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Publié par | ludwig-maximilians-universitat_munchen |
Publié le | 01 janvier 2002 |
Nombre de lectures | 12 |
Langue | English |
Poids de l'ouvrage | 1 Mo |
Extrait
Characterization of heme oxygenase-1 induction
in human endothelial cells
Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München
vorgelegt von
Nicole Bildner
aus Berlin
2002
Erklärung
Diese Dissertation wurde im Sinne von § 13 Absatz 3 bzw. 4 der Promotionsordnung
vom 29. Januar 1998 von Frau Prof. Dr. Angelika. M. Vollmar betreut.
Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig ohne unerlaubte Hilfe erarbeitet.
München, im November 2002
.........................................
(Unterschrift des Autors)
Dissertation eingereicht am
1. Gutachter: Frau Prof. Dr. A. M. Vollmar
2. Gutachter: Herr Prof. Dr. A. Pfeifer
Mündliche Prüfung am: 03.12.02
Meinen Eltern und meinem Freund Karl
CONTENTS
C ONTENTS
1 Introduction 7
1.1 Background and aim of the work 9
1.2 The heme oxygenase-1 11
1.2.1 The family of heat shock proteins 11
1.2.2 The heme oxygenase system 12
1.2.2.1 Physiological function 12
1.2.2.2 The three heme oxygenases 13
1.2.2.3 Regulation of heme oxygenase-1 13
1.2.2.4 Cytoprotective potential 14
1.3 Forms of cell death 15
1.3.1 Necrosis 15
1.3.2 Apoptosis 16
1.3.2.1 Overview 16
1.3.2.2 Mechanisms
1.3.2.3 Apoptosis in vascular disease 19
1.4 Tumor necrosis factor 20
1.4.1 Overview 20
1.4.2 Signaling by TNF- α 20
1.4.3 TNF- α and the endothelium 22
1.5 The natriuretic peptides 23
1.5.1 History 23
1.5.2 Structure 23
1.5.3 Biosynthesis 24
1.5.4 NP Receptors and Signaling 25
1.5.4.1 History 25
1.5.4.2 Structure and ligand selectivity
1.5.4.3 Signal transduction 26
1.5.5 Distribution and known effects of the natriuretic peptides 27
1.5.6 Cytoprotective effects of ANP 28
1.6 Aspirin 29
1.6.1 History 29
1.6.2 Known effects 30
1.6.3 New therapeutic aspects and limits of the prostaglandin hypothesis 31
CONTENTS
1.7 The MAPK family 32
1.7.1 Extracellular signal-regulated kinase (ERK) 33
1.7.2 The c-jun N-terminal kinase (JNK) 34
1.7.3 p38 MAPK pathway 35
1.7.4 Regulation of the transcription factor AP-1 by MAP kinases 36
2 Materials and methods 39
2.1 Materials 41
2.2 Cell culture 42
2.2.1 Solutions2
2.2.2 Cell isolation of HUVEC 42
2.2.3 Cultivation of HUVEC 43
2.2.3.1 Passaging 43
2.2.3.2 Freezing, storage and thawing
2.3 Characterization of HUVEC by means of von Willebrand factor 44
2.3.1 Immunfluorescence 44
2.3.1.1 Solutions 44
2.3.1.2 Antibody
2.3.1.3 Experimental procedure 44
2.3.2 Flow cytometry 44
2.3.2.1 Solutions 44
2.3.2.2 Experimental procedure 45
2.4 Western blot analysis of proteins 46
2.4.1 Solutions and antibodies 46
2.4.2 Sample preparation 48
2.4.3 SDS polyacrylamide electrophoresis 49
2.4.4 Semidry blotting 49
2.4.5 Incubation with antibodies 50
2.5 cGMP measurement 50
2.5.1 Solutions 50
2.5.2 Experimental procedure1
2.6 Detection of mRNA 52
2.6.1 Isolation of RNA2
2.6.2 Determination of RNA concentration 53
2.6.3 Agarose gel electrophoresis 53
2.6.4 Reverse transcription 54