Investigation of the role of {PKCα [PKC-alpha] for influenza A virus-induced signalling and of the inhibitory effect of verapamil on virus replication [Elektronische Ressource] / vorgelegt von Mohammad Intakhab Alam

Sujets

• Gesundheit

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2008
• Nombre de lectures: 32
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Aus dem Institut für Medizinische Virologie
der Justus-Liebig-Universität Gießen
Betreuer: Prof . Dr. Stephan Pleschka



Investigation of the Role of PKC for Influenza A Virus-
Induced Signalling and of the Inhibitory Effect of
Verapamil on Virus Replication


INAUGURAL-DISSERTATION
zur
Erlangung des Doktorgrades
der Naturwissenschaftlichen Fachbereiche
der Justus-Liebig-Universität Gießen
Dr. rer. nat.

vorgelegt von
Mohammad Intakhab Alam
New Delhi, India


Gießen, Germany, November 2007







Mit Genehmigung des Fachbereichs Biologie
der Justus-Liebig-Universität Gießen




Dekan: Prof. Peter R. Schreiner


1. Gutachter: Prof. Dr. Stephan Pleschka
Institute of Medical Virology
Justus-Liebig-University Giessen

2. Gutachter: Prof. Dr. Trinad Chakraborty
Institute for Medical Microbiology
Justus-Liebig-University Giessen

3. Gutachter: Prof. Dr. Albrecht Bindereif
Institute for Biochemistry
Justus-Liebig-University Giessen
i
Table of Contents
1. Introduction 1
1.1 The Causative Agent............................................................................................1
1.1.1 Influenza .....................................................................................................1
1.1.2 History of Influenza.....................................................................................1
1.1.3 Human influenza and transmission ..............................................................1
1.1.4 Clinical symptoms of influenza virus infection ............................................3
1.1.5 Different types of influenza viruses .............................................................3
1.2 Influenza A virus .................................................................................................3
1.2.1 Morphology and genome structure...............................................................3
1.2.2 Genome replication and propagation............................................................9
1.2.3 Antigenic variation of influenza virus infection (antigenic shift and drift)..15
1.3 Avian influenza viruses......................................................................................16
1.3.1 History of avian influenza .........................................................................16
1.3.2 Direct transmission....................................................................................17
1.4 The progress of reverse genetic systems for influenza viruses ............................18
1.5 Influenza vaccines and antivirals........................................................................19
1.6 Signal transduction and influenza viruses...........................................................20
1.6.1 Mechanisms of intracellular signal transduction.........................................20
1.6.2 The Raf/MEK/ERK pathway (MAPK signaling cascade) ..........................22
1.6.3 Virus-induced Raf/MEK/ERK (MAPK) signaling cascade is essential
host function for influenza A virus propagation25
1.6.4 Role of virus induced calcium dependent PKC signal transmission..........26
1.6.5 Protein kinase C as a therapeutic target......................................................26
1.6.6 Calcium channel blocker (Verapamil)........................................................27
1.7 Aim of the project..............................................................................................27
2. Materials and Methods 29
2.1 Materials ............................................................................................................29
2.1.1 Chemicals and reagents.............................................................................29
2.1.2 Instruments................................................................................................30
2.1.3 Enzymes and enzyme inhibitor ..................................................................31
2.1.4 Nucleotides and reaction buffer .................................................................31
2.1.5 Plasmids....................................................................................................32
2.1.6 Kits32
2.1.7 Solutions for plasmid DNA isolation .........................................................32
2.1.8 Materials for cell culture............................................................................33
ii
2.1.9 Methylcellulose (MC) media, 100 ml (1.75%) ...........................................33
2.1.10 Preparation of all kind of buffers..............................................................33
2.1.11 Preparation of TLB buffer........................................................................33
2.1.12 Lysis Buffer.............................................................................................34
2.1.13 5x SDS-PAGE buffer ..............................................................................34
2.1.14 Transfer buffer (Semi-dry).......................................................................34
2.1.15 10x TBS (Tris Buffer Saline)...................................................................34
2.1.16 1x TBST buffer .......................................................................................34
2.1.17 Blocking buffer........................................................................................35
2.1.18 SDS-PAGE buffer and gel35
2.1.19 Primer extension sequencing gel (6%) .....................................................36
2.1.20 Materials for cell viability (MTT) test......................................................36
2.2 E. coli strains and cell lines and virus strains......................................................36
2.3 Agarose gel electrophoresis ...............................................................................37
2.4 Monoclonal and polyclonal antibodies...............................................................37
2.5 Buffers for Immunoflorescence assay ................................................................38
2.6 Mowiol DABCO ...............................................................................................38
2.7 Stimulators and Inhibitors..................................................................................39
2.8 Other materials ..................................................................................................39
2.9 Methods .............................................................................................................39
2.9.1 Maintenance of cell culture .......................................................................39
2.9.2 Storage and thawing cell cultures ..............................................................39
2.9.3 Infection of cells .......................................................................................40
2.9.4 Preparation of cell lysates for Western blot analysis...................................41
2.9.5 Western blotting (Semi-dry) ......................................................................41
2.9.5.1 Measurement of protein concentration (Bio-Rad protein assay)...............41
2.9.5.2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)...........................41
2.9.5.3 Transfer membrane in "Semi-dry" electroblotter.....................................42
2.9.5.4 Immunodetection of proteins on PVDF-Membrane.................................42
2.9.5.5 Enhanced Chemiluminescence (ECL) reaction........................................43
2.9.5.6 Striping of bound antibodies from the PVDF Membrane.........................43
2.9.5.7 Quantification of protein bands...............................................................43
2.9.6 Detection of influenza A viral proteins ......................................................44
2.9.6.1 Western blot analysis of viral proteins ....................................................45
2.9.7 Immunocomplex kinase assay (ICA) .........................................................45
2.9.8 Cell viability test (MTT Assay) .................................................................46
2.9.9 Analysis of infectious virus titres47
2.9.9.1 Immunohistochemistry (Focus forming units, FFU)................................47
2.9.10 Immunofluorescence assay (IFA)/Laser scanning confocal microscopy ...49 iii
2.9.11 Preparation of plasmid DNA....................................................................49
2.9.11.1 Measurement of plasmid DNA concentration........................................51
2.9.11.2 Restriction endonuclease digestion........................................................51
2.9.11.3 Agarose gel electrophoresis ..................................................................51
2.9.12 DNA-transfection of eucaryotic cell cultures............................................51
2.9.12.1 Transfection of adherent 293T cells ......................................................51
2.9.12.2 Transfection of suspended MDCK cells ................................................52
2.9.13 Primer Extension .....................................................