Die funktionelle Rolle von NFATc1 in der Kontrolle von Leben und Tod von Lymphozyten [Elektronische Ressource] / vorgelegt von Wen Chen

Wen Chen

Sujets

• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Aus dem Institut für Pathologie
der Universität Würzburg
Vorstand: Prof. Dr. H.K. Müller-Hermelink


Die Funktionelle Rolle von NFATc1 in der Kontrolle
von Leben und Tod von Lymphozyten






Inaugural- Dissertation
zur Erlangung der Doktorwürde der
Medizinischen Fakultät
der
Bayerischen Julius-Maximilians-Universität zu Würzburg


vorgelegt von
Wen Chen
HUBEI, PR. CHINA

Würzburg, Oktober 2007
Referent: Prof. Dr. E. Serfling
Korreferent: PD Dr. Ch. Kneitz
Dekan: Prof. Dr. M. Frosch










Tag der mündlichen Prüfung:






Der Promovend ist Arzt




- ii -
Chen,Wen
Molecular Pathology,
Institute of Pathology of Würzburg University
Josef-Schneider-Str. 2 Am Schwarzenberg 8
D-97080 Wuerzburg,Germany D-97078 Wuerzburg
Tel.: [phone] 0049 931 2705081
Fax: [phone] [e-mail]

Declaration


I submit the dissertation for Dr. med. I declare that the submitted dissertation
was completed by myself and no other. I have not used any sources or
materials other than those enclosed.
Moreover, I declare that the dissertation has not been submitted further in this
form or any other form, and has not been used to obtain any other equivalent
qualification or degree at any other organization.
Additionally, I have not applied for, nor will I attempt to apply for any other
degree or qualification in relation to this work.

This work was completed from July 2005 to August 2007 at the Department of
molecular Pathology, Institute for Pathology, Bayerische Julius-Maximilians-
University, Würzburg, under the supervision of Professor Dr. Edgar Serfling
(Faculty of Medicine).


Examiner: Prof. Dr. E. Serfling

Coexaminer: PD Dr. Ch. Kneitz



Würzburg Wen Chen



- iii - Table of Contents

Table of Contents


Declaration ..........................................................................................................................................iii
Table of Contents ................................................................................................................................iv
1. Introduction ......................................................................................................................................1
1.1 The life and death of lymphocytes..............................................................................................1
1.1.1 Maturation and activation of lymphocytes through crucial signaling pathways .................1
1.2 NFATc1: Structure and Function of its Isoforms .......................................................................4
1.2.1 NFAT family members........................................................................................................4
1.2.2 The structure of the NFATC1 gene and its 6 protein isoforms............................................4
1.3 Current problems: NFATc1function and the goal of this study .................................................7
2. MATERIALS AND METHODS .....................................................................................................9
2.1 Materials.....................................................................................................................................9
2.1.1 General materials.................................................................................................................9
2.1.2 Chemicals ............................................................................................................................9
2.1.3 Instruments ........................................................................................................................11
2.1.4 Kits ....................................................................................................................................12
2.1.5 Reagents ............................................................................................................................12
2.1.6 Antibodies..........................................................................................................................12
2.1.7 Oligonucleotides................................................................................................................12
2.1.8 Antibiotics .........................................................................................................................13
2.1.9 Solutions and buffers.........................................................................................................14
Mili-Q grade water was used to prepare all the solutions. The solutions were sterilized by......14
2.1.10 Growth medium...............................................................................................................19
2.1.11 Bacterial strains ...............................................................................................................21
2.1.12 Mammalian cell lines.......................................................................................................21
2.1.13 Mice.................................................................................................................................21
2.2 Methods ....................................................................................................................................22
2.2.1 Bacterial manipulation.......................................................................................................22
2.2.2 DNA methods....................................................................................................................23
2.2.3 Protein methods .................................................................................................................27
2.2.4 Cell culture methods..........................................................................................................32
2.2.5 FACS staining and apoptosis analysis...............................................................................33
2.2.6 ES cell culture and manipulation.......................................................................................33
3. Results ............................................................................................................................................38
3.1 Creation of Murine Embryonic Stem Cells Containing a Targeted NFATc1 Allele for its
Conditional Inactivation in Mice....................................................................................................38
3.1.1 The DNA construct for targeting the NFATc1 P1 promoter .............................................38
3.1.2 Electroporation of targeting vector DNA and double selection of recombinant ES cells..39
3.1.3 Preliminary screening of ES cells for site-specific recombination by long distance PCR 41
3.1.4 Confirmation of positive clones by Southern blot assays..................................................41
- iv - Table of Contents
3.1.5 Cre-recombinase expression in ES cells carrying a mutated Nfatc1 gene.........................43
3.1.6 Chimeras............................................................................................................................45
3.2 Analysis of NFATc1 Function in Chicken B cell Line DT40 ..................................................46
3.2.1 Disruption of the Nfatc1 gene in chicken DT40 B lymphocytes and re-expression of
human NFATc1/αΑ or αC..........................................................................................................46
3.2.2 Expression of human NFATc1/αΑ or human NFATc1/αC and apoptosis induction .......48
3.2.3 The pkc-θ promoter is a NFATc1/αA target .....................................................................50
3.2.4 NFATc1/αΑ cooperates with NF-κB to regulate transcription of Nfatc1 in lymphocytes56
4. Discussion ......................................................................................................................................60
4.1 The role of NFATc1 factors in the apoptosis induction of lymphocytes..................................60
4.1.1 NFATc1/αΑ protects lymphocytes against apoptosis .......................................................60
4.1.2 NFATc1/αΑ cooperates with NF-kB to determine the fate of lymphocytes upon
stimulation through immunoreceptors........................................................................................62
4.2 Experimental models for the conditional disruption of the Nfatc1 gene ..................................65
4.3 The significance of this study...................................................................................................66
Summary ............................................................................................................................................67
References ..........................................................................................................................................71
Abbreviations .................................................