Inhalt

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   1 Abstract
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  2 Introduction
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  2.1 The influenza A virus
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   2.1.1 Pathogen-host interactions
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   2.1.2 General properties of the influenza A virus
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   2.1.3 The influenza A replication cycle
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   2.1.4 Influenza A and apoptosis
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  2.2 Annexin A2
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   2.2.1 The annexin protein family
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  2.2.2 Specific properties and functions of annexin A2
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   2.2.2.1 The annexin A2/p11 complex
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   2.2.2.2 Annexin A2 localisation
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   2.2.2.3 Binding of annexin A2 to membrane microdomains and the cytoskeleton
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   2.2.2.4 Posttranslational modifications of annexin A2
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   2.2.2.5 Annexin A2 and pathogens
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   2.3 Aim of this thesis
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  3 Materials and Methods
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  3.1 Materials
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   3.1.1 Virus strains
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   3.1.2 Bacterial strains
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   3.1.3 Eukaryotic cell lines
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   3.1.4 Plasmids
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   3.1.5 Oligonucleotides
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   3.1.6 Enzymes
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   3.1.7 Antibodies
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   3.1.8 Kits
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   3.1.9 Chemicals and reagents
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   3.1.10 Other materials and equipment
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  3.2 Cellbiological methods
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   3.2.1 Cell culture
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   3.2.2 Freezing cells
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   3.2.3 Thawing cells
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  3.2.4 Transient transfection of eukaryotic cells
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   3.2.4.1 Plasmid transfection
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   3.2.4.2 siRNA transfection
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   3.2.5 Immunofluorescence
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   3.2.6 Induction and microscopy of PMSs
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   3.2.7.1 Tissue culture for microscopy
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   3.2.7.2 Microscopy
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   3.2.7.3 Image analysis
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   3.2.8 Flow cytometry
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  3.3 Proteinbiochemical methods
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   3.3.1 Preparation of cellular protein extracts
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   3.3.2 Triton solubilisation
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   3.3.3 DRM flotation gradient
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   3.3.4 Protein quantification
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   3.3.5 Protein precipitation with TCA
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   3.3.6 SDS-PAGE
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   3.3.7 Western blotting
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   3.3.8 Stripping of blot membranes
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  3.4 Molecular biological methods
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  3.4.1 Bacterial methods
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   3.4.1.1 Cultivation and long term storage of bacteria
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   3.4.1.2 Preparation of CaCl2-competent E. coli DH5
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   3.4.1.3 Transformation of CaCl2 competent E. coli DH5
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  3.4.2 Methods of DNA preparation
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   3.4.2.1 Isolation and determination of plasmid DNA
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   3.4.2.2 Agarose gel electrophoresis
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   3.4.2.3 Elution of DNA from agarose gels
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  3.4.3 Enzymatic DNA methods
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   3.4.3.1 Restriction digests
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   3.4.3.2 Ligation of DNA fragments
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   3.4.3.3 PCR
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   3.4.3.4 Colony PCR
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   3.4.3.5 Sequencing of DNA
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   3.4.3.6 Real-time PCR
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   3.4.4 Cloning of mCherry labelled annexin A2 constructs
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  3.5 Virological methods
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   3.5.1 Viral infection of cells
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   3.5.2 Plaque assay
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  3.5.3 Influenza virus propagation
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   3.5.3.1 Propagation of FPV in MDCK
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   3.5.3.2 Propagation of PR8 in embryonated chicken eggs
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   3.5.4 Influenza virus purification
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  4 Results
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  4.1 Role of annexin A2 in IAV infection
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  4.1.1 Annexin A2 dependency of IAV replication
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   4.1.1.1 IAV replication in XM expressing cells
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   4.1.1.2 IAV replication in annexin A2 siRNA transfected cells
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   4.1.1.3 IAV replication in CMECs from an annexin A2 knockout mouse
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   4.1.2 Real-time PCR analysis of annexin A1 and annexin A2 after IAV infection
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   4.1.3 Hemagglutinin localisation in annexin A2 depleted cells
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   4.1.4 Annexin A2 and FPV localisation in infected cells
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   4.1.5 Annexin A2 incorporation in IAV particles
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   4.2 Involvement of annexin A2 in apoptotic processes
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  4.3 Association of annexin A2 with membrane microdomains
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   4.3.1 Cell fractionation by DRM flotation gradient
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   4.3.2 Influence of Tyr23 phosphorylation and PI(4,5)P2-binding on the association of annexin A2 with membrane microdomains
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   4.3.3 Influence of p11- and Ca2+- binding on the association of annexin A2 with membrane microdomains
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   4.3.4 Laurdan 2photon microscopy of annexin A2 expressing cells
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  5 Discussion
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   5.1 Annexin A2 in IAV infection
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   5.2 Annexin A2 in apoptotic processes
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  5.3 Annexin A2 association with membrane microdomains
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   5.3.1 DRM association of annexin A2 mutants
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   5.3.2 Localisation of annexin A2 in the GM1 phase of PMSs
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   5.3.3 Laurdan 2photon analysis of cells expressing annexin A2 variants
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   6 References
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  7 Appendix
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   7.1 Abbreviations
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   7.2 Supplementary data
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   Conference Presentations
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   Curriculum Vitae
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   Acknowledgements