XXth Gliwice Scientific Meetings 2016

Gliwice, November 18-19, 2016 http://gsn.io.gliwice.pl/

. .

Organizers of Gliwice Scientific Meetings:

Association for the Support of Cancer Research Maria Skłodowska-Curie Memorial Cancer Center and and Institute of Oncology, Gliwice Branch Silesian University of Technology

Patronage and Co-organizers:

Marshal’s Office of the Silesian Voivodeship Ministry of Science and Higher Education Polish Academy of Sciences Gliwice Municipal Office

. Scientific Committee of Gliwice Scientific Meetings 2016:

Andrzej Bednarek, Łódź Mieczysław Chorąży, Gliwice Franck Delaunay, Nice Witold Konopka, Warszawa Marek Los, Linköping Joanna Rzeszowska-Wolny, Gliwice – President Dorota Ścieglinska, Gliwice Andrzej Świerniak, Gliwice Piotr Widłak, Gliwice

Organizing Committee:

Iwona Domińczyk Agnieszka Dudło Roman Jaksik Joanna Łanuszewska Lucyna Ponge Jacek Rogoliński Magdalena Skonieczna Aleksander Sochanik

. 20th Anniversary of Gliwice Scientific Meetings 1997-2016

.

.

Scientific Programs of Gliwice Scientific Meetings in years 1997-2015 (I) Gliwickie Spotkania Naukowe 1997 Oddziaływania białko – DNA 21-22.11.1997

Modelowanie układów biologicznych Uszkadzanie i śmierć komórek Bogdan Lesyng: Jak modelować układy Ewa Sikora: Mechanizmy śmierci komórek biomolekularne; od miareczkowania indukowanej kurkuminą (składnik curry); do dynamiki kwantowej. udział czynników transkrypcyjnych. Andrzej Polański: Niesymetryczna segregacja Krzysztof Szyfter: Ryzyko genetyczne w raku w modelach genetyki popularnej. krtani określone na podstawie rozkładu Rafał Tarnawski: Modele radiobiologiczne genotypów baterii enzymów stosowane w praktyce klinicznej. odpowiedzialnych za metabolizm kacerogenów.

Marcin Kruszewski: Rekombinacja Oddziaływania białko-DNA w procesach homologiczna DNA w komórkach ssaków transkrypcji i replikacji upośledzonych w naprawie uszkodzeń Ewa Bartnik: TBP - Struktura i rola DNA. w transkrypcji Grzegorz Węgrzyn: Rola podjednostki alfa Nowe techniki polimerazy RNA E. coli w inicjacji i Wojciech Markiewicz: Syntetyczne antyterminacji transkrypcji. oligonukleotydowe biblioteki Jolanta Zakrzewska: Oddziaływania białka kombinatoryczne i ich zastosowanie. DnaA z regionem inicjującym replikację Valentin Shick: Analysis of DNA and protein DNA. microchips. Jarosław Marszałek: Rola nukleotydów adeninowych w oddziaływaniach białka inicjatorowego DnaA z DNA. Jerzy Majka: Badanie oddziaływania białka DnaA z regionem inicjującym replikację DNA za pomocą techniki SPR. Igor Konieczny: Oddziaływania białek replikacyjnych a zmiany konformacji DNA podczas inicjacji replikacji plazmidu RK2. Dagmara Jakimowicz: Izolacja regionu oriC za pomocą chromatografii powinowactwa.

Chromatyna i białka jądrowe Anna Goc: Gen hydroksylazy tyrozynowej - okolica i sąsiedzi. Eliza Wyszko: Białka specyficznie wiążące DNA i RNA. Małgorzata Giel-Pietraszyk: Oddziaływanie peptydowych palców cynkowych z kwasami nukleinowymi. Ewa Markiewicz: Nadekspresja białka 14-3-3 w komórkach bakteryjnych i ziemniaku. Grzegorz Wilczyński: Technologia antysensu w badaniach fizjologicznej funkcji białek 14-3-3. (II) Gliwice Scientific Meetings 1998 Structure and Function of the Genome 50th Anniversary of Institute of Oncology in Gliwice 27-28.11.1998

Mathematical Models in Biology DNA Damage, Repair and Cellular Death Jerzy Nakielski: Tensorial model of growth Regen Drouin: UV – induced DNA damage and cell divisions in the root apex distribution at the DNA sequence level. Andrzej Polański: DNA statistics Ryszard Oliński: Oxidatively damaged DNA and the history of the population. bases - relevance to mutagenesis Andrzej Świerniak Control of aminoacids and cancer. concentration in cell populations Anna Kulma: Generation of transgenic potato as a potential technique of cancer cell- plans differing in carbohydrate level specific therapy. by manipulation of 14-3-3 protein expression. Chromatin Structure and Gene Expression Magdalena Milcarz: Apoptotic-like effect of 14-3-3 protein overexpression in E. coli. Wolfgang Henning: Histone replacement variants. Piotr Widłak: DFF, human endonuclease activated during apoptosis. Andrzej Jerzmanowski: Chromatin structures in transcriptional regulation. Krzysztof Staroń: Eukaryotic topoisomerase I. Ryszard Rzepecki: Interactions of D. melanogaster karyoskeletal proteins - lamins and topoisomerase II with nucleic acids in vivo. Wolf H. Strätling: The nuclear protein ARBP/MeCP2. Jan Filipski: Nuclease hypersensitive sites and meiotic double-strand breaks in human DNA cloned in yeast.

Genetic Polymorphism and Cancer Jerzy Ihnatowicz: The quantitative analysis of cancer epidemiology problems. Barbara Jarząb: Mutations of protooncogene RET and medullary thyroid carcinoma. Marek Rusin: Polymorphism of DNA repair genes. Maria Wideł: Individual radiosensitivity and DNA damage.

(III) Gliwickie Spotkania Naukowe 1999 Struktura i funkcje genomu 26-27.11.1999

Uszkodzenia i naprawa DNA: Maria Wideł: Uszkodzenia DNA indukowane Mechanizmy promieniami jonizującymi w limfocytach Małgorzata Zdzienicka: Radiowrażliwość chorych na raka szyjki macicy i nowotwory a naprawa dwuniciowych pęknięć DNA. głowy i szyi. Ryszard Oliński: Indukcja i reperacje Ryszard Rzepecki: Regulacja aktywności oksydacyjnych uszkodzeń DNA. topoizomerazy II i jej wiązanie do kwasów nukleinowych podczas cyklu Marcin Kruszewski: Liczba wywoływanych komórkowego. przez H2O2 uszkodzeń DNA zależy od poziomu wolnych jonów żelaza. Bogdan Smołka: Metoda kometkowa; nowy sposób analizy obrazu. Wanda Baer-Dubowska: Addukty policyklicznych węglowodorów aromatycznych z DNA i ich modyfikacja Zastosowanie biologii molekularnej przez potencjalne czynniki chemo- w medycynie prewencyjne. Jan Lubiński: Stan obecny i perspektywy Barbara Tudek: Lokalizacja cyklicznych diagnostyki molekularnej w medycynie. adduktów zasad DNA w obrębie ludzkiego Włodzimierz Krzyżosiak: Strategie wykrywania genu p53. mutacji w genach. Krzysztof Szyfter: Charakterystyka zmian Marek Mirowski: Zastosowanie liczby kopii DNA na poziomie mikroprocesorów genetycznych chromosomowym w guzach pierwotnych i w diagnostyce raka tarczycy. przerzutach w płaskonabłonkowym raku krtani. Janusz Limon: Znaczenie techniki FISH w diagnostyce białaczek oraz litych guzów Maria Grąziewicz: Fapy-adenina; wpływ nowotworowych człowieka. oksydacyjnej pochodnej adeniny na replikację DNA i jej właściwości Barbara Jarząb: Onkogen RET; znaczenie mutagenne. w patogenezie i diagnostyce raka tarczycy. Joanna Rzeszowska-Wolny: Białka rozpoznające uszkodzony DNA. Dorota Butkiewicz: Polimorfizm genów NER i ryzyko zachorowania na niedrobnokomórkowego raka płuc.

Uszkodzenia i naprawa DNA: Efekty biologiczne Marek Gniazdowski: Leki przeciwnowotworowe a czynniki transkrypcyjne. Andrzej Pawlak: Genetyczna podatność na raka płuca i związane z nim mutacje somatyczne. Marek Rusin: Nowe polimorfizmy genów naprawy DNA-MGMT i MPG. Antonina Cebulska-Wasilewska: Porównanie uszkodzeń DNA i chromosomowych oraz podatność na promieniowanie UV i X w limfocytach ludzi zdrowych i pacjentów z nowotworem skóry. (IV) Gliwickie Spotkania Naukowe 2000 Współczesna terapia nowotworów: od badań molekularnych do zastosowań klinicznych 24-25.11.2000

Biologia komórek nowotworowych Janusz Siedlecki: Rola 3' niekodującej części Kazimierz Dux: Biologia nowotworów. RNA w ekspresji genów. Mieczysław Chorąży: Genetyka nowotworów - Piotr Widłak: Białka chromatyny rozpoznające udział genów w nowotworzeniu. uszkodzony DNA. Jan Steffen: Dziedziczne predyspozycje do rozwoju nowotworu. Modele matematyczne w biologii Wenancjusz Domagała: Biologia komórki i medycynie nowotworowej: czym różni się komórka Marek Kimmel: Wczesna detekcja raka płuc nowotworowa od prawidłowej? w oparciu o tomografię komputerową Przemysław Janik: Angiogeneza i analizę biomarkerów. w nowotworach. Andrzej Płucienniczak: Wykorzystanie zjawiska Czesław Radzikowski: Powstawanie supresji PCR, czyli patelnie w biologii przerzutów. molekularnej. Krzysztof Fujarewicz: Klasyfikacja w oparciu Uszkodzenia i śmierć komórki o analizę ekspresji genów. Andrzej Polański: Analiza stabilności bi- Józefa Wesierska-Gadek: Role of poly (ADP - liniowego modelu chemioterapii nowotworów ribose) polymerase-1 in the regulation uwzględniającego fazoczułość leków. of p35 response to genotoxic stimuli. Ryszard Oliński: Analiza zawartości 8-okso-deoksyguanozyny i 8-okso-guaniny

w moczu człowieka; czy ich poziom odzwierciedla aktywność enzymów reperacyjnych? Ewa Sikora: Kurkumina jako czynnik indukujący nietypową apoptozę komórek prawidłowych i nowotworowych. Andrzej Pawlak: Wzajemne oddziaływania niedotlenienia i ekspozycji na policykliczne węglowodory aromatyczne. Barbara Jarząb: Analiza ekspresji genu tyreoglobuliny: zastosowanie w monitorowaniu chorych na raka tarczycy.

Genome Structure and Regulation of Gene Expression Nicolai Sjakste: Two-directional transcription of the chicken alpha-globin gene domain. Jean-Marc Matter: Functional interactions between bHLH transcription factors regulate neural determination in the CNS. Wojciech Makałowski: Organizacja i ewolucja genomu człowieka.

(V) Gliwice Scientific Meetings 2001 Molecular Biology and Cancer Research 23-24.11.2001

Chromatin structure and function DNA damage and repair Ronald Hancock: Molecular crowding Jacques Nino: Mutation strategies. and assembly of nuclear compartments. Ryszard Oliński: Oxidative stress and cancer Nocolais Sjakste: Proteasome role development. in pathology, first studies on proteasome Krzysztof Szyfter: A link between chromosome gene polymorphism. instability, DNA damage and repair capacity Jan Filipski: Localization of the hot spots and cancer risk factor. of recombination in segments of human Piotr Widłak: Nucleases engaged in cell death. DNA cloned in YACs. Marek Rusin: DNA damage recognition protein Jiri Fajkus: Chromatin structure of plant XPA – regulation of its expression telomeres and subtelomeres. and function. Dorota Dziadkowiec: DNA repair and mating Cellular stress and death type switching in Schizosaccharomyces Irena Szumiel: The celluar response to DNA pombe. damage; the importance of being p35 positive. Maciej Żylicz: Role of the heat shock proteins in cell transformation. Ewa Nino: Oxidized low density lipoproteins in the process of atherosclerosis. Józefa Gadek-Wesierski: Inducion of apoptosis in human cervix carcinoma cells during therapy by cisplatin. Andrzej Pawlak: Mutations of aryl hydrocarbon responsive elements (AHRE) determine the tissue specific genotoxic consequences of PAH exposure. Tatiana Kałuża: Real - Time PCR.

Mathematical modelling of biological processes Marek Kimmel: SNP haplotypes and cancer. Bogdan Lesyng: Modelling of biomolecular systems and processes using microscopic and mezoscopic approaches. Mirosław Lachowicz: Mathematical modeling of non-local phenomena in tumor development. Krzysztof Fujarewicz: Improved classification of microarray gene expression data using support vector machine. Cezary Labuda: New trends in molecular diagnostics.

