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Assessment of the Effect of Mitotane on Expression Profile

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Assessment of the Effect of Mitotane on Expression Profile ASSESSMENT OF THE EFFECT OF MITOTANE ON EXPRESSION PROFILE OF THE GENES INVOLVED IN MITOCHONDRIAL METABOLISM IN HUMAN ADRENOCORTICAL, LUNG, BREAST AND COLON CANCER CELL LINES PhD thesis Author: Urszula Waszut Supervisor: dr hab. n. med. Dorota Dworakowska Department of Nuclear Medicine Chair of Nuclear Medicine and Radiology Informatics Faculty of Health Sciences with Subfaculty of Nursing And Institute of Maritime and Tropical Medicine Head of the Department: Prof. dr hab. n. med. Piotr Lass Medical University of Gda!sk Gda sk 2017 OCENA WP"YWU MITOTANU NA EKSPRESJ# GEN$W ZAANGA%OWANYCH W REGULACJ# METABOLIZMU MITOCHONDRIALNEGO W LUDZKICH LINIACH KOM$RKOWYCH RAKA KORY NADNERCZA, P"UC, PIERSI I JELITA GRUBEGO Praca Doktorska Autor: Urszula Waszut Promotor: dr hab. n. med. Dorota Dworakowska Zak&ad Medycyny Nuklearnej Katedra Medycyny Nuklearnej i Informatyki Radiologicznej Wydzia& Nauk o Zdrowiu z Oddzia&em Piel'gniarstwa i Instytutem Medycyny Morskiej i Tropikalnej Kierownik: prof. dr hab. n. med. Piotr Lass Gda!ski Uniwersytet Medyczny Gda sk!2017! This PhD project has been carried out within the grant “Bridge” (POMOST/2012-5/3) awarded to Dr Dorota Dworakowska MD (Hons) PhD, entitled "Pre-clinical targeting of PI3K/Akt/mTOR and RAF/MEK/ERK signalling pathways in adrenocortical cancer: impact on steroidogenesis, cell proliferation and apoptosis", co-financed by the Foundation for Polish Science and European Union. From my supervisor Dr Dorota Dworakowska MD (Hons) PhD, I received a scholarship for doctoral students, financed within the grant of the Foundation of Polish Science, and also from the "Endocrine Cancer Research Fund", Kings College Hospital Charity, managed by Dr Dworakowska. The research study described here has been conducted at the Department of Molecular Pathology, King’s College Hospital, London, The UK in collaboration with the Richard Dimbleby Department of Cancer Research, King's College London, London. UK. Results have been presented during international congresses including the Endocrine Society's 97th Annual Meeting in San Diego, USA (March 5–8th , 2015) and the 21st Convention of the Polish Society of Endocrinology in Katowice, Poland (September 15– 17th). Projekt doktorski zosta! wykonany w ramach grantu POMOST/2012-5/3 przyznanego dr hab. med. Dorocie Dworakowskiej, zatytu!owanego “Ocena wp!ywu terapii celowanej szlakw PI3K/Akt/mTOR oraz RAF/MEK/ERK na steroidogenez", proliferacj" komrkową oraz apoptoz" w raku kory nadnerczy – badania przedkliniczne”, wspłfinansowanego przez Fundacj" na Rzecz Nauki Polskiej i Uni" Europejsk#. Miejsce wykonania bada$: King’s College Hospital London “Molecular Pathology Department” we wspłpracy z Richard Dimbleby Department of Cancer Research, King's College London. Otrzymywa!am od pani promotor dr hab. n. med. Doroty Dworakowskiej stypendium dla doktorantw, finansowanego w ramach grantu Fundacji na Rzecz Nauki Polskiej oraz stypednium ze środkw finansowanych z zarz#dzanego przez ni# funduszu charytatywnego “Endocrine Cancer Reserach Fund” Kings College Hospital Charity, Londyn, Wielka Brytania. Wyniki zawarte w niemniejszej pracy by!y prezentowane na mi"dzynarodowych konferencjach naukowych wliczaj#c: Endocrine Society's 97th Annual Meeting w San Diego, USA (5–8 marca 2015) oraz XXI Zjazd Polskiego Towarzystwa Endokrynologicznego w Katowicach (15– 17 wrze%nia 2016). Acknowledgements : I owe my main debt of gratitude to the grant awardee and my supervisor Dorota Dworakowska MD (Hons) PhD for this opportunity to take part in the Foundation for Polish Science-funded grant, enabling me to conduct interesting research and providing support. I also wish to express my appreciation of research fellows within the (Viapath) and Molecular Pathology and Clinical Biochemistry Labs, Kings College Hospital London, and research labs of Guy's Hospital, who helped in both practical ways and by their friendship and moral support. I would especially like to thank Mel Smith (PhD) of the Department of Molecular Pathology for providing facilities and advice and checking my descriptions of the molecular biology, Salvador Diaz-Cano (MD PhD) and Jane Moorhead (PhD) of the Department of Histopathology for providing histological material and advice and Norman Taylor (PhD) of the Department of Clinical Biochemistry for checking my English and finding texts of references that I could not access. I wish to thank Dr Piotr W#& who provided statistical analysis under this grant, and especially to my colleague working under the same grant, Paulina Szyszka, for collaboration and mutual support during our research project. My grateful thanks are due to Magdalena D#browski for financial help and her constant friendship, and