In vitro and in vivo analysis of corticotropin releasing hormone receptor dependent signal transduction using microarray technology [Elektronische Ressource] / Cornelia Graf

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Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 35
• Langue: Deutsch
• Poids de l'ouvrage: 9 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Entwicklungsgenetik







In vitro and in vivo analysis of corticotropin-
releasing hormone receptor-dependent signal
transduction using microarray technology




Cornelia Graf



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. A. Gierl
Prüfer der Dissertation: 1. Univ.-Prof. Dr. W. Wurst
2. Univ.-Prof. Dr. D. Langosch

Die Dissertation wurde am 28.01.2009 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 25.06.2009 angenommen.




























TRUTH IS EVER TO BE FOUND IN SIMPLICITY,
AND NOT IN THE MULTIPLICITY AND CONFUSION OF THINGS.

- Sir Isaac Newton -

TABLE OF CONTENTS

Table of Contents
TABLE OF CONTENTS...........................................................................................................1
ABBREVIATIONS....................................................................................................................2
ABSTRACT..............................................................................................................................2
1. INTRODUCTION ..................................................................................................................2
1.1 STRESS AND THE HYPOTHALAMIC-PITUITARY-ADRENAL AXIS...............................................2
1.2 DYSREGULATION OF THE HPA AXIS AND PSYCHIATRIC DISORDERS .....................................2
1.3 CRH-LIKE PEPTIDES AND THEIR RECEPTORS .....................................................................2
1.4 G PROTEIN-COUPLED RECEPTOR-DEPENDENT SIGNALING CASCADES..................................2
1.5 ANIMAL MODELS OF ANXIETY AND DEPRESSION BASED ON THE CRH SYSTEM......................2
1.6 CRHR1 ANTAGONISTS AS NOVEL CLASS OF ANTIDEPRESSANTS .........................................2
1.7 MICROARRAY TECHNOLOGY ..............................................................................................2
1.7.1 The role of the transcriptome...................................................................................2
1.7.2 Microarray technology .............................................................................................2
1.7.3 Microarrays in psychiatric research .........................................................................2
2. OBJECTIVE OF THIS STUDY2
3. MATERIAL AND METHODS ...............................................................................................2
3.1 MATERIAL.........................................................................................................................2
3.1.1 Buffers and solutions2
3.1.2 Media for Escherichia coli (E. coli)...........................................................................2
3.1.3 Media and materials for cell culture .........................................................................2
3.1.4 PCR Primer..............................................................................................................2
3.1.5 Plasmids ..................................................................................................................2
3.1.6 Cell lines ..................................................................................................................2
3.1.7 Animals ....................................................................................................................2
3.2 METHODS.........................................................................................................................2
3.2.1 Microbiological methods ..........................................................................................2
3.2.2 Preparation and analysis of nucleic acids................................................................2
3.2.3 Polymerase chain reaction (PCR)............................................................................2
3.2.4 Cloning...2
3.2.5 Cell culture...............................................................................................................2
3.2.6 In vivo experiment....................................................................................................2
3.2.7 Expression analysis using microarray technology: in vitro.......................................2
3.2.8 Expression analysis using miovivo2
3.2.9 In situ hybridization ..................................................................................................2
3.2.10 Statistical analysis .................................................................................................2
4. RESULTS.............................................................................................................................2
1 TABLE OF CONTENTS

4.1 NEW TARGET GENES OF CRHR1-DEPENDENT SIGNALING PATHWAYS IN VITRO AND IN
VIVO.......................................................................................................................................2
4.1.1 Analysis of CRH-stimulated signaling pathways in AtT-20 cells..............................2
4.1.2 Analysis of stress-stimulated CRHR1-dependent signaling pathways in vivo .........2
4.1.3 Validation of regulation of selected candidate genes in vitro and in vivo.................2
4.2 COMMON REGULATED GENES IN VITRO AND IN VIVO............................................................2
4.3 FUNCTIONAL ANALYSIS OF CANDIDATE GENES....................................................................2
4.3.1 Expression of CRH receptors ..................................................................................2
4.3.2 Reporter assays: AtT-20 cells2
4.3.3 Reporter assays: HN9 cells .....................................................................................2
4.4 INTERACTION OF CRHR1- AND ERBB-ACTIVATED SIGNALING MECHANISMS.........................2
4.4.1 Expression of ErbB receptors2
4.4.2 Effects of ErbB3 overexpression on CRHR1-dependent signaling..........................2
4.5 ERRFI1 EXPRESSION IN VIVO: IN SITU HYBRIDIZATION .........................................................2
5. DISCUSSION .......................................................................................................................2
5.1 RELIABILITY OF NEW CANDIDATE GENES.............................................................................2
5.1.1 CRHR1 stimulation ..................................................................................................2
5.1.2 Microarray analyses.................................................................................................2
5.1.3 Selection of candidate genes in vitro .......................................................................2
5.1.4 Validation of differential gene expression ................................................................2
5.2 COMPARISON OF CRHR1-DEPENDENTLY REGULATED GENES IN VITRO AND IN VIVO ............2
5.3 POTENTIAL ROLE OF NEW CANDIDATES IN CRH/CRHR1-REGULATED SIGNAL
TRANSDUCTION IN CORTICOTROPE AND NEURONAL CELLS........................................................2
5.4 INTERACTION OF CRH/CRHR1- AND ERBB-STIMULATED SIGNALING CASCADES .................2
5.5 ERRFI1 EXPRESSION IN THE MURINE BRAIN ........................................................................2
6. CONCLUSION......................................................................................................................2
7. SUPPLEMENTARY DATA...................................................................................................2
8. REFERENCES...2
9. ACKNOWLEDGEMENTS ....................................................................................................2
10. CURRICULUM VITAE........................................................................................................2
11. LIST OF PUBLICATIONS..................................................................................................2
12. DECLARATION / ERKLÄRUNG........................................................................................2


2 ABBREVIATIONS

Abbreviations
AC adenylyl cyclase
ACTH adrenocorticotropin
ATP adenosine triphosphate
aRNA amplified ribonucleic acid
AVP arginine vasopressin
b.w. body weight
BLA basolateral amygdala
bp basepairs
BSA bovine serum albumin
cAMP cyclic adenosine monophosphate
cDNA copyDNA
CeA central amygdala
CNS central nervous system
CORT corticosterone
Cp crossing point
CRH cotropin-releasing hormone
CRHR CRH receptor
CRIB Cdc42/Rac-interaction and binding
CSF cerebrospinal fluid
Cy3 /