(VI) Gliwice Scientific Meetings 2002 22-23.11.2002

New Trends in Cancer Therapy DNA Repair, Radiobiology and Medicine Beata Utracka-Hutka: Chemotherapy - Marcel Blaese: Role of cytoplasmic retinoic where are you going? Principles, acid binding proteins CRABPI and II new targets, challenges for the future. in regulating sensitivity of tumor cells Jan Walewski: Therapy for lymphoma: to retinoids and ionizing radiation. current concepts and new approaches. Kai Rothkamm: Cell-cycle-dependent Jerzy Konopa: Mechanisms of action contribution of non-homologous end joining as a basis for the development and homologous recombination to the of new antitumor compounds. repair of radiation-induced DNA double- strand breaks in mammalian cells. Maciej Siewiński: Inhibitorotherapy of cancer. Ingo Brammer: Individual radiosensitivity: Rafał Tarnawski: How molecular biology molecular mechanisms and clinical impact. may improve outcome of radiation oncology interactions. Elisabeth Ortmann: Modulation of radiation induced apoptosis by antioxidant vitamins.

Antonina Cebulska-Wasilewska: Predictive Transgenic Organisms in assays - comparison between various Agrobiotechnology and Medicine biological end-point and various doses. Mieczysław Chorąży: Genetically modified Nadzeya Rabakon: Genetic efficiency organisms - introduction. of low-dose ionizing radiation in small Marcin Łukaszewicz: Transgenic plants mammals under chronic irradiation. overexpressing biologically active Barbara Tudek: Long-chain adducts of fatty compounds. acids derivatives to DNA bases, mutations Agnieszka Sirko: The potential of transgenic and repair. plants for subunit vaccine production. Krzysztof Szyfter: Phenotypic and genotypic Jerzy Jurka: Transposable elements: indications for an impaired DNA repair horizontal transfer and mechanisms capacity in laryngeal cancer subjects. of integration. Ryszard Oliński: Oxidative DNA damage and repair; insight from determination Genomics in Medicine of 8-oxoguanine and 8-oxo-2'- deoxoguanosine in extracellular fluids. Helena Sztajer: Genomics and proteomics as a tools for analysis of phospholipid Grażyna Motykiewicz: DNA repair capacity hydroperoxide glutathione peroxidase in lymphoblasts from sisters discordant expression and transformation for breast cancer. during spermatogenesis. Włodzimierz Krzyżosiak: Translational Miscellanea regulation of the BRCA1 gene. Ludmila Drobot: Ruk adaptor proteins - Marek Figlerowicz: Discovery of RNAi structural and functional features. phenomenon - turning-point in functional Wiesława Widłak: Why spermatogenic cells genomics. are sensitive to elevated temperature. Catharina Larsson: Positional cloning of tumor The role of heat shock proteins. suppressor genes in parathyroid Dorota Rybaczek: Premature mitosis (PCC) – tumorigenesis. a preserved ability of chromatin to replicate Patrick Gaudray: Deciphering Menin's the DNA. interaction networks as a tool to tackle Olgierd Unold: Towards DNA based computer its biological role. – the initial state. Barbara Jarząb: Expression profiling by DNA Sergey Razin: Chicken domain of alpha-globin microarray in endocrine - related cancer. genes: organization and regulation of globin genes expression.

(VII) Gliwice Scientific Meetings 2003 21-22.11.2003

Structure and Functions of the Genome DNA repair and their role in carcinogenesis Sergey Razin: Domain organization of human Ryszard Oliński: The molecular links between dystrophin gene. oxidative damage to DNA and cancer. Jan Filipski: Mapping of the hot spots of Marcin Kruszewski: Labile iron pool, oxidative recombination in human DNA cloned DNA damage and carcinogenesis. in S.cerevisiae. Miroslav Chovanec: DNA double-strand break Elena Youdinkova: Organization of the chicken repair and its implication in cancer. domain of alpha-globin genes. Rosa Goncharova: Some aspects of applying Vlada Philmonienko: Nuclear myosin I antimutagens as anticarcinogens. and actin are required for transcription Joanna Rzeszowska-Wolny: Nucleotide of ribosomal genes. excision repair – new role in oxidative Maria Malanga: Poly(ADP-ribose) reactivates damage repair? stalled DNA topoisomerase I: relevance Marek Rusin: DNA repair genes; to genomic stability and cancer therapy. polymorphisms and risk of cancer. Marek Gniazdowski: Covalent binding of amidine analogues of anthracyclines Mathematical Models of in Biology to DNA. and Medicine Jacek Leluk: Wrong assumptions and Transgenic Organisms in misinterpretations in molecular biology, Agrobiotechnology and Medicine - biochemistry and bioinformatics. Centre of Excellence PAGEN Workshop on Modifications of commercially Valuable Plants Aleksander Masny, Andrzej Płucienniczak: Bernd Müller-Röber: Transcription factor PCR performed at low denaturation function search: combining genomics and temperatures – PCR melting. single-gene approaches to discover new Joanna Polańska: Using Gaussian mixtures regulatory networks in plant biology. to infer structure in microarray data. Holger Hesse: Transgenic plants as a tool Tomasz Magdziarz: Designing drugs to improve sulfur amino acid content in by docking ligands into proteins - chemical plants. problems and technical challenges. Urte Schlüter: Manipulation of flax for improved Marcin Pacholczyk: Analysis of differences fibre extractability. in protein substitution patterns based Jan Szopa: Flax engineering for fiber improvement. on statistical tests. Marcin Łukaszewicz: The antioxidant potential Satellite workshop: Cytogenetic of transgenic plants. Markers in Assessment of Human Jacek Hennig: Functional analysis of gluB Exposure to Carcinogenic Substances gene in Solanum tuberosum plants. Maria Sąsiadek: Chromosomal aberrations Janusz Zimny: Transgenic crops in the world. and cancer risk. Modern Trends in Diagnosis and Maria Dušinská: Assessment of biomarkers Therapy of Cancer of exposure, effect and individual susceptibility in workers exposed to mineral Beata Utracka-Hutka: Targeted therapy. fibres. Adam Maciejewski, Bogusław Mąka: Francesca Marcon, Riccardo Crebelli: Reconstructive surgery. Can current biomarkers be applied in the Rafał Tarnawski: Radiosurgery. monitoring of low environmental exposures? Brygida Białas: Real-time brachytherapy of Results from an investigation on traffic prostate cancer. wardens of Rome city. Barbara Jarząb: Expression profiles in papillary Grażyna Motykiewicz: Application of thyroid cancer analysed by DNA microarrays. cytogenetic markers in the polluted region of Upper Silesia. Maciej Małecki, Przemysław Janik: Angiogenic gene therapy: a bicistronic strategy. Elżbieta Skrzydlewska: Green tea in cancer prevention. Małgorzata Wierzbicka: Biological implications of failures in solid tumours treatment. (VIII) Gliwice Scientific Meetings 2004

19-20.11.2004

Mathematical Models in Genomics Ulla Kasten-Pisula: Molecular mechanisms and Cancer Biology of the individual radiosensitivity: impact Session supported by Apple IMC Poland SAD on DNA repair. Michał Dąbrowski: Identification of potential Paweł Jałoszyński: Fidelity or survival? cis-regulatory features associated with a How unrepaired DNA is replicated mode of gene expression common to when polymerases encounter problems. neuronal differentiation and hippocampal Barbara Tudek: Processing of lipid development. peroxidation-derived DNA adducts Krzysztof Fujarewicz: Identification of models by repair enzymes and DNA polymerases. of cell signaling pathways. Miroslav Chovanec: Repair of DNA double- Piotr Formanowicz: Isothermic sequencing strand breaks of different origin by hybridisation. in Saccharomyces cerevisiae. Krzysztof Simek: SVD as a tool for pattern discovery in gene expression data. Cellular Stress, Signaling Pathways Bogdan Smołka: Fast denoising of cDNA and Cancer microarray images. Session supported by Committee for Human Genetics and Molecular Pathology, PAS Structure and Function of the Genome Maciej Żylicz: Hsp70 and MDM2 are important for the transcriptional activity of wild type Sergey Razin: The spatial organization of DNA p53. in eukaryotic cell nuclei plays an important role in determining positions of Joanna Rzeszowska-Wolny: Gene expression recombination hot-spots. profiles and cellular stress. Jan Filipski: Epigenetic regulation of Svetlana Sidorenko: CD150-mediated transcription and the origin of isochors. signaling. Piotr Widłak: The other face of histone H1. Lyudmila Sidorik: The role of molecular chaperons in cancer. Vasyl F. Chekhun: New trends in experimental oncology in Ukraine. Jekaterina Erenpreisa: Expression of meiotic genes and phenotype by irradiated tumour New concepts and methods cells. Lise L. Hansen: PCR amplification of highly Functional Genomics in Medicine GC-rich templates. An efficient tool Session supported by State Committee for Scientific in the search for new prognostic markers Research (Grant PBZ-KBN-040/PO4/2001) in cancer. Barbara Jarząb: Genomic approach to thyroid Janusz M. Bujnicki: Protein structure prediction cancer. by consensus fold recognition Małgorzata Wiench: RET protooncogene and assembly of fragments. and gene expression profile of thyroid Gabriel Wcisło: From molecular studies cancer. to clinical practice. An EGFR (epidermal Joanna Polańska: Applications of mixture growth factor receptor) story. models to gene expression profiles. Katarzyna Lisowska: Gene expression profiling DNA Repair Mechanisms and their Role in hereditary breast cancer; preliminary in Carcinogenesis results. Antonina Cebulska-Wasilewska: Response Michał Jarząb: From microarray data to the to challenging dose of x-rays as biomarker biological relevance. of susceptibility in molecular epidemiology.

Wolfgang Goedecke: The fate of DNA double- strand breaks: aspects of mitotic and meiotic cells. Andrzej Wójcik: Do interindividual differences in lymphocyte chromosome radiosensitivity exist?

(IX) Gliwice Scientific Meetings 2005 The 80th Anniversary of Professor Mieczysław Chorąży 18-19.11.2005

Structure and Function of the Genome - Andrzej Świerniak: Antiangiogenic therapy as a Epigenetic Mechanisms control problem William T. Garrard: Mechanisms of activation Anna Marciniak-Czochra: A simple and silencing of the immunoglobulin kappa mathematical model for cell signaling locus. in radiation experiment Andrzej Jerzmanowski: Chromatin remodeling Piotr Czerski: Sekwencjonowanie i synteza and linker histones in plant development. DNA Sergey Razin: Assembly of nuclear matrix - bound protein complexes involved in non-homologous end joining is induced Sympozjum satelitarne: by inhibition of DNA topoisomerase II. Nowe leki i strategie terapeutyczne we Harry Sherthan: Satellite DNA, meiotic współczesnej onkologii telomeres and chromosome evolution. Piotr Wysocki: Efekt przeciwnowotworowy Jan Barciszewski: Diagnosis and therapy kaptoprilu – druga strona medalu. of brain tumors. Elżbieta Pajtasz-Piasecka: Aktywność Response to Stress and Alterations przeciwnowotworowa szczepionki na bazie komórek dendrytycznych transdukowanych of Genome and Transcriptome in genami mysiej IL-12 lub IL-2 u myszy Cellular Pathology z zaawansowanym nowotworem MC38. Lise Lotte Hansen: Genomic instability Dominika Nowis: Badanie mechanizmów of the HCP region in somatic breast cancer. niszczenia komórek nowotworowych przez Ryszard Oliński: Clinical significance terapię fotodynamiczną oraz próby of oxidative DNA damage. potęgowania jej efektywności. Krzysztof Szyfter: Progression of head Danuta Duś: Białka oporności wielolekowej and neck cancer studied by molecular w komórkach macierzystych krwi. cytogenetics. Maciej Ugorski: Wykorzystanie RNAi do Stanisław Cebrat: The role of crossing-over hamowania ekspresji glikozylotransferaz. in the computer simulated populations. Joanna Wietrzyk: Toksyczność nowych analogów witaminy D i ich aktywność in Protein Sciences in Biology and Medicine vitro w terapii kombinowanej z Alfred Pingoud: Engineering of meganucleases. cytostatykami w modelu raka gardła. Janusz M. Bujnicki: Protein structure prediction Katarzyna Szczaurska: Aktywność by combination of fold-recognition and de przeciwnowotworowa bakteriofagów novo holding. w terapii skojarzonej z cytostatykami. Jerzy Ostrowski: Strengths and limitations of Janusz Boratyński: Koniugaty metotreksat – microarray- and mass spectrometry-based nośnik – właściwości chemiczne i methods in clinical research. biologiczne. Michał Dadlez: Application of mass Aleksandra Rusin: Przeciwnowotworowe i spectrometry for the studies of proteomes. przeciwzapalne właściwości nowych Krzysztof Puszyński: Logical analysis of koniugatów niesteroidowych leków proteomic data. przeciwzapalnych i oligomerów 3- hydroksymaślanu. Marcin Pacholczyk: Some algorithms of molecular docking. Tomasz Cichoń: Kombinacja cyklofosfamid z cytokinami indukowanymi przez sekwencje Therapeutic Strategies and Mathematical CpG DNA w hamowaniu wzrostu doświadczalnych przerzutów czerniaka Models in Contemporary Oncology B16(F10) w płucach myszy. Pedro de Campos-Lima: New strategies in therapy of virus-caused tumors Marek Los: Apoptin - a novel, cancer-selective killer

