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Functional Characterisation of ANKRD1 and Its Regulation by RASSF1A and YAP1 Signalling

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Functional Characterisation of ANKRD1 and Its Regulation by RASSF1A and YAP1 Signalling Functional characterisation of ANKRD1 and its regulation by RASSF1A and YAP1 signalling DISSERTATION Zur Erlangung des Doktorgrades der Naturwissenschaften (Doctor rerum naturalium Dr. rer. nat.) Angefertigt am Institut für Genetik Fachbereich Biologie und Chemie der Justus-Liebig-Universität Giessen Vorgelegt von Adriana Patricia Jiménez Carrera Giessen, Januar 2017 1. Gutachter: Prof. Dr. Reinhard Dammann Institut für Genetik Fachbereich Biologie und Chemie der Justus-Liebig-Universität Giessen 2. Gutachter: Prof. Dr. M. Lienhard Schmitz Institut für Biochemie Fachbereich Medizin der Justus-Liebig-Universität Giessen ERKLÄRUNG Hiermit erkläre ich, dass ich die vorliegende Doktorarbeit selbständig und ohne unzulässige fremde Hilfe oder Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Alle Textstellen, die wörtlich oder sinngemäß aus veröffentlichten Schriften entnommen sind, und alle Angaben, die auf mündlichen Auskünfte beruhen, sind als solche kenntlich gemacht. Bei den von mir durchgeführten und in der Dissertation erwähnten Untersuchungen habe ich die Grundsätze guter wissenschaftlicher Praxis, wie sie in der “Satzung der Justus-Liebig-Universität Gießen zur Sicherung guter wissenschaftlicher Praxis” niedergelegt sind, eingehalten. Giessen, den 31.01.2017 …………………………………………………………. Adriana Patricia Jiménez Carrera Während dieser Arbeit ist eine Publikation mit dem Titel “The tumor suppressor RASSF1A induces the YAP1 target gene ANKRD1 that is epigenetically inactivated in human cancers and inhibits tumor growth” entstanden. Die Publikation ist in der Revisionsphase bei der Zeitschrift “Oncotarget”. Eine weitere Publikation als Ko-Autorin wurde 2016 veröffentlicht mit dem Titel “The dual specificity phosphatase 2 gene is hypermethylated in human cancer and regulated by epigenetic mechanisms”. T. Haag, A.M. Richter, M.B. Schneider, A.P. Jiménez, and R.H. Dammann. BMC Cancer 2016. Die Ergebnisse dieser Publikation wurden bei dieser Arbeit nicht mit einbezogen. For Claude Index ABBREVIATIONS ........................................................................................................ 1 SUMMARY ................................................................................................................... 4 ZUSAMMENFASSUNG ............................................................................................... 5 1. INTRODUCTION ...................................................................................................... 7 1.1 Cancer ....................................................................................................................... 7 1.2 The Hippo pathway .................................................................................................. 8 1.3 Deregulation of the Hippo pathway in cancer ........................................................ 11 1.4 Yes-associated protein 1 (YAP1) ........................................................................... 13 1.5 RAS association domain family (RASSF) ............................................................. 15 1.6 Ankyrin repeat domain protein 1 (ANKRD1) ........................................................ 17 Aims of this study ........................................................................................................ 19 2. MATERIALS AND METHODS ............................................................................. 20 2.1 MATERIALS ......................................................................................................... 20 2.1.1 List of Materials .................................................................................................. 20 2.1.1.1 Chemicals ......................................................................................................... 20 2.1.1.2 Size and molecular weight standards ............................................................... 22 2.1.1.3 Antibiotics ........................................................................................................ 22 2.1.1.4 Transfection reagents ....................................................................................... 22 2.1.1.5 Enzymes ........................................................................................................... 22 2.1.1.6 Kits and microarrays ........................................................................................ 23 2.1.1.7 Antibodies for western blots ............................................................................ 24 2.1.1.8 Vectors ............................................................................................................. 24 2.1.1.9 Chemically- competent E. coli strains .............................................................. 27 2.1.1.10 Human cell lines ............................................................................................. 27 2.1.1.11 Hepatic primary tumors and normal liver tissues ........................................... 28 2.1.2 Primers ................................................................................................................ 28 2.1.3 Media ................................................................................................................... 30 2.1.4 General buffers and solutions .............................................................................. 31 2.1.5 Buffers for SDS-PAGE and western blot ............................................................ 32 2.1.6 Basic commodities .............................................................................................. 33 2.1.6.1 Consumables .................................................................................................... 33 2.1.6.2 Equipment ........................................................................................................ 33 2.1.6.3 Centrifuges ....................................................................................................... 34 2.1.6.4 Incubators ......................................................................................................... 34 2.1.6.5 Microscopes and camera .................................................................................. 34 2.1.6.6 Shakers ............................................................................................................. 34 2.1.6.7 PCR Thermocyclers ......................................................................................... 35 2.1.6.8 Water baths ....................................................................................................... 35 2.1.7 Software .............................................................................................................. 35 2.2. METHODS ............................................................................................................ 36 2.2.1 Molecular cloning ............................................................................................... 36 2.2.1.1 Restriction digestion ......................................................................................... 36 2.2.1.2 Ligation and transformation ............................................................................. 36 2.2.1.3 Plasmid preparation and glycerol stock cultures .............................................. 37 2.2.2 Mutagenesis ......................................................................................................... 37 2.2.3 Cell culture .......................................................................................................... 38 2.2.3.1 Cell lines and transfections .............................................................................. 38 2.2.3.2 Promoter assays ................................................................................................ 38 2.2.3.3 Colony formation and growth curves ............................................................... 39 2.2.3.4 Generation of stable cell line ............................................................................ 40 2.2.3.5 Knockdown assays by small interfering RNA ................................................. 41 2.2.4 DNA isolation ..................................................................................................... 42 2.2.5 RNA isolation ...................................................................................................... 42 2.2.6 Methylation analysis ........................................................................................... 43 2.2.6.1 In vitro methylation (ivm) ................................................................................ 43 2.2.6.2 DNA bisulfite conversion ................................................................................. 43 2.2.6.3 Combined bisulfite restriction analysis (CoBRA) ........................................... 44 2.2.7 Agarose gel electrophoresis (AGE) ..................................................................... 44 2.2.8 Polymerase chain reaction (PCR) ....................................................................... 44 2.2.8.1 Reverse transcriptase PCR (RT-PCR) .............................................................. 45 2.2.8.2 Semiquantitative PCR ...................................................................................... 46 2.2.8.3 Quantitative real time PCR (qRT-PCR) ........................................................... 47 2.2.8.4 PCR for CoBRA ............................................................................................... 48 2.2.8.5 Mutagenesis PCR ............................................................................................
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