Department of Internal Medicine I, University Hospital Ulm Prof. Dr. Seufferlein The Role of Inherited Keratin Variants in Liver Disease Development Dissertation presented to the Medical Faculty of Ulm University to obtain the degree Doctor of Human Biology Özlem Kücükoglu From Gaziantep, Turkey 2013 Current Dean: Prof. Dr. Thomas Wirth Thesis reviewers: 1st reviewer: PD. Dr. Pavel Strnad 2nd reviewer: Prof. Dr. biol. hum. Uwe Knippschild Date of doctorate awarded: May 3, 2013 Parts of this dissertation have been published in the following journal article: Strnad P*, Kucukoglu O*, Lunova M, Guldiken N, Lienau TC, Stickel F, Omary MB., “Non-Coding Keratin Variants Associate With Liver Fibrosis Progression in Patients With Hemochromatosis” PloS One 2012;7(3). *These authors contributed equally to this work TABLE OF CONTENTS ABBREVIATIONS............................................................................................................IV 1. INTRODUCTION .......................................................................................................... 1 1.1. THE COMPOSITION AND FUNCTION OF CYTOSKELETON ......................................... 1 1.2. INTERMEDIATE FILAMENTS AND THEIR DISEASE ASSOCIATION ............................. 2 1.3. KERATINS............................................................................................................. 5 1.4. FUNCTION OF KERATINS: ...................................................................................... 5 1.5. REGULATION OF KERATINS (POSTTRANSLATIONAL MODIFICATIONS AND IFAPS) 7 1.6. THE ROLE OF KERATIN 8 AND 18 IN DISEASES DEVELOPMENT............................. 11 1.6.1. ANIMAL MODELS ................................................................................................ 11 1.6.2. HUMAN STUDIES................................................................................................. 13 1.7. HEPATITIS C INFECTION AND HALT-C TRIAL.................................................... 14 1.8. HEREDITARY HEMOCHROMATOSIS..................................................................... 18 1.9. LIVER FIBROSIS .................................................................................................. 19 AIM OF THE STUDY ................................................................................................ 21 2. MATERIALS AND METHODS................................................................................. 22 2.1. MATERIALS .............................................................................................................. 22 2.1.1. CHEMICALS AND REAGENTS ............................................................................... 22 2.1.2. INSTRUMENTS AND EQUIPMENTS ........................................................................ 25 2.1.3. CONSUMABLES .................................................................................................. 26 2.1.4. REACTION KITS AND ENZYMES ........................................................................... 27 2.1.5. ANTIBODIES USED FOR WESTERN BLOT AND IMMUNOFLUORESCENCE ................ 28 2.1.5.1. PRIMARY ANTIBODIES ........................................................................................ 28 2.1.5.2. SECONDARY ANTIBODIES.................................................................................... 28 2.1.6. CELL LINES......................................................................................................... 29 2.1.7. PLASMID CLONE LIST .......................................................................................... 29 2.1.8. BACTERIAL STRAIN............................................................................................. 29 2.1.9. OLIGONUCLEOTIDES (PRIMERS) ......................................................................... 29 2.1.10. ANIMALS ............................................................................................................ 31 2.1.11. BUFFER............................................................................................................... 32 2.2. METHODS ................................................................................................................. 34 2.2.1. HUMAN SUBJECTS............................................................................................... 34 2.2.2. ANIMAL EXPERIMENTS ....................................................................................... 37 2.2.2.1. LIVER FIBROSIS MODELS..................................................................................... 38 2.2.2.2. MICE IRON OVERLOAD EXPERIMENT ................................................................... 39 2.2.2.3. CLINICAL CHEMISTRY, SERUM AND HEPATIC IRON MEASUREMENTS ................... 