(X) Gliwice Scientific Meetings 2006 The 10th Anniversary of Gliwice Scientific Meetings 17-18.11.2006

Cell Signaling and Responses to Stress Hanna Rokita: Immunotherapy of Carmel Mothersill: Radiation induced neuroblastoma with peptide vaccines based bystander effects in vivo – are they on molecular mimicry of GD2 gangliosides. protective or destructive? Aleksander Sieroń: Zinc-dependent Maciej Żylicz: Role of molecular chaperones metalloproteases and their inhibitors in cell transformation. in biology and diagnostics of cancer. Anna von Mikecz: Quality control in the cell Rafał Tarnawski: Modern radio-oncology. nucleus by the ubiquitin-proteasome Frank Koltrowitz: The experimental power system. of MACSmolecular gene expression Ryszard Oliński: Oxidative DNA damage; analysis products. cause or consequence of cancer? Barbara Tudek: Modulation of oxidative DNA Biotechnologia w Polsce – perspektywy damage repair by oxidative stress and rozwoju i problemy dydaktyczne neoplastic transformation. Technologia: Jolanta Barańska: Nucleotide receptors and their role in cellular signaling in glioma Andrzej Pilc: Nowe leki antypsychotyczne C6 cells. Stanisław Bielecki: Możliwości zewnętrznego Joanna Rzeszowska-Wolny: Intercellular i wewnętrznego stosowania celulozy communication in X-irradiated cells and its mikrobiologicznej w medycynie influence on gene expression profiles. Andrzej Płócienniczak: Produkcja ludzkiego Jarosław Śmieja: What mathematical modeling hormonu wzrostu can help with in analysis of signaling Jan Szopa: Genetyczne modyfikacje roślin pathways? (Lessons from modeling uprawnych of IFN- pathway). Bernard Korzeniewski: Analiza kontroli metabolicznej a biotechnologia Cell Death and Aging OLYMPUS Optical: FluoView 1000 – nowa Ding Xue: Coordination of apoptotic DNA jakość w mikroskopii konfokalnej degradation and cell corpse engulfment Edukacja: in C. elegans. Jacek Leluk: Zdalne nauczanie wczoraj, dziś Ewa Sikora: What if not apoptosis? i jutro. Andrzej M. Składanowski: Too many ways Amalia Guzdek: Biotechnologia w to die: cell death-cell survival dilemma Uniwersytecie Jagiellońskim - dekada in cancer chemotherapy. nauczania. Gregor Meiss: Structure-function relationships Andrzej C. Składanowski: Patologia in DNA-fragmentation factor. molekularna - jak są nauczani studenci Piotr Widłak: Does active DFF need regulation? Międzyuczelnianego Wydziału (How many steps for regulation of apoptotic Biotechnologii Uniwersytetu Gdańskiego nucleases?) i Akademii Medycznej w Gdańsku - analiza SWOT Modern Trends in Cancer Diagnostics Perspektywy: and Therapy Korneliusz Miksch: Rola biotechnologii w Barbara Jarząb: Cancer transcriptome: factors zrównoważonym rozwoju województwa determining gene expression profiles. śląskiego Marek Jakóbisiak: Prospects of cancer Ludwik Tomiałojć: Ekologiczne i ekonomiczne immunotherapy. źródła obaw przed szybkim wprowadzaniem Marek Los: Hijacking cell survival pathways upraw GMO to selectively kill cancer cells. Mieczysław Chorąży: Modyfikacje genetyczne – nadzieje i obawy

XIth Gliwice Scientific Meetings 2007 Molecular Biology and Bioinformatics in Cancer Diagnostics and Therapy 16-17.11.2007 Modeling and Synthetic Approaches Maria Obolenskaya: Novel approach in the in Drug Discovery study of IFN alpha activity. Johann Gasteiger: Explorations into Krzysztof Puszyński: Deterministic model biochemical pathways. of p53|Mdm2 signaling pathway. Beata Walczak: A start-to-end approach to the analysis of proteomic data. Ion Channels in Biology and Medicine Jaroslaw Polanski: The design and discovery Mustafa B. A. Djamgoz: Voltage-gated Na of HIV integrase inhibitors. channel upregulation and potentiation of cellular behaviours in metastatic disease. Alicja Ratuszna: The new photosensitizers and their application in Photodynamic Zbigniew Grzywna: Wound healing as a Therapy. diffusional sorption process. Timothy Madden: An approach to drug Steven White: Making sense of voltage sensors. development in an academic environment. Jerzy Mozrzymas: Pharmacokinetic description Waldemar Priebe: Design and development of GABAergic current modulation by of novel anticancer drugs for treatment benzodiazepines in neurons. of brain tumors. Maria Mycielska: Metabolic pathways in human Bogdan Lesyng: Oncogenic JAK/STAT prostate cancer cells: regulation by voltage- signalling pathways and design of JAK- gated Na channel activity. kinase inhibitors. Krzysztof Dołowy: Ion channels and genetic Grzegorz Grynkiewicz: Sugars in new drug disorders – the cystic fibrosis case. design – a lesson from natural products with anticancer activity. Genomics and Proteomics in Medicine Wiesław Szeja: Glycoconjugates. Development Adam Godzik: Metagenomics provides new of a new biologically active compounds. perspectives on human health. Daria Handkiewicz-Junak: Molecular PET New Trends in Cancer Diagnostics and imaging and gene expression profiling Therapy of malignant tumors. Thoralf Christoffersen: Mechanisms integrating Krzysztof Fujarewicz: Monte Carlo methods signalling from G protein-coupled receptors for genomic data analysis. and EGF receptors in normal and malignant Piotr Widłak: Mass Spectrometry analyses of gastrointestinal cells. the serum proteome - an important tool of Dagny Sandness: Role of different prostanoid clinical proteomics. receptors in the growth-stimulatory effects Joanna Polańska: Application of the Gaussian of prostaglandins. mixture model to proteomic MALDI-ToF Barbara Tudek: DNA repair in the pathology mass spectra. of lung and colon cancer. Tomasz Bieńkowski: Discovery and validation Carmel Mothersill: Harnessing low dose of biomarkers by Mass Spectrometry. radiation responses for radiation protection. Jacek Leluk: Theoretical background and utility Colin Seymour: Natural products as radiation of commonly used sequence multiple response modifiers. alignment tools. Raisa Smolyakova: Prognostic molecular David Hulmes: Controlling the assembly of the biologic markers in breast cancer. cellular microenvironment and applications Walter Mier: The potential of endoradiotherapy in tissue regeneration and repair. for the development of highly specific Aleksander Sieroń: Genetic engineering of ECM cytostatics. compounds for regenerative medicine. Dariusz Iżycki: Genetic antimelanoma vaccines Marek Kowalczuk: Biopolyesters and their - promises and obstacles. synthetic analogues of controlled structure at Marek Los: Targeted cancer therapies the molecular level for regenerative medicine. using derivates of natural products – Anna Szydło: Copolymers of synthetic Brevinin-2R and Apoptin as examples. biodegradable scaffold with collagen type I. Maciej Giefing: Array-GCH based identification Ksymena Urbanek: Characterization of of oncogenes and tumor suppressor genes nonadherent rat bone marrow stem cells involved in laryngeal cancer. (VSEL cells). XIIth Gliwice Scientific Meetings 2008

21-22.11.2008

Responses to Ionizing Radiation and Jerzy Jurka: Mobile DNA elements throughout DNA Damage evolution. Kevin Prise: New insights on bystander Maria Obolenskaya: Folate-related metabolism signalling: The role of DNA damage in human placenta. sensing and repair. Jan Poleszczuk: Tumor development model Carmel Mothersill: Bystander effects and under angiogenic signaling with adapative responses induced by radiation dependence on vessel impairment. exposure. Marie Boyd: Bystander effects and targeted New Drugs and Treatments radiotherapy for gliomas. Ryszard Oliński: Oxidative processes and Joanna Rzeszowska-Wolny: Transcription cancer. profile change after irradiation or in Jean Francois Mouscadet: in silico study bystander cells; differences between cell suggests that Raltegravir-resistant lines. mutations modify the DNA recognition Rachel Martin: Characterizing protein post properties of HIV-1 integrase. translational modifications using MALDI Marek Los: Apoptin and its derivatives as mass spectrometry. molecular templates for the development of Beata Walczak: Proteomics: automated Bcr-Abl kinase inhibitors. electro-phoregram analysis. Alexa Schieck: Identification of the determinant Chryso Kanthou: Response of the tumour of hepatitis B virus liver tropism and its vasculature to radiation and vascular implications for hepatocyte-specific drug disrupting agents. targeting. Walter Mier: The targeting of melanoma Martin Dolezal: Biologically active pyrazines, for endoradiotherapy. the past and the future. Micheline Giphart-Gassler: Towards Experimental Biotechnology a gene expression profile to classify patients for normal tissue toxicity. Wiesław Szeja: Synthetic glycomix for mining natural products as drug leads. Part I. Molecular Channels Grzegorz Grynkiewicz: Synthetic glycomix Mustafa B.A. Djamgoz: Voltage-gated ion for mining natural products as drug leads. channels as mediators of growth factor Part II. effects in metastatic disease. Aleksandra Rusin: Genistein derivatives Adam Szewczyk: Mitochondrial potassium and their biological activity. channels. Krystiana Krzysko: Binding of genisteine Maria Mycielska: Citrate transport in human derivatives to abl and lck protein kinases, prostate cancer cells: regulation by and to microtubules - modelling studies. functional voltage-gated Na+ channel Andrzej Gamian: Glycation of proteins expression. as their modification in physiology Krzysztof Dołowy: The role of mitochondrial and disease. ion channels in ischemic preconditioning. Olena Palyvoda: Molecular organization in self-assembled monolayers used for Systems Biology, Bioinformatics neuronal cell growth. and Signaling Pathways Joanna Polańska: Statistical processing of Miguel Rubi: Somatic exocytosis of serotonin DNA microarray data - detecting subtle mediated by molecular motors. changes of gene expressions. Jerzy Mozrzymas: Impact of acidosis on modulation of GABAergic IPSCs by benzodiazepine receptor agonists.