finally my family for their love and support and specifically to Bogdan for making it possible for me to work in London and write up at home and my father for help with layout of figures and tables and checking text for technical accuracy. I take full responsibility for any remaining errors. Podzi kowania:! Mj d!ug wdzi"czno%ci g!ownie nale&y si" Pani Promotor dr hab. med. Dorocie Dworakowskiej za mo&liwo%* uczestnictwa w grancie Fundacji na Rzecz Nauki Polskiej, po%wi"conym ciekawemu obszarowi bada$ i za okazane mi wsparcie. Sk!adnam podziekowania dla Pani Promotor za pomoc merytoryczna w cz"%ci badowczej i statystycznej oraz finansow# (udzielenie stypendiium FNP oraz środkw z funduszu Kings College Hospital Charity ‘Endocrine Cancer Research Fund’. Pragn" tak&e wyrazi* moj# wdzi"czno%* dla pracownikw naukowych (Viapath) Departamentu Patologii Molekularnej i Biochemii, Szpitala Kings College w Londynie, ktrzy pomogli mi w sposb praktyczny, a tak&e okazali przyja+$ i wsparcie. Chcia!abym szczeglnie podzi"kowa* Dr Mel Smith za udost"pnienie laboratoriw i sprz"tu badawczego oraz za udzielone doradztwo i sprawdzenie moich tekstw zwi#zanych z zagadnieniami biologii molekularnej, a tak&e Dr Salvador Diaz-Cano z Departamentu Histopatologii w King College Hospital za udzielon# pomoc. Dr Normanowi Taylorowi z Zak!adu Biochemii Klinicznej pragn" podzi"kowa* za korekt" pisowni, wyszukanie i udostepnienie tekstw, do ktrych nie mia!am otwartego dost"pu. Pragn" tak&e podzi"kowa*, dr hab. Piotrowi W"&owi jako osobie, ktra wykonala statystyke w ramach grantu, oraz szczegunie dr Paulinie Szyszce za wspłprac" i wzajemn# pomoc podczas projektu badawczego. Z Wdzieczno%ci# podzi"kowania dla Magdaleny D#browski za przyja+$ oraz wsparcie finansowe, ko$cz#c dzi"kuje mojej rodzinie za ich mi!o%* i wsparcie, wspominaj#c szczeglnie mojego ojca, ktremu chc" podzi"kowa* za pomoc w uk!adzie rysunkw, tabel oraz sprawdzenie tekstu pod wzgl"dem poprawno%ci technicznej. Bior" pe!n# odpowiedzialno%* za wszelkie pozosta!e b!"dy. Table of contents List of abbreviations: .................................................................................................................. 10 Abstract ...................................................................................................................................... 13 Streszczenie ................................................................................................................................ 14 I. Introduction ...................................................................................................... 15 1.1 Mitotane: basis for understanding possible mechanisms of action. ......................................... 15 1.1.1 Historical background ....................................................................................................... 15 1.1.2 Clinical effectiveness and clinical monitoring .................................................................. 16 1.1.3 Effects on adrenal tissue and cells ..................................................................................... 18 1.1.4 Mechanisms of adrenocortical cell death and stasis in response to mitotane. ................... 19 1.1.5 Activation of mitotane ....................................................................................................... 20 1.1.6 Proteins involved in response to mitotane ......................................................................... 22 1.1.7 Mitochondrial effects ........................................................................................................ 23 1.1.8 Mitotane effects on mitochondria that may lead to apoptosis ........................................... 25 1.1.9 Calcium signalling at the MAM, a crucial apoptosis trigger ............................................. 28 1.1.10 Effects of mitotane on cellular biochemistry and steroidogenesis................................... 29 1.2. Mitochondria as semiautonomic structures with major roles in energy metabolism and apoptosis................................................................................................................................................ 30 1.3 Cell lines used in the experiments ............................................................................................. 34 1.4 Heterogeneity in context of ACC study and gene expression study from formalin-fixed, paraffin-embedded adrenocortical carcinoma tissue ............................................................................ 37 II. Aims of the study.................................................................................................... 39 III. Materials and methods.......................................................................................... 40 3.1 Biological material, reagents, equipment and software ..........................................................
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