Investigations of measles virus regulation on activation and function of antigen presenting cells [Elektronische Ressource] / Yoana Shishkova

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Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 38
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Investigations of Measles virus regulation
on activation and function
of antigen presenting cells




Thesis submitted to

the Julius-Maximilians-University

Würzburg, Germany


towards fulfillment of the requirements for the degree of
Dr. rer. nat.
Yoana Shishkova
Bulgaria, Kazanlak


Würzburg, Germany
18.04.2008




Eingereicht am:

18.04.2008 .............................................................................................................................





Mitglieder der Promotionskommission:

...............................................................................................................................................

Vorsitzender: Prof. Dr. G. Krohne (vom Dekan beauftragt)

...............................................................................................................................................

Gutachter : Prof. Dr. S. Schneider-Schaulies

...............................................................................................................................................

Gutachter: Prof. Dr. T. Raabe

...............................................................................................................................................







Tag des Promotionskolloquiums: 02.07.2008

...............................................................................................................................................




Doktorurkunde ausgehändigt am:

...............................................................................................................................................



Declaration





I declare that the submitted dissertation was completed by myself and none other, and 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 and has not been used for
obtaining any other equivalent qualification in any other organization. Additionally, other
than this degree I have not applied or will not attempt to apply for any other degree, title or
qualification in relation to this work.










Würzburg 18.04.2008 Shishkova Y.
Yoana Shishkova





Die vorliegende Arbeit wurde in der Zeit vom 01.Oktober 2003 bis 31. Januar 2007
am Institut für Virologie und Immunbiologie der Bayerischen Julius-Maximilians-Universität
Würzburg unter der Anleitung von Professor Sibylle Schneider-Schaulies und
Herr Prof. Dr. Thomas Raabe gemacht.



Acknowledgement



Here, I thank my tutor,
professor Sibylle Schneider-Schaulies, for directing my research project and helpful
suggestions on my manuscript
professor Georg Krohne, Dr. Elita Avota for scientific discussion,
Dr. Harms for his help with cell imaging,
Elisabeth Meier-Natus for the technical help
and Steffen Schwab for his german translation of the summary,
and our entire group members for their kind help in both lab work and daily life.
Thank you !
Contents
1. Introduction ...................................................................................................................................................... 1
1.1. Measles virus .................................................................................................................................. ....... .1...........
1.1.1 . Pathogenesis ............................................................................. . 1.....................
1.1.2 . MV organization .......................................................................... .................... 3
1.2 . Dendritic cells ................................................................................. .... .5..........
1.2.1 . Dendritic cell biology: differentiation, migraantdio nf unctions .................................. ......... 5
1.2.2 . MV and DCs ............................................................................ . 7.....................
1.2.2.1. Infection of dendritic cells b.y. ..M.V.. .................................................. . 7.....................
1.2.2.2. Activation of DCs by MV ........................................................................................................ .8..................
1.2.2.3. Impairment of DC function by .M..V. .................................................... ................... 9
1.2.2.4. MV induced immunosupprassion ....................................................... ................ 10
1.3. DC/T Cell interaction ..................................................................................................................... .1.1................
1.3.1 . Adhesion, signaling and integrins ........................................................ ............... 11
1.3.2 . Cytoskeletal rearrangements in DCs and T .c.e.ll.s. ........................................... .......... 14
1.3.3 . Immunological synapse (IS) ............................................................... ............... 15
1.3.3.1. Organisation of IS .................................................................................................................. . .1.5..............
1.3.3.2. Different types of IS ................................................................. ..1.6..............
1.4. Aim of the work ............................................................................ ..1.8..............
2. Materials ..........................................................................................................................................................19
2.1 . Cell lines ...................................................................................... ... .1.9........
2.3 . Viruses .............................................................................................................................................. ..... .1.9........
2.4. Cell culture media ............................................................................ .2..0..............
2.5 . Solutions and buffers used for cell separ.a.t.io..n. ................................................ ............... 22
2.6 . Antibodies for purity determination of the isocelallt edpopulations.................................................... ......... 23
2.7 . Immunofluorescence ................................................................................................................ .2. .4...................
2.7.1 . Antibodies for immunofluorescence ........................................................ ............ 24
2.7.2 . FACS buffers ...........................................................................2..7. ...................
2.8 . In situ-immunlocalisation ........................................................................................................... .2. .7..................
2.8.1 . Buffers for in situ-immunolocalisation ..................................................... ............. 27
2.8.2 . Reagents for in situ-immunolocalisation ................................................... ............ 28
2.9 . FN/ICAM-1 adhesion assays .................................................................... ................... 28
2.9.1 . Buffers for adhesion assays .............................................................. ................ 28
2.9.2 . Reagents for adhesion assays ............................................................ ............... 28
2.10 . Kit for fast activated cell ELISA-based adhseasyio n. ..a.s............................................ .............. 28
2.11 . Cell lysis ...................................................................................... .... .2..9......
2.12 . Determination of protein concentration......................................................... ............... 29
2.13 . Western Immunoblotting of proteins .......................................................... ................ 29
2.13.1 . Buffers for WB .........................................................................9. ................... 2
2.13.2 . Antibodies for WB ....................................................................... .................. 30
2.13.3 . SDS-polyacrylamide gel electrophoresis (SDS-PA.G.E.). ......................................... ........ 31
2.14. Kinase assay ................................................................................................................................... ... .3.2............
2.14.1 . Kinase buffer............................................................................3.2.. ...................
2.14.2 . Washing solutions ........................................................................ .................. 33
2.15 . Plasmids .......................................................................................................................................... .... .3.3..........
2.15.1 . Human MHCII-GFP plasmid ................................................................ .............. 33
2.15.2 . Human GTP-ases –