XIIIth Gliwice Scientific Meetings 2009

20-21.11.2009

2nd Polish-German Cancer Workshop analyses for predicting oncogenic signaling in colon tumors. Molecular Epidemiology of Cancer Anna Fiszer-Kierzkowska: Molecular profiling Kari Hemminki: Genetic basis of familial of histopatologically normal prostate tissue aggregation of cancer. adjacent to cancer. Jan Lubiński: The latest advances in clinical Piotr Widłak: MALDI-TOF MS-based serum genetics of tumors including gastric cancer. proteome pattern analysis in molecular diagnostics of breast cancer. Ute Hamann: Search for modifiers of hereditary breast cancer risk. Molecular Biology, Biotechnology and Asta Försti: Evidence for genetic basis in Bioinformatics in Cancer Diagnostics breast cancer survival. and Therapy. Ewa Grzybowska: Molecular epidemiology of hereditary breast and ovary cancer Bystander Mechanisms as Targets in Silesia, clinical course of breast for Cancer Radiotherapy and ovary cancer in respect to polymorphisms of PGR and MDR1 genes. Carmel Mothersill: Targeting bystander signalling and response pathways for Annekatrin Lukanova: Pregnancy hormones cancer drug development. and maternal cancer. Marie Boyd: Bystander effects elicited by Dorota Butkiewicz: Genetic influences on different radiation qualities - therapeutic survival in lung cancer. opportunities? Federico Canzian: Genome-wide studies Elisabeth Schueltke: Synchrotron radiotherapy in cancer. for human glioma: role of bystander Krzysztof Szyfter: Identification of laryngeal mechanisms. cancer-related genes using high resolution Mansoor Ahmed: High-dose lattice radiation array-CGH technique. therapy: clinical, physics and biological perspective of bystander effects. Functional Genomics in Cancer Research Wojciech Jurczak: Haematologic toxicity Stefan Wiemann: Modeling and testing of cell of radioimmunotherapy.Colin Seymour: cycle regulation via the ErbB protein and Radiation-induced bystander effects in vivo: miRNA network in breast cancer. possible novel targets. Aurelio Teleman: Identification of a James Murphy: Low LET radiation and phosphatase regulating insulin signaling. bystander factor damage to mammalian Marcin Szaumkessel: Pyrosequencing-based mitochondria. DNA methylation profiling of Fanconi Bruce C. McKay: Targeting transcription- anemia/BRCA pathway genes in head and coupled repair as an anti-cancer strategy. neck squamous cell carcinoma. Anna Marciniak-Czochra: Characterization Sandra Steinbrink: Genome-wide functional of the hematopoietic stem cells using screens to identify novel factors in cancer mathematical models. pathways. Krzysztof Puszyński: Crosstalk between p53 Joanna Polańska: New mathematical and nuclear factor-kappaB systems: pro- approaches in analyses of genomic and and anti-apoptotic functions of NF-kB. proteomic data. Joanna Rzeszowska-Wolny: Blood cells, can Barbara Jarząb: Thyroid carcinoma as a model they be important in bystander effects? for gene expression profiling of cancer. Jörg Hoheisel: Functional genomics and Ion Channels and Cancer proteomics in pancreatic cancer research. Miquel Rubi: A thermodynamic description Jolanta Kupryjańczyk: Prognostic and of active transport through channel proteins. predictive factors in ovarian cancer – Walter Stühmer: A potassium channel involved results of verification of DNA microarray in cancer. data. Maria Mycielska: Cloning and characterization Magdalena Skrzypczak, Jerzy Ostrowski: of citrate transporter and its K regulatory Potential and challenges of microarray data component in prostate and prostate cancer. XIVth Gliwice Scientific Meetings 2010

26-27.11.2010

Stress Response Mechanisms and Mateusz Gałuszka: CLC bio-specialized Their Usefulness in Prediction of software solutions for genetics and Cancer Response to Therapy genomics. Luciene Zanchetta: Simulated sunlight-induced William Amos: Exploring patterns of non- damage to mitochondria and mtDNA randomness in human mutations. in human skin cells. Olivier Lichtarge: Evolution: a guide to protein Geza Sáfrány: Low dose radiation induced function and its redesign. transcriptional alterations in bystander Marek Kimmel: Patterns of evolution of human primary human fibroblast cells. genetic disease with implications for deep Carmel Mothersill: Issues in the interpretation sequencing methods. of low dose effects in radiobiology and environmental radiation protection. Colin Seymour: Update on communication of bystander signals between organisms. Rob Mairs: Potent bystander effects are induced by targeted radionuclides. Joanna Rzeszowska-Wolny: Oxidative stress and bystander effect. Lyudmila Drobot: The oncogenic potential of adaptor protein Ruk/CIN85 in human breast adenocarcinoma.

New Drugs and Treatments Ross Hannan: Transcription by RNA polymerase I as target for anticancer therapy. Wiesław Szeja: Synthesis and biological activity of genistein glycosides and glycoconjugates. Ilona Wandzik: Synthesis of complex derivatives of uridine as potential inhibitors of glycosyltransferases. Aleksandra Rusin: Biological activities of synthetic genistein derivatives.

Molecular Modeling and Bioinformatics Tools Andrzej Polanski: Large scale searching for exact tandem repeats in genomes based on the Burrows - Wheeler transform algorithm. Krzysztof Fujarewicz: Genomic data analysis with bootstrapping. Jacek Koronacki: A reliable and simple approach to feature selection and interdependency discovery in supervised classification.

XVth Gliwice Scientific Meetings 2011 18-19.11.2011

Mechanisms of Metastasis Andrzej Świerniak: Bystander effect modeling session co-organized by EACR members using evolutionary games. Claudine Kieda: Tumour angiogenesis Tomasz Wojdyła: Investigating the genealogical normalization in the prevention of relationship among Slavs, Balts and Finns using metastasis. demographic network model. Angels Sierra: Brain metastasis proteins and Maria Wideł: Protective bystander effect. their interactions: a rational predictive biomarkers research. Piotr Widłak: Radiation-related changes in serum proteome profiles detected by MS in blood of Ingeborg Tinhofer: Incidence and prognostic patients treated with RT due to larynx cancer. role of circulating tumor cells in squamous cell carcinoma of the head and neck region. Colin Seymour: The Ego, The Id, and Radiotherapy. Lyudmila B. Drobot: The biological role of Polish-German-American Workshop on adaptor/scaffold protein ruk/cin85 in breast carcinogenesis. Progress in Neurooncology: From the Bench to the Clinic Marek Los: Attempts to target cancer stem cells. Salinomycin as an example. Brain tumours biology Maciej Ugorski: The role of ceramide Bożena Kamińska-Kaczmarek: Glioma initiating cells galactosy-ceramide (UGT8), a new and immune microenvironment of gliomas. molecular marker of breast cancer Marcel Kool: Integrated genomics on molecular malignancy, in cancer progression. subtypes in medulloblastoma. Stanisław Szala: Tumor vasculature in malignant Regulation of Gene Expression gliomas. at the Transcript Level Frank Winkler: New insights into brain tumor Martin Simard: Understanding RNA silencing biology by intravital microscopy. pathways through the Argonaute proteins. Klaus-Rudiger Trott: Why gliomas are radioresistant? Gunter Meister: Analysis of Argonaute Amir Abdollahi: Next Generation NeuroRadioOncology. interactions in mammalian cells. Diagnostics and Therapy Petr Svoboda: Unique regulation of small David Capper: Visualizing mutations: application of RNAs during oocyte-to-embryo transition IDH1 and BRAF mutation specific antibodies in in mammals. neuropathology. Anna Kurzyńska-Kokorniak, Marek Figlerowicz: Barbara Bobek-Billewicz: Brain tumor properties to be Modulation of microRNA biogenesis by using identified by MRI - not always a success story. short oligo-RNA molecules. Henryk Majchrzak: Progress in surgical treatment Krystian Jażdżewski: The role of microRNA of low grade gliomas. sequence variation in thyroid cancer. Pawel Nauman: Different aspects of surgery for Witold Konopka: Hypothalamic miRNA primary brain tumours. suppresses obesity in mice. Volker Budach: Radiosurgery and fractionated Ana Kozomara: The miRbase and the deep- radiotherapy of benign brain tumours. sequencing data. Rafał Tarnawski: Prophylactic cranial irradiation Kathrin Leppek: Roquin promotes rapid TNF with dose reduction in regions of active mRNA degradation via a stem-loop neurogennesis - a pilot study for SCLC patients recognition element. with metastases in brain. Joanna Rzeszowska-Wolny: MicroRNA and Novel Therapeutic Modalities RNA oxidative damage. Volker Budach: Nano-Thermo-Radiotherapy or Genes and Response to Radiation: recurrent gliomas – results of a new therapeutic modality. Low Doses, Bystander Effects Timothy Madden: Translating innovative therapies Carmel Mothersill: Emerging issues in radio- for CNS malignancies - overcoming impediments. biology – the impact of non-targeted effects. Charles Conrad: Oncolytic adenovirus therapy for Marek K. Janiak: Antineoplastic effects of low- brain tumors: a guide for development from the level exposures to ionizing radiation. bench to the bedside in the academic setting. Marek Kimmel: Spatial and stochastic effects Waldemar Priebe: Discovery and development of in models of cell interaction novel therapies for brain cancer. XVIth Gliwice Scientific Meetings 2012 16-17.11.2012

Structure of the Nucleus and Proteomics Regulation of Gene Expression session co-organized by Polish Society of Proteomics Jan Brzeski: Maize in the maze: a rough guide Andreas Rőmpp: High resolution mass to the puzzling epigenetics of paramutation. spectrometry imaging – comprehensive and specific histological information Sergey Razin: Folded chromatin domain instead at cellular resolution. of an active chromatin hub: a model based on reconsidered essentials of the chromosome Jacek R. Wiśniewski: Quantitative study conformation capture procedure. of colorectal cancer to a depth of 10,000 proteins using laser microdissected formalin Sławomir Kumala: The influence of chromatin fixed and paraffin embedded tissue. topology on the accessibility of DNA to damage. Marcus Macht: Proteomics through integrated MALDI and ESI. Ronald Hancock: New models of the nucleus and chromosomes. Jerzy Silberring: Rapid analysis of drugs of abuse as an initial step towards Cellular Responses to Ionizing predictive toxicology. Radiation and Stressing Factors Łukasz Marczak: Mass spectrometry session co-organized by EACR members based analysis of protein Mary Helen Barcellos-Hoff: Microenvironment N-homocysteinylation. matters: contributions to radiation Joanna Polańska: Detection and quantification carcinogenesis. of MALDI ToF spectral peaks by using Carmel Mothersill: Transmission of signals Gaussian mixture decomposition. from irradiated rats to cage mates: Piotr Widłak: Mass profiling of cancer serum an inter-animal bystander effect. proteome - does it provide any useful Colin Seymour: The first cut is the deepest. information? Barbara Tudek: Repair of oxidative DNA Systems Biology damage during development of colon cancer. Mieczysław Chorąży: The systems biology: introductory remarks. Selectively Cytotoxic Proteins Izabela Makałowska: Retrogenes – Mathieu H. Noteborn: Apoptin induces cell death trash or treasure? by targeting various tumor processes. Marek Rusin: Cross-talk between p53 Mahvash Tavassoli: Human Gyrovirus Apoptin and Akt kinase pathways. shows a similar function to VP3/Apoptin. Joanna Janiszewska: microRNAs and their Stian Knappskog: Inactivation of the TP53 importance in laryngeal carcinoma. gene or the p53 regulators Chk2 / ATM Marek Kimmel: Variability and homeostasis predicts resistance to anthracyclines in cycling cell populations. in breast cancer. Andrzej Świerniak: Extended model Marek Los: Modeling of interaction between of interaction between tumour cells. BCR-ABL and apoptin - novel way for argeting the deregulated Abelson kinase activity.

XVIIth Gliwice Scientific Meetings 2013 15-16.11.2013

Regulation of Gene Expression and André Goffeau: Targeting selectively the Replication unique energy metabolism of cancer with a small molecule inhibitor. Anke van den Berg: The role of microRNAs in B cell Hodgkin and non-Hodgkin Andrzej Swinarew: The instrumental analysis of lymphoma. novel polymeric materials for bioapplications. Joost Kluiver: Long noncoding (lnc)RNAs: novel players in B-cell lymphoma. Dietary Factors in Cancer Prevention session co-organized by EACR members Małgorzata Czyż: Heterogeneity of Wanda Baer-Dubowska: Diet and cancer: from melanospheres: therapeutic implications. epidemiological data to mechanism-based Krzysztof Puszyński: Dynamics of intracellular cancer prevention. processes. Adriana Albini: The tumor microenvironment as target for cancer therapy and prevention Cellular Pathways Driven by Reactive by dietary components. Oxygen Species Karen Brown: Development of resveratrol for Sebastian Student: Reactive oxygen species cancer chemoprevention. in the nucleus and cell death. Albena Dinkova-Kostova: Chemoprevention Carmel Mothersill: Radiation-induced non- against cancer by isothiocyanates. targeted effects: horizontal and vertical Jędrzej Antosiewicz: Chemoprevention of transmission of genetic change mediated prostate cancer by organosulfur by oxidative stress? compounds from garlic. Barbara Tudek: Lipid peroxidation modulates Wim Van den Berghe: Promises & challenges DNA repair and sensitizes cells to in epigenetic remodeling of breast cancer genotoxic factors. metastasis by natural withanolides from Natasha Kopitar: Deletion of stefin B gene Withania somnifera (Ashwagandha). enhances mitochondrial ROS formation Agnieszka Bartoszek: Discoveries of and Nlrp3 inflammasome activation. experimental chemoprevention - how to exploit them in rational design of health DNA Repair in Aging and quality and therapeutic foods. Cancerogenesis Leon H Mullenders: Spot on the DNA damage Cancer Killing response: from lesion recognition to human Sun Xiao-Feng: Biomarkers in colorectal disease and therapy. cancer. Wojciech Niedźwiedź: BLM collaborates with Marek Los: Salinomycin – experimental drug TOPBP1 in promoting checkpoint activation with preferred toxicity towards cancer stem and replication fork stability. cells, the role of autophagy in salinomycin Miroslav Pirsel: DNA repair helicase – the story toxicity. with an unexpected end. Nikolaos Sfakianakis: Numerical study of Grażyna Mosieniak: DNA damage response cancer invasion of extracellular matrix. in cancer cell senescence. Katrina Erenpreisa: Senescence, polyploidy, and stemness – three components of the Cancer Proteomics and Metabolomics response of tumour cells to DNA damage. session co-organized by Polish Society of Proteomics Kourosh Lotfi: P-glycoprotein transport Corinna Henkel: MALDI imaging in clinical of the active imatinib metabolite, CGP- research: searching for the Holy Grail. 74588, in chronic myeloid leukemia cells. Angels Sierra: Proteins and protein-protein Jerzy Pieczykolan: Evaluation of novel, interacting motifs to therapeutic target anticancer, targeted fusion biomolecule of metastasis. with high antiangiogenic and cytotoxic Tone F. Bathen: Metabolic profiling of breast activity – summary of preclinical findings. cancer. XVIIIth Gliwice Scientific Meetings 2014 21-22.11.2014

