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Functional Characterization and Pathophysiological Evaluation of the Olfactomedin-Like 2B Gene

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Functional Characterization and Pathophysiological Evaluation of the Olfactomedin-Like 2B Gene TECHNISCHE UNIVERSITÄT MÜNCHEN FAKULTÄT FÜR MEDIZIN Functional characterization and pathophysiological evaluation of the Olfactomedin-like 2B gene Tanja Angelika Plӧtz Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation. Vorsitzender: Prof. Dr. Radu Roland Rad Prüfer der Dissertation: 1. Priv.-Doz. Dr. Arne Pfeufer 2. Prof. Dr. Hans-Werner Mewes Die Dissertation wurde am 20.07.2020 bei der Technischen Universität München eingereicht und durch die Fakultät für Medizin am 16.03.2021 angenommen. Table of Content TABLE OF CONTENT Table of Content ............................................................................................................... 2 Abbreviations ................................................................................................................... 5 I. Abstract .................................................................................................................... 8 Zusammenfassung ............................................................................................................ 9 II. Introduction .............................................................................................................10 1. Cardiomyopathies and Arrhythmias ............................................................................ 10 2. Genome-wide Association Studies ............................................................................... 15 3. GWAS for Cardiomyopathies........................................................................................ 16 4. The Olfactomedins ....................................................................................................... 20 5. Aims of the Study ......................................................................................................... 22 III. Material and Methods ..............................................................................................23 1. Material ........................................................................................................................ 23 1.1. Cohorts ..................................................................................................................... 23 1.2. Molecular Biology ..................................................................................................... 23 1.3. Cell Culture ............................................................................................................... 25 1.4. Human Heart Tissue ................................................................................................. 26 1.5. Antibodies ................................................................................................................ 26 1.6. Laboratory Chemicals and Reagents ........................................................................ 28 1.7. Kits ............................................................................................................................ 30 1.8. Consumables ............................................................................................................ 31 1.9. Technical Devices ..................................................................................................... 31 2. Methods ....................................................................................................................... 33 2.1. Molecular Biology ..................................................................................................... 33 2.1.1. Molecular Cloning ................................................................................................ 33 2.1.2. RNA Isolation, Purification and Reverse Transcription including qPCR ............... 34 2.2. Cell Culture ............................................................................................................... 35 2.2.1. Growth and Maintenance of HEK293 Cells .......................................................... 35 2 Table of Content 2.2.2. Transient Transfection and Lysis of HEK293 Cells ................................................ 36 2.2.3. Cryo-stocks ........................................................................................................... 37 2.3. Protein Preparation .................................................................................................. 37 2.3.1. Isolation of Protein from HEK293 Cells ................................................................ 37 2.3.2. Isolation of Protein from Human Heart Tissue .................................................... 37 2.3.3. Isolation of Protein from Human Whole Blood ................................................... 38 2.3.4. Protein Purification by FLAG Tag .......................................................................... 38 2.3.5. Protein Purification by Protein G Conjugated Sepharose Beads ......................... 39 2.3.6. Protein Purification by Compat-AbleTM Protein Assay ......................................... 39 2.3.7. Protein Quantitation by BCA ................................................................................ 39 2.4. Protein Analysis ........................................................................................................ 39 2.4.1. Sodium Dodecyl Sulphate-polyacrylamide Gel Electrophoresis .......................... 39 2.4.2. Western Blot Analysis .......................................................................................... 39 2.4.3. Colloidal Coomassie Staining................................................................................ 40 2.4.4. Mass Spectrometry .............................................................................................. 40 2.5. Immunoassays .......................................................................................................... 41 2.5.1. Dot Blot Analysis................................................................................................... 41 2.5.2. Enzyme-linked Immunosorbent Assay ................................................................. 41 IV. Results .................................................................................................................43 1. Characterization of the OLFML2B Protein ................................................................... 43 1.1. Validation of Monoclonal Antibodies ...................................................................... 43 1.2. Intracellular Presence of Wildtype and 23 Mutant Proteins ................................... 44 1.3. In-silico Modelling of the Structure.......................................................................... 45 1.4. Structural Changes ................................................................................................... 46 1.5. Secretional Behaviour .............................................................................................. 47 1.6. Dominant Effect ....................................................................................................... 49 1.7. Temperature-Dependent Secretion ......................................................................... 50 2. Detection of OLFML2B in Human Heart Tissue ............................................................ 53 3. Proteomics of OLFML2B by LC-MSMS .......................................................................... 54 4. Tenascin C as Interaction Partner of OLFML2B ............................................................ 56 3 Table of Content 5. Establishment of two Sandwich ELISAs for Soluble OLFML2B ..................................... 57 V. Discussion ................................................................................................................59 1. Characterization of the OLFML2B Protein ................................................................... 59 2. Interaction Partners of the OLFML2B Protein.............................................................. 64 3. Generation of a Biomarker Assay to Detect Circulating OLFML2B Protein ................. 66 4. Conclusion .................................................................................................................... 69 VI. References ...........................................................................................................70 VII. List of Figures .......................................................................................................84 VIII. List of Tables .........................................................................................................87 IX. Acknowledgements ..............................................................................................89 4 Abbreviations ABBREVIATIONS aa Amino acid ab Antibody AF Atrial fibrillation ARIC Atherosclerosis Risk in Communities BCA Bicinchoninic acid bp Base pair BrS Brugada syndrome BSA Bovine serum albumin C-terminus Carboxy-terminus CaCl2 Calcium chloride CAD Coronary artery disease CPVT Catecholaminergic polymorphic ventricular tachycardia DCM Dilated cardiomyopathy ddH2O Double distilled water DMEM Dulbecco’s modified eagle medium DNA Deoxyribonucleic acid ECG Electrocardiogram ECL Enhanced chemiluminescence ECM Extracellular matrix EDTA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent assay ER Endoplasmic reticulum ExAC Exome aggregation consortium FBS Fetal bovine serum GAPDH Glycerinaldehyd-3-phosphat-Dehydrogenase GenNOVA Genetic Epidemiology Network of Atherosclerosis
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