Gene expression analysis reveals novel functions of vitamin D and glucocorticoids [Elektronische Ressource] / Florian Graedler

Eleonora.Minina

Sujets

• Gesundheit

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 20
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

Institut für Experimentelle Genetik
GSF-Forschungzentrum für Umwelt und Gesundheit,
Neuherberg






Gene expression analysis reveals novel functions of
vitamin D and glucocorticoids



Florian Graedler






Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan
für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines


Doktors der Naturwissenschaften


genehmigten Dissertation.




Vorsitzender: Univ.-Prof. Dr. Heinrich H.D. Meyer


Prüfer der Dissertation: 1. Priv.-Doz. Dr. Jerzy Adamski
2. Univ.-Prof. Dr. Hans-Rudolf Fries


Die Dissertation wurde am 21.10.2004 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 31.01.2005 angenommen.

Index
Summary ................................................................................................ 7
Zusammenfassung................................................................................ 9
1. Introduction...................................................................................... 11
1.1. Gene expression profiling: Technology development of cDNA microarrays.................... 11
1.1.1 Functional genomics ...........................................................................................................11
1.1.2 Modern analytical methods in functional genomics ............................................................11
1.1.3. DNA microarray technology ...............................................................................................12
1.1.3.1 The principle .............................................................................................................12
1.1.3.2 Oligonucleotide versus cDNA arrays........................................................................13
1.1.3.3 Protocol development and technical sources of interference...................................14
1.1.3.4 Processing microarray data......................................................................................15
1.1.3.5 Data interpretation: from single genes to pathways .................................................18
1.1.3.6 Applications of microarray technology and outlook ..................................................19
1.1.3.7 Microarray Technology as a tool in Molecular Endocrinology..................................20
1.2 Vitamin D.................................................................................................................................... 21
1.2.1 Vitamin D synthesis and metabolism ..................................................................................21
1.2.2 Functions of the vitamin D system ......................................................................................24
1.2.2.1 Molecular mechanism of vitamin D action................................................................24
1.2.2.2 Classical functions of vitamin D: Calcium / Phosphate homeostasis.......................25
1.2.2.3 Convergent actions of VDR and RXR signalling ......................................................27
1.2.2.4 Novel implications of vitamin D action......................................................................27
1.3 Functions of vitamin D-inducible Cyr 61 in human fetal osteoblasts (hfOB) ..................... 29
1.4 Glucocorticoid treatment of insulin-secreting pancreatic β-cells ....................................... 30
1.5 Aims of the project ................................................................................................................... 31
2. Establishing gene expression profiling with cDNA microarrays;
experiments with cell culture systems .............................................. 32
2.1 Establishing the Technology................................................................................................... 32
2
Index

2.1.1 Microarray production..........................................................................................................32
2.1.1.1 Clone selection .........................................................................................................33
2.1.1.2 Printing microarrays..................................................................................................33
2.1.2 Sample preparation .............................................................................................................39
2.1.2.1 Isolation and purification of RNA ..............................................................................39
2.1.2.2 RNA transcription and labeling .................................................................................39
2.1.3 Testing the method with cell culture systems: Proof of principle ........................................40
2.2 Gene expression profiling of human fetal osteoblasts after treatment with Cyr61 ........... 45
2.3 The mouse cDNA array ............................................................................................................ 50
2.4 Expression profiling of glucocorticoid-treated pancreatic β−cells ..................................... 52
2.4.1 Experimental set-up ............................................................................................................52
2.4.2 Data analysis.......................................................................................................................52
2.4.3 Genes, differentially expressed after glucocorticoid treatment: Discussion........................54
3. Gene expression profiling of vitamin D receptor knockout
(VDRKO) against wild type mouse kidney ........................................ 57
3.1 Gene expression profiling with cDNA microarrays............................................................... 57
3.1.1 Gene Expression profiles of kidneys from male ND mice: VDRKO compared to WT ........57
3.1.2 Gender and diet specific influences on gene expression....................................................62
3.1.2.1 Experimental Design.................................................................................................62
3.1.2.2 Data processing of single experiments.....................................................................64
3.1.2.3 Different approaches of gene clustering reveal gender-specific effects...................65
3.1.2.3.1 Hierarchical clustering ...........................................................................................65
3.1.2.3.2 Significance analysis of microarray data (SAM)....................................................69
3.1.3 Genes, differentially expressed in kidneys of male RD mice: VDRKO compared to WT ...70
3.2 Validation by quantitative PCR and interpretation................................................................ 73
3.2.1 Genes involed in ion transport and calcium homeostasis...................................................74
3.2.2 Nuclear hormone receptors and steorid/cholesterol metabolism........................................78
3.2.3 Cross-talk between nuclear hormone receptors .................................................................83
3.2.4 Lipid metabolism and energy supply...................................................................................84
3.2.5 Blood pressure control ........................................................................................................90
3 Index

3.2.6 Cell growth, proliferation, apoptosis and cancer .................................................................93
4. Expression analysis of VDRKO and WT mouse heart.................. 97
4.1 Microarray experiments: Experimental set-up....................................................................... 97
4.2 Analysis of microarray data..................................................................................................... 98
4.3 Annotation of candidate genes ............................................................................................. 107
4.4 Confirmation of candidate genes by qPCR.......................................................................... 109
4.5 Discussion of gene expression profiles and assignment of candidate genes to
physiological functions and pathways....................................................................................... 112
4.5.1 Candidate genes involved in the immune system.............................................................113
4.5.2 Lipid metabolism and energy mobilization ........................................................................115
4.5.3 Ion transport processes.....................................................................................................118
4.5.4 Marker genes for hypertension and cardiac hypertrophy..................................................119
4.5.5 Genes induced in ischemic insult and cardiac hypoxia.....................................................121
5. Conclusions ................................................................................... 123
5.1 Establishing microarray technology...................................................................