Transcriptional Responses to Stress Franck Delaunay: Multispectral fluorescent Allan R. Brasier: NF-kB signaling and the single live cell imaging to analyse the cell innate immune response. cycle and the circadian clock dynamics. Neil D. Perkins: Regulation of cancer cell Carmel Mothersill: Radiation-induced bystander proliferation and survival by NF-kB. signals: evidence for a role of secondary UVA emission in the generation of oxidative stress Bożena Kamińska: Transcriptional and and bystander effects by beta irradiation of epigenetic mechanisms controlling human keratinocytes. inflammation. Piotr Formanowicz: Petri net based approach Marek Kimmel: Simplicity and complexity for modeling and analysis of selected in models of cell signaling. aspects of atherosclerosis. Tomasz Lipniacki: NF-kB and IRF3 crosstalk Krzysztof Kucharczyk: Minor genetic variants signaling in MEFs. discovery and detection down to 0,1% level Mariusz Hartman: Immediate and long-term at the heterogenic tumor material by the molecular response of melanoma cells MSSCP method. to changes in microenvironment. Michal Braczkowski: Application of ROCHE Diagnostics products for gene expression Biomaterials and Nanomedicine studies. Mehrdad Rafat: Bioengineered collagen constructs for cell-based regeneration of the cornea. Katerina Chlichlia: Magnetic and biogenic Satellite Workshop: nanoparticles for cancer therapy. Genes – Environment Interactions Ramunas Valiokas: Biopatterning techniques Maria Dusińska: Challenges with gene- in tissue engineering. environment interactions: Where we are Łukasz Zarodkiewicz: AFM-IR: Combining and where we need to go? atomic force microscopy and infrared Sofia Pavanello: Monitoring of exposures to spectroscopy for multifunctional carcinogens: Does the procedure need measurements – nanoscale chemical a rethink? characterization. Karin Broberg-Palmgren: Epigenetics of metals. Glycobiology in Medicine Natalia Pawlas: Genetic susceptibility to metals. Anna Lityńska: The role of aberrant Süreyya Meriç Pagano: Multi-bioassay toxicity glycosylation in human melanoma evaluation of a mixture of different group of progression to more malignant phenotype. pharmaceuticals. Marcin Czerwiński: Glycosyltranferases Norbert Kreuzinger: Wastewater treatment and blood group antigens. plants as a hub between clinical and Wojciech Jachymek: Bacterial glycomics: environmental antibiotic resistance. new approach to vaccine production. Aneta Łuczkiewicz: Human-associated Yanusz Wegrowski: Matrix proteoglycans bacteria and their mobile genetic elements - as regulators of tumor progression. important minority in wastewater and wastewater impacted ecosystems. Bioinformatics and Regulatory Aleksandra Ziembińska-Buczyńska: Antibiotics Mechanisms and their resistance genes in the environment – bacterial opportunity or session co-organized by EACR members threat? Till Bretschneider: Image-based modeling of cell motility. Sascha Ott: Analysis of DNase-seq data. XIXth Gliwice Scientific Meetings 2015 20-21.11.2015

Intra-Tumor Heterogeneity and Evolution Klaus H. Maier-Hein: Data-driven oncologic session co-organized by EACR members image analysis. Urszula Hibner: Cancer and evolution. David P. Kreil: Power and limitations of RNA- Anna Golebiewska: Clonal evolution meets Seq: findings from the SEQC consortium. cancer stem cells: in-depth analysis of genetic and phenotypic heterogeneity Roles of Noncoding RNAs in glioblastoma. Gunter Meister: RNA-binding proteins Gareth Wilson: Deciphering cancer genome as regulators of coding and non-coding gene evolution and intra-tumour heterogeneity. expression. Trevor Graham: Intra-tumour heterogeneity Joanna Rzeszowska-Wolny: X-ray induced as a pan-cancer prognostic biomarker. changes in RNA interference suggest independent modulation of mRNA and protein Bożena Kamińska-Kaczmarek: Cancer stem levels. cells and immune microenvironment. Joost Kluiver: Non-coding RNAs as oncogenic Anke van den Berg: Genomic inter- and intra- components of the MYC regulatory network tumor heterogeneity in primary lung cancer in Burkitt lymphoma. and its metastasis. Gabor Szabo: DNA/RNA hybrids in the chromatin. Kathrina Erenpreisa: Sub-cellular heterogeneity and asymmetry in fate choice of irradiated HeLa and etoposide-treated ovarian Mitochondrial Channels and Reactive teratocarcinoma PA1 cells. Oxygen Species Marek Los: Generation of limbal epithelial cell Adam Szewczyk: What we don't know about progenitors by two methods: by differentiation mitochondrial potassium channels. from iPS cells and by direct trans- Krzysztof Dołowy: Ion channels in mitochondria: differentiation from human dermal fibroblasts. the matter of life and death. Piotr Bednarczyk: Regulation of mitochondrial Radiation Biology potassium channels. Serge Candeias: Effects of low dose radiation on Przemysław Borys: Estimation of the maximum the T lymphocyte repertoire in the mouse: flux through ion channel. analysis from a TCR point of view Magdalena Skonieczna: Inhibition of the voltage- Ingeborg Tinhofer-Keilholz: Mutational profiling dependent anion channels and their influence using next-generation sequencing reveals on cell functioning. distinct molecular mechanisms of Jakub Hanus: Induction of necroptosis by radioresistance in HPV+ and HPV- head and oxidative stress in retinal pigment epithelial neck cancer. cells. Piotr Widłak: Signature of serum proteome in Katarzyna Szołtysek: Role of reactive oxygen patients exposed to ionizing radiation. species in crosstalk between UV and cytokine Karol Jelonek: Characterization of exosomes activated signaling. released from irradiated cells. Szymon Borek: Modification in modern tools for Andrzej Swinarew: MALDI-ToF and UHPLC cell based assays including mitochondria and in peptide and protein characterization - reactive oxygen species analysis. potential application in radiation research. Bioinformatics Jacek Koronacki: Discovering interdependencies of features in disease-related genomic data. Jan Komorowski: Genes causing many cancers are located near tumor-mutated motifs for the CTCF and other transcription factors. Marek Kimmel: Modeling lung cancer drivers based on the cancer genome atlas data. Lecturers that contributed to Gliwice Scientific Meetings in years 1997-2015

