The influence of receptor ligand interactions on gene expression and phagosome functions in mouse macrophages [Elektronische Ressource] / vorgelegt von Eik Hoffmann

Sujets

• Biologie

Informations

• Publié par: universitat_rostock
• Publié le: 01 janvier 2007
• Nombre de lectures: 20
• Langue: Deutsch
• Poids de l'ouvrage: 13 Mo

Extrait

The influence of receptor-ligand interactions on
gene expression and phagosome functions in
mouse macrophages
Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)
der Mathematisch-Naturwissenschaftlichen Fakultät
der Universität Rostock
urn:nbn:de:gbv:28-diss2008-0050-9
vorgelegt von
Eik Hoffmann
aus Siggelkow
Rostock, Dezember 2007





































Gutachter: PD Dr. Sergei A. Kuznetsov, Universität Rostock
Prof. Albert Haas, Universität Bonn


Datum des Wissenschaftlichen Kolloquiums: 10.03.2008






Für meinen Opa













“Das entspricht vollkommen meinen an Daphnien gewonnenen Angaben und lässt sich einfach
dahin formuliren, dass Riesenzellen Bacillen auffressen und sie dann ertödten. Dass daraus
noch nicht der definitive Sieg der Phagocyten unbedingt folgt, leuchtet von selbst ein.”

Elias Metschnikoff, 1883,
Archiv für pathologische Anatomie und Physiologie und für klinische Medicin
Table of contents
Summary 1
1 Introduction 2
1.1 Phagocytosis ...................................................................................................................................................................2
1.1.1 Phagocytic receptors...4
1.1.2 Phagocytic internalisation and signalling .......................................................................................9
1.1.3 Phagosome maturation..........................................................................................................................13
1.1.4 Degradation and antigen presentation .........................................................................................18
1.1.5 Alteration of phagosome functions by pathogens .................................................................19
1.2 Latex beads as a phagosome model.............................................................................................................21
1.3 Aims of the study........................................................................................................................................................23
2 Material and Methods 24
2.1 Cells and bacteria.......................24
2.1.1 Used material, reagents and instruments...................................................................................24
2.1.2 Cultivation of cells......24
2.1.3 Cultivation of bacteria .............................................................................................................................25
2.2 Preparation and coupling of latex beads to ligands ..............................................................................25
2.2.1 Used material and reagents................................................................................................................25
2.2.2 Coupling of latex beads to fish skin gelatin (FSG) ................................................................26
2.2.3 Coupling of latex beads to avidin.....................................................................................................26
2.2.4 Coupling of latex beads to IgG (whole molecule or Fc fragment)................................26
2.2.5 Coupling of latex beads to mouse complement......................................................................27
2.2.6 Coupling of latex beads to lipopolysaccharides (LPS) .......................................................27
2.2.7 Coupling of latex beads to mannan................................................................................................27
2.3 Isolation of phagosomes and cytosol ............................................................................................................28
2.3.1 Used material, reagents and instruments...................................................................................28
2.3.2 Isolation of latex bead containing phagosomes (LBP).......................................................28
2.3.3 Isolation of bacteria containing phagosomes...........................................................................29
2.3.4 Isolation of macrophage cytosol.......................................................................................................30
2.4 Isolation of G actin from rabbit muscle .........................................................................................................31
2.4.1 Used material, reagents and instruments31
2.4.2 Isolation of Gactin....................................................................................................................................31
2.5 In vitro actin binding assay...32
List of used lipids and inhibitors in binding assay studies ................................................34
2.6 Light microscopy studies........34
2.6.1 Fusion assay and analysis of phagocytic uptake rates......................................................34
List of markers and antibodies ..........................................................................................................35
2.6.2 Fixation and labelling of cells .............................................................................................................36
2.6.3 Conventional and confocal microscopy .......................................................................................36
2.7 Investigation of macrophage gene expression by microarray analysis....................................37
2.7.1 Used material, reagents and instruments...................................................................................37
Table of contents
2.7.2 RNA isolation, preparation of cRNA and hybridization.......................................................37
2.7.3 Data processing and comparison....................................................................................................38
2.8 Onedimensional SDSPAGE and Immunoblotting...............................................................................39
2.8.1 Used reagents and instruments .......................................................................................................39
2.8.2 1D SDSPAGE ............................................................................................................................................39
2.8.3 Immunoblotting............40
2.8.4 Quantification of stained gels, NC membranes and films.................................................40
2.9 Twodimensional SDSPAGE.............................................................................................................................40
2.9.1 Used material, reagents and instruments...................................................................................40
2.9.2 2D SDSPAGE.............41
2.10 Liquid chromatography and tandem mass spectroscopy (LC MS/MS) ....................................41
3 Results 43
3.1 Characterization of the binding of phagosomes to F actin
using an in vitro binding assay ..........................................................................................................................43
3.1.1 Binding of isolated phagosomes containing latex beads ..................................................44
3.1.2 Binding of isolated phagosomes containing pathogenic
and nonpathogenic bacteria..............................................................................................................50
3.1.3 Binding of isolated phagosomes containing latex beads
coupled to different ligands .................................................................................................................53
3.2 Investigation of phagocytosis of latex beads coupled to
different ligands in mouse macrophages .....................................................................................................57
3.2.1 Phagocytic uptake and fusion with lysosomal compartments........................................57
3.2.2 Analysis of receptor expression and tyrosine phosphorylation events
during phagocytosis .................................................................................................................................60
3.3 Influence of phagocytosis of ligand coupled latex beads on macrophage
gene expression..........................63
3.3.1 Global study of changes in gene expression determined by
microarray analysis...63
3.3.2 Upregulated genes common to all latex bead ligands........................................................65
3.3.3 Upregulated genes unique to each latex bead ligand.........................................................67
3.3.4 Analysis of altered gene expression on the protein level .................................................70
3.4 Proteomic analysis of phagosomes containing latex beads coupled to
different ligands...........................................................................................................................................................71
3.4.1 Two dimensional SDS PAGE of phagosomal proteins ......................................................71
3