Ratten-iNKT-Zellen [Elektronische Ressource] : Phänotyp und Funktion = Rat iNKT Cells / submitted by Elisa Monzón Casanova

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 18 Mo

Extrait

Ratten iNKT Zellen: Phänotyp und Funktion

Rat iNKT Cells: Phenotype and Function



Doctoral thesis for a doctoral degree at the Graduate School of
Life Sciences, Julius-Maximilians-Universität Würzburg,
Section of Infection and Immunity

Submitted by
Elisa Monzón Casanova
from
Huesca, Spain

Würzburg, 2010
















Submitted on: ………………………………………………………………………
Office stamp




Members of the Promotionskomitee:
Chairperson: Prof. Dr. Thomas Hünig
Primary Supervisor: Prof. Dr. Thomas Herrmann
Supervisor (Second): Prof. Dr. Thomas Müller
Supervisor (Third): Prof. Dr. Thomas Dandekar



Date of Public Defence: …………………………………………….…………
Date of receipt of Certificates: ……………………………………………….























A mi madre y a mi hermano

Lo orgullosos que están de mí es la mejor recompensa de todas















ACKNOWLEDGMENTS

Foremost I am very thankful to Prof. Dr. Thomas Herrmann for giving me the
opportunity to perform my PhD thesis under his supervision. I am very grateful for his
support, his encouragement, the freedom he gave me and the stimulating discussions we
had. All this was fundamental for overcoming the challenges which appeared during the
course of this work.

I am also deeply grateful to Prof. Dr. Thomas Hünig for all his support and for the
possibility to be part of the Graduate College Immunomodulation. The ongoing
exchange of knowledge between Graduate Students and Project Leaders was very
inspiring, constructive and joyful.

I also want to thank Prof. Dr. Birte Steiniger for her tremendous help in addressing the
distribution of CD1d and the members of my Supervisory Committee: Prof. Dr. Thomas
Müller and Prof. Dr. Thomas Dandekar for their support and suggestions.

I would also like to express my gratitude to the members of the Herrmann’s laboratory.
Especially to Ingrid Müller, from whom I learnt so much, and also to Lisa Starick; their
work is a big part of this thesis. Thanks as well to Jianqiang, Stefanie, Martina,
Mohindar, Daniel, Ronald, Tian and all the people who I met during the last six years at
the institute. I always experienced a very nice working atmosphere and I especially
appreciate the patience that everyone had with me when I had a “rat day”.

Finally, I want to use the lasts sentences to thank my family and friends for their love
and support. Unfortunately, there is no enough space to write every name. Nonetheless,
I want to especially thank Maria and Ivan, for their friendship and pointing the North
when cannot find it; Ronald, for his support, understanding and company and my
mother and my brother, simply, this would have not been possible without them.
TABLE OF CONTENTS

LIST OF FIGURES.......................................................................................................... 6
LIST OF TABLE.S............................................................................................................ 7
ABSTRACT...................................................................................................................... 8
ZUSAMMENFASSUNG.............................................................................................. 11
1 INTRODUCTION ................................................................................................. 14
1.1 MHC-restricted and non-MHC-restricted T cells.................................................. 15
1.2 CD1d...................................................................................................................... 17
1.2.1 CD1 family........................................................................................................... 17
1.2.2 Expression of CD1d ............................................................................................... 18
1.2.3 CD1d function: lipid antigen presenting molecule ........................................................ 19
1.3 CD1d-restricted T cells.......................................................................................... 21
1.4 Recognition of α-Gal-CD1d by semi-invariant TCRs........................................... 22
1.5 iNKT cell development ......................................................................................... 23
1.6 iNKT cell function................................................................................................. 24
1.7 iNKT cells in the rat .............................................................................................. 25
2 MATERIALS AND METHODS....................................................................... 28
2.1 Materials ................................................................................................................ 28
2.1.1 Chemical reagents.................................................................................................. 28
2.1.2 Media, buffers, solutions ......................................................................................... 30
2.1.3 Cell lines.............................................................................................................. 32
2.1.4 Animals ............................................................................................................... 33
2.1.5 Vectors ................................................................................................................ 33
2.1.6 Oligonucleotides.................................................................................................... 35
2.1.7 Antibodies and secondary reagents ............................................................................ 39
2.1.8 Enzymes and inhibitors ........................................................................................... 40
2.1.9 Commercially available kits and mixtures................................................................... 40
2.1.10 Consumables ....................................................................................................... 41
2.2 Methods ................................................................................................................. 42
2.2.1 Routine molecular biology methods........................................................................... 42
2.2.2 Cloning................................................................................................................ 48
1 2.2.2.1 Cloning of Mus musculus CD1d2 and Mus spretus CD1d1 into pczCFG5 IEGN........... 48
2.2.2.2 Cloning of rat CD1d cDNA into pczCFG5 IZ......................................................... 48
2.2.2.3 Cloning of rat β2-microglobulin into the retroviral expression vector pczCFG5 IZ ........ 48
2.2.2.4 Cloning of mouse CD1d into pXIg....................................................................... 48
2.2.2.5 Cloning of rat CD1d into pXIg............................................................................ 49
2.2.2.6 Cloning of rat β2-microglobulin-CD1d fusion protein into the pFUSE-hIgG1-Fc2 vector 49
b
2.2.2.7 Cloning of rat CD1d into the pMT/BioHis-IA α vector ............................................ 50
2.2.2.8 Cloning of rat β2-microglobulin-CD1d into the pcDNA3.1/V5 His A Bio vector .......... 50
b2.2.2.9 Cloning of rat β2-microglobulin-CD1d into the pMT/BiP/His/Strep TagIII-IA α vector . 50
2.2.2.10 Cloning of AV14-containing TCRα chains........................................................... 51
2.2.3 Production and purification of CD1d oligomers ........................................................... 51
2.2.3.1 Mouse and rat CD1d-IgG dimers ......................................................................... 51
2.2.3.2 Rat β2-microglobulin CD1d human Fc dimers........................................................ 51
2.2.3.3 Rat CD1d production in SC2 drosophila cells......................................................... 52
2.2.3.4 Rat β2-microglobulin-CD1d production in 293T human cells.................................... 52
2.2.3.5 Rat β2-microglobulin-CD1d linked to a streptag production by SC2 drosophila cells ..... 53
2.2.3.6 Protein G affinity chromatography....................................................................... 53
2.2.3.7 Photometric determination of protein concentration................................................. 53
2.2.3.8 Coomassie Blue staining.................................................................................... 54
2.2.3.9 Specific biotinylation ........................................................................................ 54
2.2.3.10 Lipid loading into CD1d molecules .................................................................... 54
2.2.3.11 Oligomerization of rat β2-microglobulin-CD1d linked to a streptag .......................... 54
2.2.