Abdollahi, Amir German Cancer Research Center, Heidelberg Albini, Adriana IRCCS MutiMedica, Milan Amos, William Cambridge University, Cambridge Antosiewicz, Jędrzej Medical University of Gdansk, Gdańsk Baer-Dubowska, Wanda Poznan University of Medical Sciences, Poznań Barańska, Jolanta Marceli Nencki Institute of Experimental Biology, PAS, Warszawa Barcellos-Hoff, Mary Helen New York University School of Medicine, New York Barciszewski, Jan Institute of Bioorganic Chemistry, PAS, Poznań Bartnik, Ewa University of Warsaw, Warszawa Bartoszek, Agnieszka Medical University of Gdansk, Gdańsk Bathen, Tone F. Norwegian University of Science and Technology, Trondheim Bednarczyk, Piotr Warsaw University of Life Sciences, Warszawa Bielecki Stanisław Technical University of Lodz, Łódź Blaese, Marcel University of Tubingen, Tubingen Bobek-Billewicz, Barbara MSC Cancer Center and Institute of Oncology, Gliwice Borys, Przemysław Silesian University of Technology, Gliwice Boyd, Marie Cancer Research UK, Glasgow Brammer, Ingo University of Hamburg, Hamburg Brasier, Allan R. University of Texas, Medical Branch, Galveston Bretschneider, Till Warwick Systems Biology Centre, Coventry Broberg-Palmgren, Karin Karolinska Institutet, Lund Brown, Karen University of Leicester, Leicester Brzeski, Jan Copernicus Science Center, Warszawa Budach, Volker Charite University, Berlin Bujnicki, Janusz M. International Institute of Molecular and Cellular Biology, Warszawa Butkiewicz, Dorota MSC Cancer Center and Institute of Oncology, Gliwice Campos-Lima, Pedro Laval University, Quebec Candeias, Serge CEA, Grenoble Canzian, Federico German Cancer Research Center, Heidelberg Capper, David German Cancer Research Center, Heidelberg Cebrat, Stanisław University of Wroclaw, Wrocław Cebulska-Wasilewska, Antonina Institute of Nuclear Physics, PAS, Kraków Chekhun, Vasyl F. Institute of Experimental Pathology and Oncology, Kiev Chlichlia, Katerina Democritus University of Thrace, Alexandroupolis Chorąży, Mieczysław MSC Cancer Center and Institute of Oncology, Gliwice Chovanec, Miroslaw Cancer Research Institute, Bratislava Christoffersen, Thoralf University of Oslo, Oslo Conrad, Charles University of Texas, MD Anderson Cancer Center, Houston Crebelli Riccardo Istituto Supriore di Sanita, Rome Czerwiński, Marcin Ludwik Hirszfeld Institute of Immunology, PAS, Wrocław Czyż, Małgorzata Medical University of Lodz, Łódź Dadlez, Michał Institute of Biochemistry and Biophysics, PAS, Warszawa Dąbrowski, Michał Marceli Nencki Institute of Experimental Biology, PAS, Warszawa Delaunay, Franck University of Nice, Nice Dinkova-Kostova, Albena University of Dundee, Dundee Djamgoz, Mustafa B.A. Imperial College, London Dolezal Martin Charles University, Prague Dołowy, Krzysztof Warsaw University of Life Sciences, Warszawa Domagala, Wenancjusz Pomeranian Medical University, Szczecin Drobot, Ludmila B. Palladin Institute of Biochemistry, NASU, Kyiv Drouin, Regen Laval University, Quebec Dusińska, Maria Norwegian Institute for Air Research, Kjeller Dux, Kazimierz MSC Cancer Center and Institute of Oncology, Warszawa Dziadkowiec, Dorota University of Wroclaw, Wrocław Erenpreisa, Katrina University of Riga, Riga Fajkus, Jiri Institute of Biophysics, Brno Figlerowicz, Marek Institute of Bioorganic Chemistry, PAS, Poznań Filipski, Jan Institute of Jacques Monod, Paris Fiszer-Kierzkowska, Anna MSC Cancer Center and Institute of Oncology, Gliwice Formanowicz, Piotr Poznan University of Technology, Poznań Försti, Asta German Cancer Research Center, Heidelberg Fujarewicz, Krzysztof Silesian University of Technology, Gliwice Gamian Andrzej Medical University of Wroclaw, Wrocław Garrard, William T. UT Southwestern Medical Center, Dallas Gasteiger, Johann Computer-Chemie-Centre, Erlangen-Nurnberg Gaudray, Patrick CNRS, Nice Getto Philippe Technische Universitat Dresden, Dresden Giefing, Maciej Institute of Human Genetics, PAS, Poznań Giel-Pietraszyk, Małgorzata Institute of Bioorganic Chemistry, PAS, Poznań Giphart-Gassler, Micheline Leiden University Medical Center, Leiden Gniazdowski, Marek Medical University of Lodz, Łódź Goc, Anna Nicolaus Copernicus University, Toruń Godzik, Adam Joint Center for Structural Genomics, La Jolla Goedecke, Wolfgang University of Essen, Essen Goffeau, André Institut des Sciences de la Vie, Louvain-la-Neuve Golebiewska Anna Luxembourg Institute of Health, Luxembourg Goncharova, Rose Institute of Genetics and Cytology, Minsk Graham, Trevor Queen Mary University of London, London Grąziewicz, Maria Institute of Biochemistry and Biophysics, PAS, Warszawa Grynkiewicz, Grzegorz Pharmaceutical Research Institute, Warszawa Guzdek Amalia Jagiellonian University, Kraków Grzybowska, Ewa MSC Cancer Center and Institute of Oncology, Gliwice Grzywna, Zbigniew Silesian University of Technology, Gliwice Hamann, Ute German Cancer Research Center, Heidelberg Hancock, Ronald Laval University Cancer Research Centre, Québec Handkiewicz-Junak, Daria MSC Cancer Center and Institute of Oncology, Gliwice Hannan, Ross Research Division, Peter MacCallum Cancer Centre, Melbourne Hansen, Lise L. University of Aarhus, Aarhus Hanus, Jakub Tulane University, New Orleans Hartman, Mariusz Medical University of Lodz, Łódź Hemminki, Kari German Cancer Research Center, Heidelberg Henkel, Corinna Ruhr University, Bochum Hennig, Jacek Institute of Biochemistry and Biophysics, PAS, Warszawa Hennig, Wolfgang Shanghai Institutes for Biological Sciences, Shanghai Hesse, Holger Max-Planck-Institute, Golm Hibner, Urszula University of Montpellier, Montpellier Hoheisel, Jörg German Cancer Research Center, Heidelberg Hulmes, David Institut de Biologie et Chimie des Protéines, CNRS, Lyon Iżycki, Dariusz Poznan University of Medical Sciences, Poznań Jachymek, Wojciech Ludwik Hirszfeld Institute of Immunology, PAS, Wrocław Jakimowicz, Dagmara Ludwik Hirszfeld Institute of Immunology, PAS, Wrocław Jakóbisiak, Marek Medical University of Warsaw, Warszawa Jałoszyński, Paweł Institute of Human Genetics, PAS, Poznań Janiak, Marek K. Military Institute of Hygiene and Epidemiology, Warszawa Janik, Przemysław MSC Cancer Center and Institute of Oncology, Warszawa Janiszewska, Joanna Institute of Human Genetics, PAS, Poznań Jarząb, Barbara MSC Cancer Center and Institute of Oncology, Gliwice Jarząb, Michał MSC Cancer Center and Institute of Oncology, Gliwice Jażdżewski, Krystian Comprehensive Cancer Center, Columbus Jelonek, Karol MSC Cancer Center and Institute of Oncology, Gliwice Jerzmanowski, Andrzej Institute of Biochemistry and Biophysics, PAS, Warszawa Jerzy Ostrowski MSC Cancer Center and Institute of Oncology, Warszawa Jurczak, Wojciech Jagiellonian University, Kraków Jurka, Jerzy Genetic Information Research Institute, Mountain View Kaminska, Bozena Marceli Nencki Institute of Experimental Biology, PAS, Warszawa Kanthou, Chryso Sheffield University, Sheffield Kasten-Pisula, Ulla Eppendorf University, Hamburg Kieda, Claudine Centre for Molecular Biophysics, UPR 4301 CNRS, Orleans Kimmel, Marek Rice University, Houston, Silesian University of Technology, Gliwice Kluiver, Joost University of Groningen, Groningen Knappskog, Stian University of Bergen, Bergen Komorowski, Jan Uppsala University, Uppsala Konieczny, Igor University of Gdansk, Gdańsk Konopa, Jerzy Gdansk University of Technology, Gdańsk Konopka, Witold German Cancer Research Center, Heidelberg Kool, Marcel German Cancer Research Center, Heidelberg Kopitar, Natasha Josef Stefan Institute, Ljubljana Koronacki, Jacek Institute of Computer Science, PAS, Warszawa Korzeniewski Bernard Jagiellonian University, Kraków Kowalczuk, Marek Centre of Polymer and Carbon Materials, PAS, Zabrze Kozomara, Ana University of Manchester, Manchestr Kreil, David P. University of Natural Resources and Life Sciences, Vienna Kreuzinger, Norbert Vienna University of Technology, Vienna Kruszewski, Marcin Institute Nuclear Chemistry and Technology, Warszawa Krzysko Krystiana University of Warsaw, Warszawa Krzyżosiak, Włodzimierz Institute of Bioorganic Chemistry, PAS, Poznań Kubicarowa, T. Institute of Biophysics, Brno Kujawski, Maciej Institute of Human Genetics, PAS, Poznań Kulma Anna University of Wroclaw, Wrocław Kumala, Sławomir McGill University, Montreal Kupryjańczyk, Jolanta MSC Cancer Center and Institute of Oncology, Warszawa Kurzyńska-Kokorniak, Anna Institute of Bioorganic Chemistry, PAS, Poznań Lachowicz, Mirosław University of Warsaw, Warszawa Larsson, Catharina Pharmacology Biovitrum, Stockholm Leluk, Jacek University of Zielona Góra, Zielona Góra Leppek, Kathrin German Cancer Research Center, Heidelberg Lesyng, Bogdan University of Warsaw, Warszawa Lichtarge, Olivier Baylor College of Medicine, Houston Limon, Janusz Medical University of Gdansk, Gdańsk Lipniacki, Tomasz Institute of Fundamental Technological Research, PAS, Warszawa Lityńska, Anna Jagiellonian University, Kraków Los, Marek Linkoping University, Linkoping Lotfi, Kourosh Linkoping University, Linkoping Lubiński, Jan Pomeranian Medical University, Szczecin Lukanova, Annekatrin German Cancer Research Center, Heidelberg Łuczkiewicz, Aneta Gdansk University of Technology, Gdańsk Łukaszewicz, Marcin University of Wroclaw, Wrocław Macht, Marcus Bruker Daltonics, Bremen Madden, Timothy University of Texas MD Anderson Cancer Center, Houston Magdziarz Tomasz University of Silesia, Katowice Maier-Hein, Klaus H. German Cancer Research Center, Heidelberg Mairs, Rob UK Beatson Laboratories, Glasgow Majchrzak, Henryk Silesian Medical University, Katowice Majka, Jerzy Ludwik Hirszfeld Institute of Immunology, PAS, Wrocław Makałowska, Izabela Adam Mickiewicz University, Poznań Makałowski, Wojciech Pennsylvania State University, University Park Malanga, Maria Institute for Veterinary Pharmacology and Toxicology, Zurich Małecki, Maciej MSC Cancer Center and Institute of Oncology, Warszawa Mansoor, Ahmed University of Miami, Miami Marciniak-Czochra, Anna University of Heidelberg, Heidelberg Marcon, Francesca Istituto Supriore di Sanita, Rome Marczak, Łukasz Institute of Bioorganic Chemistry, PAS, Poznań Markiewicz, Ewa University of Wroclaw, Wrocław Markiewicz, Wojciech Institute of Bioorganic Chemistry, PAS, Poznań Martin Rachel Shimadzu Biotech Kratos Analytical Marszałek, Jarosław University of Gdańsk, Gdańsk Masny, Aleksander Institute of Biotechnology and Antibiotics, Warszawa Matter, Jean-Marc University of Lausanne, Lausanne McKay, Bruce C. Ottawa Hospital Research Institute, Ottawa Meiss, Gregor Justus Liebig University, Giessen Meister, Gunter Regensburg University, Regensburg Meriç Pagano, Süreyya Namik Kemal University, Tekirdag Mier, Walter Universitätsklinikum Heidelberg, Heidelberg Milcarz, Magdalena University of Wroclaw, Wrocław Mirowski, Marek Medical University of Lodz, Łódź Mosieniak, Grażyna Marceli Nencki Institute of Experimental Biology, PAS, Warszawa Mothersill, Carmel McMaster University, Hamilton Mouscadet, Jean Francois Ecole Normale Supérieure de Cachan CNRS, Cachan Motykiewicz, Grażyna MSC Cancer Center and Institute of Oncology, Gliwice Mozrzymas, Jerzy Wrocław Medical University, Wrocław Mullenders, Leon H. Leiden University, Leiden Muller-Rober, Bernd University of Potsdam, Potsdam Murphy, James Institute of Technology Sligo, Ballinode Sligo Mycielska, Maria Imperial College, London Nakielski, Jerzy University of Silesia, Katowice Nauman, Paweł Institute of Psychiatry and Neurology, Warszawa Niedźwiedź, Wojciech Oxford University, Oxford Nino, Ewa INSERM, Curie University, Paris Nino, Jacques Ecole Normale Superieure, Paris Noteborn, Mathieu H. Biological Chemistry, Leiden University, Leiden Obolenskaya, Maria Institute of Molecular Biology and Genetics, NAS, Kiev Oliński, Ryszard Nicolaus Copernicus University, Bydgoszcz Ortmann, Elisabeth University of Vienna, Vienna Ostrowski, Jerzy MSC Cancer Center and Institute of Oncology, Warszawa Ott, Sascha University of Warwick, Coventry Palyvoda, Olena Wayne State University, Detroit Pavanello, Sofia University of Padova, Padova Pawlak, Andrzej Institute of Human Genetics, Poznań Pawlas, Natalia Institute of Occupational Medicine and Environmental Health, Sosnowiec Perkins, Neil D. Newcastle University, Newcastle Philmonienko, Vlada Institute of Experimental Medicine, Prague Pilc, Andrzej Institute of Pharmacology, PAS, Kraków Pingoud, Alfred Justus Liebig University, Giessen Pirsel, Miroslav Cancer Research Institute, SAS, Bratislava Płucienniczak, Andrzej Institute of Biotechnology and Antibiotics, Warszawa Polanski, Andrzej Silesian University of Technology, Gliwice Polanski, Jaroslaw University of Silesia, Katowice Polańska, Joanna Silesian University of Technology, Gliwice Poleszczuk, Jan University of Warsaw, Warszawa Priebe, Waldemar University of Texas, MD Anderson Cancer Center, Houston Prise, Kevin Gray Cancer Institute, Northwood Rabakon Nadzeya Institute of Genetics and Cytology, Minsk Puszyński, Krzysztof Silesian University of Technology, Gliwice Radzikowski, Czesław Institute of Immunology and Experimental Therapy, Wrocław Rafat, Mehrdad Linköping University, Linköping Ratuszna, Alicja University of Silesia, Katowice Razin, Sergey Institute of Gene Biology, RAS, Moscow Rokita, Hanna Jagiellonian University, Kraków Rőmpp, Andreas Justus Liebig University, Giessen Rothkamm, Kai University of Saar, Homburg Rubi, Miguel Universitat de Barcelona, Barcelona Rybaczek Dorota Universityof Lodz, Lódź Rubi, Miquel Universitat de Barcelona, Barcelona Rusin, Aleksandra MSC Cancer Center and Institute of Oncology, Gliwice Rusin, Marek MSC Cancer Center and Institute of Oncology, Gliwice Rzepecki, Ryszard University of Wroclaw, Wrocław Rzeszowska-Wolny, Joanna Silesian University of Technology, Gliwice Sáfrány, Geza National Research Institute for Radiobiology and Radiohygiene, Budapest Sandness, Dagny University of Oslo, Oslo Sąsiadek, Maria Medical University of Wroclaw, Wrocław Schieck, Alexa Universitätsklinikum Heidelberg, Heidelberg Schluter, Urte Riso National Laboratory, Copenhagen Schueltke, Elisabeth Albert Ludwigs University, Freiburg Seymour, Colin McMaster University, Hamilton Sfakianakis, Nikolaos University of Mainz, Mainz Sherthan, Harry Institute of Radiobiology, Muenhen Shieck, Valentin Institute of Molecular Biology, RAS, Moscov Sidorenko, Svetlana Institute of Experimental Pathology and Oncology, Kiev Sidorik, Lyudmila Institute of Molecular Biology and Genetics, Kiev Siedlecki, Janusz MSC Cancer Center and Institute of Oncology, Warszawa Sieroń, Aleksander Medical University of Silesia, Katowice Sierra, Angels Bellvitge Biomedical Research Institute, IDIBELL, Barcelona Siewiński, Maciej Medical University of Wroclaw, Wrocław Sikora, Ewa Marceli Nencki Institute of Experimental Biology, Warszawa Silberring, Jerzy AGH University of Science and Technology, Kraków Simard, Martin Laval University, Quebec Simek, Krzysztof Silesian University of Technology, Gliwice Sirko, Agnieszka Institute of Biochemistry and Biophysics, PAS, Warszawa Sjakste, Nicolai University of Latvia, Riga Składanowski, Andrzej M. Medical University of Gdańsk, Gdańsk Skonieczna, Magdalena Silesian University of Technology, Gliwice Skrzydlewska, Ewa Medical University of Bialystok, Białystok Skrzypczak, Magdalena MSC Cancer Center and Institute of Oncology, Warszawa Smolyakova, Raisa Alexandrov Research Institute of Oncology and Medical Radiology, Minsk Smołka, Bogdan Silesian University of Technology, Gliwice Staroń, Krzysztof University of Warsaw, Warszawa Steffen, Jan MSC Cancer Center and Institute of Oncology, Warsaw Steinbrink, Sandra German Cancer Research Center, Heidelberg Strätling, Wolf H. Eppendorf University, Hamburg Student, Sebastian Silesian University of Technology, Gliwice Stühmer, Walter Max-Planck Institute of Experimental Medicine, Göttingen Svoboda, Petr Institute of Molecular Genetics, ASCR, Prague Swinarew, Andrzej University of Silesia, Katowice Sykorowa, Ewa Institute of Biophysics, Brno Szabo, Gabor University of Debrecen, Debrecen Szala, Stanisław MSC Cancer Center and Institute of Oncology, Gliwice Szaumkessel, Marcin Institute of Human Genetics, PAS, Poznan Szeja, Wiesław Silesian University of Technology, Gliwice Szewczyk, Adam Marceli Nencki Institute of Experimental Biology, PAS, Warszawa Szołtysek, Katarzyna MSC Cancer Center and Institute of Oncology, Gliwice Szopa, Jan University of Wroclaw, Wrocław Sztajer, Helena Technical University of Braunschweig, Braunschweig Szumiel, Irena Institute Nuclear Chemistry and Technology, Warszawa Szydło, Anna Medical University of Silesia, Katowice Szyfter, Krzysztof Institute of Human Genetics, PAS, Poznań Świerniak, Andrzej Silesian University of Technology, Gliwice Tarnawski, Rafał MSC Cancer Center and Institute of Oncology, Gliwice Tavassoli, Mahvash King's College London, Guy's Hospital Campus, London Teleman, Aurelio German Cancer Research Center, Heidelberg Tinhofer-Keilholz, Ingeborg Charite University, Berlin Tomiałojć, Ludwik University of Wroclaw, Wroclaw Trott, Klaus-Rudiger Technical University of Munich, Munich Tudek, Barbara Institute of Biochemistry and Biophysics, PAS, Warszawa Ugorski, Maciej Ludwik Hirszfeld Institute of Immunology, PAS, Wrocław Unold, Olgierd Technical University of Wroclaw, Wrocław Urbanek, Ksymena Medical University of Silesia, Katowice Valiokas, Ramunas Center for Physical Sciences and Technology, Vilnius Van den Berg, Anke University of Groningen, Groningen Van den Berghe, Wim University Antwerp, Antwerp Von Mikecz, Anna Heinrich Heine University, Dusseldorf Walczak, Beata University of Silesia, Katowice Walewski, Jan MSC Cancer Center and Institute of Oncology, Warsaw Wcisło, Gabriel Military Medical Institute, Warszawa Wandzik, Ilona Silesian University of Technology, Gliwice Wegrowski, Yanusz CNRS MEDyC, Reims University, Reims Węgrzyn, Grzegorz Gdańsk University of Technology, Gdańsk Wesierski-Gadek, Józefa Medical University of Vienna, Vienna White, Steven University of California, Irvine Wideł, Maria Silesian University of Technology, Gliwice Widłak, Piotr MSC Cancer Center and Institute of Oncology, Gliwice Widłak, Wiesława MSC Cancer Center and Institute of Oncology, Gliwice Wiemann, Stefan German Cancer Research Center, Heidelberg Wierzbicka, Małgorzata Institute of Human Genetics, PAS, Poznań Wilczyński, Grzegorz University of Wroclaw, Wrocław Wilson, Gareth Cancer Research UK, London Research Institute London, London Winkler, Frank German Cancer Research Center, Heidelberg Wiśniewski, Jacek R. Max-Planck-Institute for Biochemistry, Martinsried Wojdyła, Tomasz Silesian University of Technology, Gliwice Wójcik, Andrzej Stockholms Universitet, Stockholm Wróbel, Magdalena University of Wrocław, Wrocław Wyszko, Eliza Institute of Bioorganic Chemistry, PAS, Poznań Youdinkova, Elena Institute of Gene Biology, RAS, Moscow Xiao-Feng, Sun Linkoping University, Linkoping Xue, Ding University of Colorado, Boulder Zakrzewska, Jolanta Ludwik Hirszfeld Institute of Immunology, PAS, Wrocław Zanchetta, Luciene Institute of Technology SligoAsh Lane, Sligo Zdzienicka, Margaret University of Leiden, Leiden Ziembińska-Buczyńska, Aleksandra Silesian University of Technology, Gliwice Zimny, Janusz A. Plant Breeding and Aclimatization Institute, Radzików Żylicz, Maciej International Institute of Molecular and Cellular Biology, Warszawa

