Analysis of RhoH function in vivo [Elektronische Ressource] / Tatjana Dorn

Tatjana Dorn

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 23
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER
FAKULTÄT DER BIOLOGIE DER LUDWIG-MAXIMILIANS-
UNIVERSITÄT MÜNCHEN

















ANALYSIS OF RHOH
FUNCTION IN VIVO



















TATJANA DORN

MÜNCHEN 2007 Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig und ohne unerlaubte
Hilfe angefertigt habe. Ich habe weder anderweitig versucht, eine Dissertation einzureichen
oder eine Doktorprüfung durchzuführen, noch habe ich diese Dissertation oder Teile
derselben einer anderen Prüfungskommission vorgelegt.



Tatjana Dorn
München, 13.06.2007























Die vorliegende Arbeit wurde zwischen Januar 2004 und Mai 2007 unter Anleitung von Prof.
Dr. Cord Brakebusch am Max-Planck-Institut für Biochemie in Martinsried und an der
Universität von Kopenhagen durchgeführt.


Wesentliche Teile dieser Arbeit sind in folgender Publikation veröffentlicht:

Dorn, T., Kuhn, U., Bungartz, G., Stiller, S., Bauer, M., Ellwart, J., Peters, T.,
Scharffetter-Kochanek, K., Semmrich, M., Laschinger, M., Holzmann, B., Klinkert,
W.E.F., Straten, P.T., Køllgaard, T., Sixt, M. and Brakebusch, C. (2007). RhoH is
important for positive thymocyte selection and T cell receptor signaling. Blood 109: 2346-
2355.















Promotionsgesuch eingereicht am: 13.06.2007
Tag der mündlichen Prüfung am: 01.08.2007

Erster Gutachter: Prof. Dr. Charles N. David
Zweiter Gutachter: Prof. Dr. Michael Schleicher
Sondergutachter: Prof. Dr. Cord Brakebusch
Table of contents
Table of contents
Abbreviations............................................................................................................................ 8
1 Summary........................................................................................................................... 10
2 Introduction...................................................................................................................... 11
2.1 Family of Rho GTPases ........................................................................................................ 11
2.1.1 Overview of Rho GTPase family ..................................................................................... 11
2.1.2 Regulation of Rho GTPases ............................................................................................. 13
2.1.2.1 Regulation by GEFs, GAPs and GDIs.................................................................... 13
2.1.2.2 Regulation by lipid modification ............................................................................ 15
2.1.2.3 Regulation by gene expression ............................................................................... 15
2.1.2.4 Regulation by phosphorylation and ubiquitinylation.............................................. 16
2.1.2.5 Crosstalk between Rho GTPases 16
2.1.3 Biological function of Rho GTPases ................................................................................ 16
2.2 Hematopoiesis........................................................................................................................ 18
2.2.1 Development of myeloid and erythroid cells.................................................................... 18
2.2.2 ment of lymphoid cells........................................................................................ 19
2.2.2.1 B cells ..................................................................................................................... 19
2.2.2.2 NK cells 20
2.2.2.3 T cells...................................................................................................................... 20
2.2.2.4 Natural killer T cells ............................................................................................... 24
2.2.2.5 γδ T cells ................................................................................................................. 25
2.3 TCR signaling........................................................................................................................ 25
2.4 Role of Rho GTPases in hematopoiesis............................................................................... 29
2.4.1 Migration and adhesion .................................................................................................... 29
2.4.2 Differentiation, proliferation and survival........................................................................ 29
2.4.3 Phagocytosis and production of reactive oxygen species................................................. 31
2.5 RhoH function..... 31
2.6 Aim of the project ................................................................................................................. 35
3 Materials and methods..................................................................................................... 36
3.1 Materials ................................................................................................................................ 36
3.2 Generation of the RhoH-deficient mice .............................................................................. 36
3.3 Mice ........................................................................................................................................ 36
3.4 Generation of the RhoH antibody ....................................................................................... 37
3.4.1 Immunization of a rabbit with the RhoH peptide ............................................................. 37
3.4.2 ELISA of the rabbit anti-serum ........................................................................................ 38
3.5 Bacteria culture..................................................................................................................... 38
3.5.1 Bacteria strains.. 38
3.5.2 Media and conditions for bacteria culture ........................................................................ 38
3.6 Molecular biology methods .................................................................................................. 39
3.6.1 Primers.............................................................................................................................. 39
3.6.2 Determination of DNA concentration .............................................................................. 39
3.6.3 Agarose gel electrophoresis.............................................................................................. 39
3.6.4 DNA digestion.................................................................................................................. 40
3.6.5 DNA ligation .................................................................................................................... 40
4 Table of contents
3.6.6 Dephosphorylation of the vector ...................................................................................... 40
3.6.7 Extraction of DNA fragments from agarose gel............................................................... 41
3.6.8 Cloning of RhoH constructs ............................................................................................. 41
3.6.9 Mini-preparation of plasmid DNA ................................................................................... 42
3.6.10 Maxi-preparation of plasm 42
3.6.11 Polymerase chain reaction................................................................................................ 42
3.6.12 DNA sequencing .............................................................................................................. 43
3.6.13 Preparation of competent bacteria .................................................................................... 43
3.6.13.1 Preparation of heat shock competent DH5α........................................................... 43
3.6.13.2 Preparation of electrocompetent BL21(DE3) ......................................................... 44
3.6.14 E.coli transformation ........................................................................................................ 44
3.6.14.1 Heat shock............................................................................................................... 44
3.6.14.2 Electroporation 44
3.6.15 Genomic DNA extraction................................................................................................. 45
3.6.15.1 DNA extraction from tail pieces ............................................................................. 45
3.6.15.2 DNA isolation from tail pieces for Southern blot................................................... 45
3.6.16 Southern blotting .............................................................................................................. 45
3.6.16.1 Genomic digestion and DNA gel electrophoresis 46
3.6.16.2 Labeling of the DNA probe .................................................................................... 46
3.6.16.3 Hybridizatio