The role of NF-B and C/EBP factors during pathogen-mediated activation of bovine interleukin 8 and beta-defensin in mammary epithelial cells Inaugural dissertation for the academic degree Doctor rerum naturalium of Mathematisch-Naturwissenschaftlichen Fakultät Universität Rostock By Shuzhen Liu (M. Sc.), born on March-02-1972, in Shanxi Province, China From the Forschungsinstitut für die Biologie landwirtschaftlicher Nutztiere in Dummerstorf Rostock 2009 URN: urn:nbn:de:gbv:28-diss2009-0185-5 Dean: Prof. Dr. Hendrik Schubert Reviewers: 1. Prof. Dr. Hans-Martin Seyfert Research Unit molecular biology, Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany 2. Prof. Dr. Dieter G. Weiss Division of animal Physiology, Institute of Cell Biology and Biosystems Technology, University of Rostock, Albert-Einstein-Strasse 3, 18059 Rostock, Germany 3. PD Dr. Ulrike Gimsa Research Unit Behavioural Physiology, Research Institute for the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, D-18196 Dummerstorf, Germany Date of defense: October 19th, 2009 Table of Contents TABLE OF CONTENTS 1. INTRODUCTION..................................................................................................................1 1.1 Mastitis as a challenge in general immunology..................................................................1 1.2 Innate immunity of the bovine mammary gland ................................................................2 1.3 Toll-like receptors (TLRs): main receptors perceiving the pathogen presence..................3 1.4 Interleukin 8 (IL-8): a chemokine ......................................................................................4 1.4.1 Inducible expression of IL-8 .......................................................................................4 1.4.2 Molecular regulation of the human IL-8 expression ...................................................5 1.5 Antimicrobial peptides: -defensins...................................................................................6 1.5.1 Classification and structures of defensins ...................................................................6 1.5.2-defensin in mammal.................................................................................................6 1.5.3 Inducible expression of -defensins............................................................................7 1.5.4 Roles of -defensins in mammalian immunity............................................................7 1.5.5 Regulation of -defensins expression..........................................................................8 1.6 Nuclear factors (NF-B).....................................................................................................8 1.6.1 Inhibition of NF-B.....................................................................................................9 1.6.2 Activation of NF-B....................................................................................................9 1.6.3 Phosphorylation and acetylation of NF-B...............................................................10 1.6.4 Transcriptional coactivators and interactive proteins of NF-B ...............................11 1.7 CCAAT-enhancer binding proteins (C/EBP)...................................................................11 1.7.1 Role of C/EBP in inflammatory and immune response ...........................................12 1.7.2 Autoregulation and phosphorylation of the C/EBP family factors............................13 1.7.3 Interaction of C/EBP with NF-B.............................................................................13 1.8 Chromatin remodeling......................................................................................................14 1.8.1 Overview of chromatin remodeling...........................................................................14 1.8.2 The link between acetylation and transcription.........................................................14 1.9 DNA Methylation.............................................................................................................15 1.9.1 Methods for DNA methylation analysis....................................................................15 1.9.2 Roles of DNA methylation........................................................................................16 1.10 Goals:..............................................................................................................................17 2. MATERIALS AND METHODS .........................................................................................18 i Table of Contents 2.1 Materials ...........................................................................................................................18 2.1.1 Instruments ................................................................................................................18 2.1.2 Reagents ....................................................................................................................18 2.1.3 Kits ............................................................................................................................19 2.1.4 Strains and plasmids ..................................................................................................19 2.1.5 Enzymes ....................................................................................................................19 2.1.6 Antibodies..................................................................................................................20 2.1.7 Tissue samples...........................................................................................................20 2.1.8 Cells and cell lines.....................................................................................................20 2.2 Molecular cloning methods ..............................................................................................21 2.2.1 Preparation of plasmid DNA .....................................................................................21 2.2.1.1 Mini-or midi-preparation of plasmid DNA.........................................................21 2.2.1.2 Preparation of endotoxin free plasmid DNA by Qiagen kit................................21 2.2.2 RNA techniques.........................................................................................................22 2.2.2.1 RNA extraction from cell culture and frozen tissue............................................22 2.2.2.2 RNA electrophoresis ...........................................................................................22 2.2.3 Polymerase Chain Reaction (PCR)............................................................................23 2.2.3.1 Standard PCR and high fidelity PCR ..................................................................23 2.2.3.2 Reverse transcription PCR (RT-PCR).................................................................23 2.2.3.3 Real-time PCR.....................................................................................................24 2.2.3.3.1 Preparation of standard curve...........................................................................25 2.2.3.3.2 Real-time PCR programs..................................................................................25 2.2.4 Plasmid construction techniques ...............................................................................26 2.2.4.1 DNA electrophoresis ...........................................................................................26 2.2.4.2 Purification of DNA from agarose gel ................................................................26 2.2.4.3 Restriction digestion of DNA fragment ..............................................................26 2.2.4.4 Preparation of vector for cloning.........................................................................27 2.2.4.5 Ligation…….. .....................................................................................................27 2.2.5 Transformation ..........................................................................................................27 2.2.5.1 Preparation of competent cells ............................................................................27 2.2.5.2 Transformation procedure ...................................................................................28 2.2.6 PCR mediated mutagenesis and deletion .................................................................28 2.2.7 Rapid Amplification of 5 cDNA Ends (5 RACE) ...................................................29 2.3 Protein techniques ............................................................................................................30 ii Table of Contents 2.3.1 Determination of total protein concentration (Bradford assay).................................30 2.3.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)..............30 2.3.3 Western blot...............................................................................................................31 2.4 Mammalian cell culture and reporter gene assay .............................................................32 2.4.1 Cell types and media .................................................................................................32 2.4.2 Cell culture and cryopreservation..............................................................................32 2.4.2.1 Cryopreservation of cells.....................................................................................32