Best Poster Presentations Awards of the Association for the Support of Cancer Research

2015 - Kristine Salmina, Anda Huna, Jekaterina Erenpreisa: Role of autophagy in survival of embryonal carcinoma cells treated with etoposide 2014 - Jolanta Guz, Daniel Gackowski, Marek Foksinski, Rafal Rozalski, Tomasz Dziaman, Ewelina Zarakowska, Anna Szpila, Ryszard Olinski: 5-methylcytosine, 5-hydroxymethylcytosine and 5- hydroxy-methyluracil in human leukocytes and sperm as epigenetic marks – comparison of the absolute level

2013 - Jadwiga Pietkiewicz, Urszula Bazylińska, Joanna Rossowska, Anna Choromańska, Andrzej Gamian, Kazimiera Anna Wilk: Biocompatibility of nanoparticles loaded with photosensibilizers for practical use in support of anticancer therapy

2012 - Karol Ruszel, Paweł Kowalczyk, Agnieszka Maciejewska, Jarosław Kuśmierek: The effect of chloracetaldehyde on M13MP18 phage replicated in Escherichia coli JM 105 ALKB strain

2011 - Małgorzata Sekuła, Katarzyna Miękus, Marcin Majka: SDF-1/ITAC – CXCR7 axis in biology of cervical carcinoma 2010 - Marek Żurawski: Generation of induced pluripotent stem cells from human mesenchymal and hematopoietic cells 2009 - Andriy Slonchak, Agata Chwieduk, Jhoanna Rzerzowska-Wolny, Maria Obolenska: Regulation of the human glutathione s-transferase P1 gene transcription 2008 - Wojciech Pigłowski, Piotr Filipczak, Zdzisław Krawczyk, Dorota Ścieglińska: Analysis of intracellular localization of the human HspA2 protein in cancer cells 2007 - Paweł Kowalczyk, Jolanta Jaworek, Michalina Kot, Beata Sokołowska, Aleksandra Bieleń, Beata Janowska, Jarosław M. Cieśla, Grzegorz Szparecki, Benita Sadom, Barbara Tudek: Inflammation increases oxidative DNA damage repair and stimulates preneoplastic changes in colons of newborn rats 2006 - Subbareddy Maddika, Emilia Wiechec, Lise-Lotte Hansen, Marek Los: Pro-apoptotic role of PI3- K/AKT pathway during apoptosis induced by selected anti-cancer drugs 2005 - Halina Waś, Tomasz Cichoń, Anna Ratajska, Anna Grochot, Barbara Węgiel, Agnieszka Łoboda, Agnieszka Jaźwa, Józef Dulak, Alicja Józkowicz: Effect of heme oxygenase-1 on proliferation, viability and angiogenic potential of melanoma cells in mice 2004 - Magdalena Kalinowska, Wojciech Garncarz, Monika Pietrowska, Piotr Widłak: Regulation of the human apoptotic nuclease endonuclease G 2003 - Ewa Ciesielska, Kazimierz Studzian, Elżbieta Żyner, Justyn Ochocki, Leszek Szmigiero: Cisplatin with aminoflavone wings as an improved apoptosis inducer of cancer cells 2002 - Wiesława Widłak, Witold Konopka, Anna Zborek, Dorota Ścieglińska, Zdzisław Krawczyk: GFP fluorescence in somatic cells of transgenic mice containing EGFP reporter gene under the control of the rat testis-specific hst70 gene promoter 2001 - Paweł Kowalczyk, Jarosław M. Cieśla, Maria-Anna Grąziewicz, Jarosław T. Kuśmierek, Barbara Tudek: DNA damage in P53 gene by chloroacetaldehyde and trans-4-hydroxy-2-nonenal 2000 - Olena Palyvoda, Piotr Widłak: Characterization of DFF40/CAD endonuclease - mediated pathway of apoptotic chromatin condensation

XXth Gliwice Scientific Meetings

November 18-19, 2016

. 20th Gliwice Scientific Meetings Gliwice, 18-19 November 2016 Program Friday; 18th November, 2016 9.00 – 9.15 Opening Ceremony 9.15 – 11.45 Session I: Crosstalk between the Heat Shock Response and Cancer Chairperson: Zdzisław Krawczyk Gabrielle Multhoff (TUM, Munich): Heat shock response and NK mediated immunity in cancer therapy Lea Sistonen (Abo Academi University, Turku): Transcriptional re-programming of genes in cell stress and cancer - synergistic and opposite effects of heat shock factors, HSFs Michael Y. Sherman (Boston University School of Medicine, Boston): Role of Hsp70 in tumor initiation and progression Maciej Żylicz (International Institute of Molecular and Cell Biology, Warsaw): Chaperone mediated cancer cell chemoresistance Wiesława Widłak (Center of Oncology, Gliwice): HSF1 contribution to cancer cell phenotype Dorota Ścieglińska (Center of Oncology, Gliwice): HSPA2 - cytoprotective or regulatory protein?

11.45 – 12.15 Coffee break

12.15 – 15.00 Session II: Molecular and Numerical Gears of Circadian Clocks Chairperson: Franck Delaunay Hans Reinke (Medical University, Dusseldorf): Crosstalk of stress response pathways and the circadian clock in mammals

Francis Lévi (Warwick Medical School, Warwick University, Coventry): Personalising cancer treatments through systems chronotherapeutics

Elham Farshaid (Erasmus Medical Center, Rotterdam): The circadian clock proteins BMAL1 and CLOCK, control G2/M cell cycle transition

Celine Feillet (Université Nice Sophia Antipolis, Nice): The circadian clock-cell cycle connection

Joanna Rzeszowska-Wolny (Silesian University of Technology, Gliwice, Poland): Reactive oxygen species and circadian rhythms Krzysztof Psiuk-Maksymowicz (Silesian University of Technology, Gliwice): Platform for remote testing and analyzing of biomedical data Marek Żurawski (NanoTemper Technologies): MicroScale Thermophoresis: quantitative interaction analysis and beyond

15.00 – 16.00 Lunch

16.00 – 18.00 Poster Session (and coffee)

18.00 – 19.00 Concert for 20th Anniversary of the Gliwice Scientific Meetings

20.00 – 23.00 Conference Dinner and Party (Karczma Rajcula, bus transportation) Saturday; 19th November, 2016 9.00 – 11.00 Session III: Chromatin Structure and Modulation of Transcription (session organized by EACR members) Chairperson: Andrzej Bednarek Marcelo Aldaz (MD Anderson Cancer Center, Houston): WWOX, the FRA16D chromosomal fragile site gene, at the crossroads of cancer and CNS pathology Rami Aqeilan (Hebrew University, Jerusalem): Role of tumour suppressor WWOX, the gene product of common fragile site FRA16D, in human diseases Andrzej Bednarek (Medical University of Łódź, Łódź): The role of Notch signaling pathway in regulation of gene expression in cancer Sergey Razin (Lomonosov University, Moscow): Dynamics and self-organization of Drosophila chromatin into topologically-associating domains 11.00 – 11.30 Coffee break

11.30 – 14.00 Session IV: Regulatory RNA Chairperson: Witold Konopka Angel Barco (Miguel Hernández University, Alicante): Interplay of transcriptional and epigenetic mechanisms in neuronal activity-driven gene expression Włodzimierz Krzyżosiak (Institute of Bioorganic Chemistry, Poznan): Small RNAs in triplet repeat diseases Krzysztof Chylinski (CRISPR-Lab, VBCF ProTech, Vienna): Sharpening the knives of CRISPR/Cas9 Marek Los (Linköping University, Linköping): Negative regulatory loop between miRNA301 and Akt - significance for tumor growth Krzysztof Fujarewicz (Silesian University of Technology, Gliwice): A model for the effects of ionizing radiation on microRNA-mediated regulation of mRNA levels in cells Witold Konopka (Nencki Institute of Experimental Biology, Warsaw): Neuronal RNA processing in memory formation 14.00 – 15.00 Lunch 15.00 – 16.45 Session VI: Biomaterials and Medical Biotechnology Chairperson: Marek Los Mehrdad Rafat (Linköping University, Linköping): LinkCor® bioeingineered comea: a viable alternative to human comea for keratoconus and comeal dystrophy patients Judith Staerk (University of Oslo, Oslo): Lineage specific regulation of Lamin B1 associated chromatin domains in the hematopoietic tissue Emilia Wiechec (Linköping University, Sweden): The rate-limiting enzyme in carbohydrate metabolism, PFKFB3 - differential exopression in IPS and cancer stem cells Jacek Kubiak (French National Centre for Scientific Research, Paris): Preventive measures against chronic rejection of heart transplants Anna Mielańczyk (Silesian University of Technology, Gliwice): Polymethacrylates as drug carriers - structure vs cell viability in MCF-7 cell lines (15’)

16.45 – 17.15 Best Poster Presentations

17.15 – 17.30 Concluding Remarks and Closing Ceremony

17.30 – Coffee and Discussions

Lecture abstracts

2 .