39 2.2.3. CELL CULTURE EXPERIMENTS............................................................................. 39 2.2.3.1. PRIMARY HEPATOCYTE ISOLATION ..................................................................... 39 2.2.3.2. CELL LINES......................................................................................................... 40 2.2.3.3. STIMULATION OF PRIMARY HEPATOCYTE CULTURE WITH IRON .......................... 40 2.2.3.4. CULTIVATION AND PASSAGING OF THE HEPATOCYTES AND CELL LINES............. 40 I 2.2.3.5. STIMULATION OF TRANSFECTED CELL LINE FOR HYPERPHOSPHORYLATION AND OXIDATIVE STRESS.............................................................................................. 41 2.2.3.6. FREEZING AND THAWING OF CELLS..................................................................... 41 2.2.3.7. TRANSFECTION OF CELLS VIA LIPOFECTION ........................................................ 41 2.2.3.8. MOLECULAR BIOLOGY METHODS........................................................................ 42 2.2.3.9. BACTERIAL CULTURE AND BACTERIAL GROWTH MEDIUM................................... 42 2.2.3.10.SITE DIRECTED MUTAGENESIS............................................................................. 42 2.2.3.11.TRANSFORMATION OF E. COLI AND PLASMID ISOLATION .................................... 43 2.2.3.12.ISOLATION OF DNA ............................................................................................ 44 2.2.3.13.ISOLATION OF RNA ............................................................................................ 45 2.2.3.14.DETERMINATION OF DNA OR RNA CONCENTRATION AND PURITY .................... 45 2.2.3.15.POLYMERASE CHAIN REACTION (PCR) ............................................................... 45 2.2.3.16.DNA PURIFICATION ............................................................................................ 47 2.2.3.17.DNA ELECTROPHORESIS SYSTEM AND DNA ISOLATION FROM AGAROSE GEL .... 47 2.2.3.18.REVERSE TRANSCRIPTION PCR (RT-PCR) ......................................................... 48 2.2.3.19.QUANTITATIVE REAL TIME PCR (QRT-PCR) ..................................................... 48 2.2.3.20.DENATURING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (DHPLC) ......... 49 2.2.3.21.DNA SEQUENCING.............................................................................................. 51 2.2.4. BIOCHEMICAL METHODS..................................................................................... 51 2.2.4.1. TOTAL PROTEIN EXTRACTION FROM LIVER TISSUE.............................................. 51 2.2.4.2. TOTAL PROTEIN EXTRACTION FROM CELL CULTURES ......................................... 52 2.2.4.3. SEPARATION OF PROTEINS BY SDS-PAGE ......................................................... 52 2.2.4.4. STAINING OF PROTEINS IN ACRYLAMIDE GELS WITH COOMASSIE BRILLIANT BLUE ........................................................................................................................... 53 2.2.4.5. BRADFORD PROTEIN ASSAY ................................................................................ 53 2.2.4.6. WESTERN BLOT .................................................................................................. 53 2.2.4.7. IMMUNOPRECIPITATION...................................................................................... 54 2.2.4.8. IMMUNOFLUORESCENCE STAINING AND IMAGING OF LIVER TISSUE AND CELL LINE (NIH-3T3).......................................................................................................... 55 2.2.4.9. HISTOLOGICAL STAINING.................................................................................... 55 2.2.4.10.KINASE ASSAY .................................................................................................... 57 2.2.4.11.CYTOTOXICITY DETECTION ASSAYS.................................................................... 57 2.2.5. ANALYSIS AND SOFTWARES................................................................................ 58 2.2.6. ETHICS STATEMENT ............................................................................................ 58 2.2.7. SAFETY MEASURES ............................................................................................. 59 3. RESULTS...................................................................................................................... 60 3.1. ANALYSIS OF THE KERATIN VARIANTS IN PATIENTS WITH CHRONIC HEPATITIS C. ........................................................................................................................... 60 3.1.1. PATIENT DEMOGRAPHICS ................................................................................... 60 3.1.2. OVERVIEW OF KERATIN VARIANTS FOUND IN PATIENTS WITH CHRONIC HEPATITIS