLE OF CONTENTS
DISCUSSION
1 GBP-1 IS A MARKER OF THE INFLAMMATORY CYTOKINE-ACTIVATED PHENOTYPE OF
ENDOTHELIAL CELLS
IN VITRO. 94
2 SUBCELLULAR OCALIZATION OF GBP-1 IN ENDOTHELIAL CELLS. 97
3 GBP-1 IS A MARKER OF NON-PROLIFERATING, INFLAMMATORY
CYTOKINE-ACTIVATED
ENDOTHELIAL CELLS IN VIVO.98
4 GBP-1 IS A SECRETED PROTEIN. 100
5 GBP-1 IN THE SERUM AS A SEROLOGICALLY ACCESSIBLE MARKER OF
IC-ACTIVATION OF
ENDOTHELIAL CELLS.105
6 POSSIBLE ROLE OF SECRETED GBP-1. 108
REFERENCES. 110
ANNEX.126
EIDESSTATTLICHE ERKLSRUNG. 127
OWN PUBLICATIONS. 128
CURRICULUM VITAE.131
AKN O WLEDGEMENT.132.
IV |
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| author_facet | Lubeseder-Martellato, Clara |
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| spelling | Lubeseder-Martellato, Clara Verfasser aut The guanylate binding protein-1 a molecular marker of the inflammatory cytokine-activated phenotype of endothelial cells von Clara Lubeseder-Martellato 2003 IV, 132 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Univ., Diss., 2003 (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=010577951&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Lubeseder-Martellato, Clara The guanylate binding protein-1 a molecular marker of the inflammatory cytokine-activated phenotype of endothelial cells |
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| title | The guanylate binding protein-1 a molecular marker of the inflammatory cytokine-activated phenotype of endothelial cells |
| title_auth | The guanylate binding protein-1 a molecular marker of the inflammatory cytokine-activated phenotype of endothelial cells |
| title_exact_search | The guanylate binding protein-1 a molecular marker of the inflammatory cytokine-activated phenotype of endothelial cells |
| title_full | The guanylate binding protein-1 a molecular marker of the inflammatory cytokine-activated phenotype of endothelial cells von Clara Lubeseder-Martellato |
| title_fullStr | The guanylate binding protein-1 a molecular marker of the inflammatory cytokine-activated phenotype of endothelial cells von Clara Lubeseder-Martellato |
| title_full_unstemmed | The guanylate binding protein-1 a molecular marker of the inflammatory cytokine-activated phenotype of endothelial cells von Clara Lubeseder-Martellato |
| title_short | The guanylate binding protein-1 |
| title_sort | the guanylate binding protein 1 a molecular marker of the inflammatory cytokine activated phenotype of endothelial cells |
| title_sub | a molecular marker of the inflammatory cytokine-activated phenotype of endothelial cells |
| topic_facet | Hochschulschrift |
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