The role of Four-and-a-half LIM domain protein 2 in dendritic cell migration [Elektronische Ressource] / Katharina König. Mathematisch-Naturwissenschaftliche Fakultät

Katharina König

Sujets

• Biologie

Informations

• Publié par: rheinische_friedrich-wilhelms-universitat_bonn
• Publié le: 01 janvier 2011
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

The role of Four-and-a-half LIM domain protein 2
in dendritic cell migration







Dissertation

zur
Erlangung des Doktorgrades (Dr. rer. nat.)
der
Mathematisch-Naturwissenschaftlichen Fakultät
der
Rheinischen Friedrich-Wilhelms-Universität Bonn



vorgelegt von
Katharina König
aus
Hamm


Bonn
August 2011


With authorization of the Faculty of Mathematics and Natural Sciences of the
Rheinische Friedrich-Wilhelms-University of Bonn






















The study presented here was conducted at the Institute of Pathology of the
University Hospital Bonn.

First Referee (Thesis Advisor): Prof. Dr. Reinhard Büttner
Second Referee: Prof. Dr. Percy Knolle
Third Referee: Prof. Dr. Waldemar Kolanus
Fourth Referee: Prof. Dr. Dieter Fürst

Tag der Promotion: 20. Dezember 2011
Erscheinungsjahr: 2012



Diese Dissertation ist auf dem Hochschulschriftenserver der ULB Bonn
http://hss.ulb.uni-bonn.de/diss_online elektronisch publiziertTable of Contents

Table of contents

Abstract ..........................................................................................1
Zusammenfassung ........................................................................2
1. Introduction..............................................................................2
1.1 Cell migration....................................................................................... 2
1.1.1 The Cytoskeleton.......................................................................... 2
1.1.2 Components and structure of the actin cytoskeleton .................... 4
1.1.3 Pushing the cell forward ............................................................... 5
1.1.4 Small Rho GTPases ..................................................................... 7
1.2 The immune system............................................................................. 8
1.2.1 Innate versus adaptive immunity .................................................. 9
1.2.2 Dendritic cells ............................................................................. 10
1.2.3 Antigen uptake and processing .................................................. 10
1.2.4 Maturation................................................................................... 11
1.2.5 The migratory phenotype of DC.................................................. 14
1.2.6 Chemokine, CC Motif, receptor 7 (CCR7) .................................. 16
1.3 Four-and-a-half lim domains 2 (FHL2)............................................... 17
1.3.1 Structure and expression of FHL2 .............................................. 17
1.3.2 Function of FHL2 ........................................................................ 18
1.3.3 FHL2 affiliates with actin associated proteins and structures ..... 19
1.4 Sphigosine-1-Phosphate Receptors (S1PR)...................................... 20
1.5 Objective............................................................................................ 23

2. Materials and Methods ..........................................................24
2.1 Materials ............................................................................................ 24
2.1.1 Equipment .................................................................................. 24
2.1.2 Consumables.............................................................................. 26
2.1.3 Chemicals (liquid) ....................................................................... 27
2.1.4 Chemicals (solid) ........................................................................ 28
2.1.5 Reagents .................................................................................... 28
2.1.6 Antibody coated beads ............................................................... 29
2.1.7 Fluorochrome labeled ligands..................................................... 30 Table of Contents

2.1.8 Fluorochromes............................................................................ 30
2.1.9 Enzymes..................................................................................... 30
2.1.10 Proteins ..................................................................................... 30
2.1.11 Inhibitors ..................................................................................... 30
2.1.12 Cytokines and Chemokines ........................................................ 31
2.1.13 Cell culture supplies.................................................................... 31
2.1.14 Western Blot supplies................................................................. 31
2.1.15 Oligonucleotides ......................................................................... 32
2.1.16 Antibodies................................................................................... 32
2.1.17 Secondary antibodies ................................................................. 33
2.1.18 Kits.............................................................................................. 34
2.1.19 Media, Solutions and Buffer........................................................ 34
2.1.20 Software ..................................................................................... 36
2.1.21 Cell lines ..................................................................................... 37
2.1.22 Mouse lines................................................................................ 37
2.2 Methods............................................................................................. 37
2.2.1 Cell Culture ...................................................................................... 37
2.2.1.1 Primary cell isolation.................................................................. 37
2.2.1.2 Production of GMCSF ............................................................... 38
2.2.1.3 Determination of cell number..................................................... 38
2.2.1.4 Generation of bone marrow derived dendritic cells.................... 38
2.2.2 Molecular Biological methods ..................................................... 39
2.2.2.1 Mycoplasma PCR...................................................................... 39
2.2.2.2 Isolation of total RNA from tissue .............................................. 40
2.2.2.3 Isolation of total RNA from cell culture....................................... 41
2.2.2.4 RT-PCR..................................................................................... 41
2.2.2.5 Semi-quantitative RT-PCR ........................................................ 41
2.2.2.6 Gel Electrophoresis ................................................................... 42
2.2.2.7 Transfection of BMDC with siRNA............................................. 42
2.2.2.8 Quantitative Real-Time PCR ..................................................... 42
2.2.3 Protein Biochemical methods ..................................................... 43
2.2.3.1 Sample Preparation for western blot ......................................... 43
2.2.3.2 Protein determination by Bradford Assay .................................. 43
2.2.3.4 SDS-PAGE................................................................................ 43 Table of Contents

2.2.3.5 Western Blot.............................................................................. 44
2.2.3.6 Detection of FHL2 and β-actin................................................... 44
2.2.3.7 Immunoprecipitation .................................................................. 44
2.2.3.8 Immunofluorescence ................................................................. 45
2.2.4 Migration assays......................................................................... 46
2.2.4.1 Timelapse-video microscopy ..................................................... 46
2.2.4.2 Transwell Assay ........................................................................ 46
2.2.4.3 In vivo migration ........................................................................ 46
2.2.4.4 Determination of survival by MTT Assay ................................... 47
2.2.5 Flow cytometry............................................................................ 47
2.2.5.1 Staining of surface markers....................................................... 47
2.2.5.2 Intracellular cytokine staining .................................................... 48
2.2.5.3 Expression of CCR7 .................................................................. 48
2.2.5.4 Quantification of F-actin by flow cytometry ................................ 48
2.2.6 Rac1 Activation Assay................................................................ 49
2.2.7 Immunological methods.............................................................. 49
2.2.7.1 Antigen uptake........................................................................... 49
2.2.7.2 Purification of CD11c positive cells out of BMDC culture .......... 50
2.2.7.3 Purification of CD8 and CD4 cells .......................................