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Interacting Protein (AIP)-Dependent Tumorigenesis in the Pituitary

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Interacting Protein (AIP)-Dependent Tumorigenesis in the Pituitary TECHNISCHE UNIVERSITÄT MÜNCHEN FAKULTÄT FÜR MEDIZIN Elucidation of the molecular mechanisms underlying aryl hydrocarbon receptor- interacting protein (AIP)-dependent tumorigenesis in the pituitary Eva-Maria Bogner Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzende: Prof. Dr. Stephanie E. Combs Prüfer der Dissertation: 1. Prof. Dr. Michael J. Atkinson 2. Prof. Dr. Nina H. Uhlenhaut Die Dissertation wurde am 06.05.2020 bei der Technischen Universität München eingereicht und durch die Fakultät für Medizin am 01.12.2020 angenommen. Statuatory Declaration/Eidesstattliche Erklärung ____________________________________________________________________________ Statutory Declaration: I declare that I have authored this thesis independently, that I have not used other than the declared sources/resources, and that I have explicitly marked all material which has been quoted either literally or by content from the used sources. Munich, ……………………………………………… Eva-Maria Bogner Eidesstattliche Erklärung: Ich erkläre an Eides statt, dass ich die vorliegende Arbeit selbstständig verfasst, keine anderen als die angegebenen Quellen/Hilfsmittel benutzt, und die den benutzten Quellen wörtlich und inhaltlich entnommene Stellen als solche kenntlich gemacht habe. München, ……………………………………………… Eva-Maria Bogner _____________________________________________________________________________________ I Table of Contents ____________________________________________________________________________ Table of Contents Statutory Declaration: ........................................................................................................................... II Eidesstattliche Erklärung: ..................................................................................................................... II Abstract ................................................................................................................................................. VI Zusammenfassung ............................................................................................................................. VII Abbreviations ........................................................................................................................................ IX 1. Introduction .................................................................................................................................... 1 1.1. Pituitary adenomas ................................................................................................................ 1 1.1.1. The pituitary gland and the endocrine system ............................................................. 1 1.1.2. Clinical manifestations of pituitary adenomas (PAs) ................................................... 3 1.1.3. Treatment of PAs and their symptoms ......................................................................... 5 1.1.4. Genetics of PAs .............................................................................................................. 7 1.2. Aryl hydrocarbon receptor interacting protein (AIP) ............................................................ 9 1.2.1. Physiological functions of AIP ..................................................................................... 10 1.2.2. Role of AIP in PAs ........................................................................................................ 12 1.3. microRNAs ........................................................................................................................... 14 1.3.1. Biosynthesis, Transcriptional Regulation and cellular functions of miRNAs ........... 15 1.3.2. miRNAs in health and disease .................................................................................... 16 1.3.3. miRNAs in PAs ............................................................................................................. 16 1.4. Hypothesis and aim of the thesis........................................................................................ 18 2. Material and Methods.................................................................................................................. 19 2.1 Material ................................................................................................................................. 19 2.1.1 Instruments ................................................................................................................... 19 2.1.2 Consumable Materials ................................................................................................. 21 2.1.3 Chemicals and reagents .............................................................................................. 23 _____________________________________________________________________________________ II Table of Contents ____________________________________________________________________________ 2.1.4 Buffers and Solutions ................................................................................................... 25 2.1.5 Commercially available Kits ........................................................................................ 27 2.1.6 Constructs ..................................................................................................................... 28 2.1.7 Antibodies ..................................................................................................................... 30 2.1.7 Primers and Oligos....................................................................................................... 31 2.1.8 Enzymes ....................................................................................................................... 33 2.1.9 Bacteria ......................................................................................................................... 33 2.1.10 Cell lines........................................................................................................................ 33 2.1.11 Human pituitary adenoma tissue ................................................................................ 34 2.1.12 Standards ...................................................................................................................... 37 2.1.13 Desinfections ................................................................................................................ 37 2.1.14 Software ........................................................................................................................ 37 2.1.15 Online tools ................................................................................................................... 38 2.2 Methods ................................................................................................................................ 38 2.2.1 Molecular biology methods .......................................................................................... 38 2.2.2 Cloning .......................................................................................................................... 41 2.2.3 Cell culture methods .................................................................................................... 43 2.2.4 Biochemical methods ................................................................................................... 47 2.2.5 Immunhistological stainings ........................................................................................ 50 2.2.6 Statistical Analysis ....................................................................................................... 52 3. Results.......................................................................................................................................... 53 3.1 Literature review of pathogenic and likely pathogenic AIP mutations in PA patients .... 53 3.2 Collection of pituitary adenoma samples and analysis of clinical parameters of the patients ............................................................................................................................................. 58 3.3 MiRNA expression in human pituitary adenomas ............................................................. 60 3.3.1 miRNA expression analysis in AIPmut+ versus AIPmut- PAs ................................. 60 _____________________________________________________________________________________ III Table of Contents ____________________________________________________________________________ 3.3.2 Validation of differential miRNA expression in somatotropinomas .......................... 64 3.4 miRNA expression depends on AIP mutation status ........................................................ 65 3.3.1 Mutant AIP increases cell proliferation of AIP -/- MEFs.................................................. 65 3.3.2 Mutant AIP is highly unstable in vitro ......................................................................... 66 3.3.3. Mutant AIP affects the expression levels of miR-145 and miR-34a in MEF AIP -/- cells 68 3.3.4. Further characterization of mutant AIP in Aip-/- MEF cells ...................................... 71 3.5 Tumorigenic behavior upon miRNA overexpression in GH3 cells ................................... 74 3.6 Therapy response upon miRNA overexpression .............................................................. 81 3.7 Involvement of the cAMP pathway ....................................................................................
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