Session I

Crosstalk between the Heat Shock Response and Cancer

. 3 4 . HEAT SHOCK RESPONSE AND NK MEDIATED IMMUNITY IN CANCER THERAPY

Gabriele Multhoff, Stefan Stangl, Maxim Shevtsov

Klinikum rechts der Isar TU München

The major stress-inducible Hsp70, a member of the HSP70 (HSPA1) family is involved in a number of chaperoning functions such as protein folding of newly synthesized polypeptides, transport of proteins across lipid membranes, prevention of protein aggregation, protein degradation, protection against apoptosis, antigen processing and cross-presentation. Hsp70 is frequently overexpressed in many different tumor types and metastases. We found that Hsp70 is also presented on the plasma membrane of tumor, but not normal cells. This tumor-specific membrane localization is enabled by an interaction of Hsp70 with tumor-specific lipids (1). We have produced fluorescently-labeled Hsp70- specific probes such as a full length cmHsp70.1 antibody (2), Hsp70-Fab fragment (3), and a tumor cell penetrating Hsp70 peptide TPP (4) which enable the imaging of mHsp70 positive tumors and metastases in different tumor mouse models (5). Since Hsp70 is not expressed on cells of the tumor microenvironment such as tumor infiltrating macrophages or fibroblasts, mHsp70 provides a tumor- specific target (4). After binding, mHsp70 rapidly gets endocytosed at 37°C (6), and thus mHsp70 not only serves as a tumor-specific target for imaging, but also could act as a vehicle for a targeted uptake of drug-conjugated, functionalized nanoparticles (NP) (7-9). Membrane-bound Hsp70 exerts dual functions, on the one hand it mediates resistance of tumor cells against radiation therapy (10), on the other hand, NK cells that have been pre-stimulated with either full length Hsp70 or a peptide derived thereof plus low dose IL-2 have the capacity to kill mHsp70 positive tumor cells via granzyme B mediated apoptosis. Following binding to mHsp70, granzyme B is rapidly taken up into tumor cells in a perforin-independent manner and thus induces tumor-specific apoptosis (11). Based on these findings a phase II clinical trial has been initiated to test the efficacy of ex vivo Hsp70 peptide plus IL-2 stimulated, autologous NK cells in NSCLC patients after radiochemotherapy (12).

1. Gehrmann M, Liebisch G, Schmitz G, Anderson R, Steinem C, De MA, et al. (2008): Tumor-specific Hsp70 plasma membrane localization is enabled by the glycosphingolipid Gb3. PLoSONE 3: e1925. 2. Stangl S, Gehrmann M, Riegger J, Kuhs K, Riederer I, Sievert W, et al. (2011): Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody. ProcNatlAcadSciUSA 108: 733-738. 3. Stangl S, Themelis G, Friedrich L, Ntziachristos V, Sarantopoulos A, Molls M, et al. (2011): Detection of irradiation-induced, membrane heat shock protein 70 (Hsp70) in mouse tumors using Hsp70 Fab fragment. RadiotherOncol 99: 313-316. 4. Stangl S, Varga J, Freysoldt B, Trajkovic-Arsic M, Siveke JT, Greten FR, et al. (2014): Selective in vivo imaging of syngeneic, spontaneous, and xenograft tumors using a novel tumor cell-specific hsp70 peptide-based probe. Cancer Res. 74: 6903-6912. 5. Stangl S, Gehrmann M, Dressel R, Alves F, Dullin C, Themelis G, et al. (2011): In vivo imaging of CT26 mouse tumors by using cmHsp70.1 monoclonal antibody. JCell MolMed 15: 874-887. 6. Gehrmann M, Stangl S, Foulds GA, Oellinger R, Breuninger S, Rad R, et al. (2014): Tumor imaging and targeting potential of an Hsp70- derived 14-mer peptide. PLoS One 9: e105344. 7. Gehrmann MK, Kimm MA, Stangl S, Schmid TE, Noel PB, Rummeny EJ, et al. (2015): Imaging of Hsp70-positive tumors with cmHsp70.1 antibody-conjugated gold nanoparticles. Int J Nanomedicine 10: 5687-5700. 8. Shevtsov MA, Nikolaev BP, Ryzhov VA, Yakovleva LY, Marchenko YY, Parr MA, et al. (2015): Ionizing radiation improves glioma-specific targeting of superparamagnetic iron oxide nanoparticles conjugated with cmHsp70.1 monoclonal antibodies (SPION-cmHsp70.1). Nanoscale 7: 20652-20664. 9. Gaca S, Reichert S, Multhoff G, Wacker M, Hehlgans S, Botzler C, et al. (2013): Targeting by cmHsp70.1-antibody coated and survivin miRNA plasmid loaded nanoparticles to radiosensitize glioblastoma cells. J Control Release 172: 201-206,. 10. Murakami N, Kühnel A, Schmid TE, Ilicic K, Stangl S, Braun IS, Gehrmann M, Molls M, Itami J, Multhoff G. (2015): Role of mHsp70 in radiation sensitivity of tumor cells. Rad Oncol 10:149. 12. Gehrmann M, Stangl S, Kirschner A, Foulds GA, Sievert W, Doss BT, et al. (2012): Immunotherapeutic targeting of membrane hsp70- expressing tumors using recombinant human granzyme B. PLoSONE 7: e41341. 13. Specht HM, Ahrens N, Blankenstein C, Duell T, Fietkau R, Gaipl US, GüntherC, Gunther S, Habl G, Hautmann M, Hiuber RM, Molls M, Offner R, Rödel F, Schütz M, Combs SE, Multhoff G. (2015): Hsp70 peptide activated NK cells for the treatment of patients with NSCLC after radiochemotherapy – from preclinical studies to a phase II trial. Front Immunol NK cell biology 162: 1-9.

. 5 TRANSCRIPTIONAL RE-PROGRAMMING OF GENES IN CELL STRESS AND CANCER – SYNERGISTIC AND OPPOSITE EFFECTS OF HEAT SHOCK FACTORS, HSFS

Lea Sistonen

Faculty of Science and Engineering Åbo Akademi University Turku, Finland

Heat shock factors (HSFs) are key transcriptional regulators in cell survival. HSF1 has been established as a driver of carcinogenesis, whereas the role of HSF2 human malignancies has remained enigmatic. We have now evidence to show that HSF2 suppresses the invasive capacity of prostate cancer cells and that many prostate cancer and other cancer patients display decreased levels of HSF2. Gene expression profiling together with functional studies suggest that HSF2 is associated with GTPase activity, cell adhesion, extracellular matrix and cytoskeleton dynamics. Plasticity of transcriptional programs is fundamental for all biological processes from cellular growth and differentiation to coordinated functions of tissues and whole organisms as the transcriptional programs define the identities of cells and their responses to various stimuli. To investigate the mechanisms by which human cells promptly and profoundly reprogram their RNA synthesis upon acute heat stress, we profiled the genome-wide nascent transcription in K562 cells exposed to a heat shock and compared the transcripts with those present under normal growth conditions. The results from Precision Run-On sequencing (PRO-seq) revealed an induction of hundreds of genes and a repression of thousands of genes in heat-shocked cells. In addition to protein-coding genes, many distal regulatory elements that possessed an open conformation prior to stress, were also actively transcribed and we named them as distal Transcribed Regulatory Elements (dTREs). Importantly, PRO-seq showed that the release of promoter-proximal RNA Polymerase to productive elongation is the decisive step for either stress-induced transcriptional upregulation or downregulation. To understand how RNA synthesis is regulated in the context of dynamic chromatin environment, we analyzed the chromatin architecture and how HSF1 interacts with the local chromatin as well as how the transcriptional stress response of genes and dTREs is coordinated across the human genome. Our results highlight the delicate spatial organization of chromatin that pre-wires gene expression and defines the directionality at the gene promoters upon acute stress.

6 . ROLE OF HSP70 IN TUMOR INITIATION AND PROGRESSION

Michael Sherman

Department of Biochemistry, Boston University Medical School, Boston, MA02118 [email protected]

The heat shock protein Hsp70 has emerged as a major player in cancer and potential drug target. Indeed, knockout of the Hsp70 gene severely delayed tumor emergence in animal models, and thus established that Hsp70 plays a critical role in cancer development. Series of genetic studies established that Hsp70 is required for tumorigenesis at several steps, including tumor initiation and metastasis. These findings were corroborated by retrospective studies of human patients, showing close correlation between high levels of Hsp70 and poor prognosis of breast and other cancers. Based on these studies that established the role of Hsp70 in cancer, efforts have been undertaken to develop Hsp70 inhibitors for cancer treatment. Using genetic methods, we established that specific effects of Hsp70 on cancer result from its direct interaction with a co-chaperone Bag3. This novel Hsp70-Bag3 module controls multiple signaling pathways that regulate cancer and normal cell physiology. In collaboration with Dr. Jason Gestwicki (UCSF) we have developed a molecular scaffold that inhibits the Hsp70-Bag3 interaction, and mimic effects of Hsp70 depletion on signaling and tumor development. In a related study we discovered that the Hsp70-Bag3 module plays physiological role in normal cells in sensing proteasome and ribosome activities and transmitting these signals to multiple signaling pathways.

. 7 CHAPERONE MEDIATED CANCER CELL CHEMORESISTANCE

Zuzanna Tracz-Gaszewska1,2, Bartosz Wawrzynow2, Przemysław Biecek4,5, Marcin Herok1,3, Marcin Kosinski5,4, Marta Klimczak1,6, Patrycja Czerwińska1,6, Maciej B. Olszewski1, Milena Wiech1, Maciej Wiznerowicz6, Alicja Żylicz1, Maciej Żylicz1

1International Institute of Molecular and Cell Biology, Warsaw, Poland; 2Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland; 3Nencki Institute of Experimental Biology, PAS, Poland; 4Faculty of Mathematics, Informatics, and Mechanics, University of Warsaw, Warsaw, Poland; 5Faculty of Mathematics and Information Science, Warsaw University of Technology, Warsaw, Poland; 6Laboratory of Gene Therapy, the Greater Poland Cancer Center, Poznan, Poland

Utilizing The Cancer Genome Atlas (TCGA) data sets, we show that a subset of cancer patients with mutated TP53 and simultaneous elevation of MDM2 exhibit statistically decreased survival post treatment. Thus, we hypothesize that in this genetic background, patients develop resistance to chemotherapy more efficiently. By employing a breast and lung cancer cell line model, we demonstrate that stable expression of p53 structural mutant (i.e. p53 R175H) gives the cells a considerable growth advantage and resistance to drug induced apoptosis. Furthermore, this gain-of-function phenotype is dependent on the molecular chaperones HSP70 (HSPA1A) and HSP40 (DNAJB1), which sequester TAp73 into a complex with p53 structural mutant. This substantially decreases TAp73-dependent apoptosis and in consequence amplifies chemoresistance to DNA damage inducing drugs, routinely used in clinics. Additionally, increased levels of MDM2 oncoprotein can replace the chaperones in the mentioned complex resulting in the formation of stable three-body p53 R175H-TAp73 -MDM2 complex, which additionally significantly amplifies cancer cell chemoresistance. The presence of MDM2 inhibitor – Nutlin-3 dissociates MDM2 from this three-body complex and partially restores drug dependent apoptosis of cancer cells. Our findings demonstrate that molecular chaperones aid cancer cells in surviving the cytotoxic effect of chemotherapeutics and have potential therapeutic implications.

8 . HSF1 CONTRIBUTION TO CANCER CELL PHENOTYPE

Natalia Vydra, Agnieszka Toma-Jonik, Joanna Korfanty, Patryk Janus, Wiesława Widłak

Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, ul. Wybrzeze Armii Krajowej 15, 44-101 Gliwice, Poland

Heat Shock Transcription Factor 1 (HSF1) is activated under stress and constitute the major mechanism of cell adaptation to unfavorable conditions. In contrast to beneficial effects in most cells, HSF1 can act to detriment of organisms by promoting oncogenesis and supporting of lethal phenotype of cancer. High expression of HSF1 was found in a broad range of tumors and tumor cell lines. In addition, HSF1 high level was associated with increased mortality of e.g. estrogen receptor (ER)- positive breast cancer patients and may contribute to tamoxifen resistance. Estrogens (especially 17β-estradiol, E2) play a major role in promoting the proliferation of many types of cells, both normal and neoplastic ones. We found that E2 treatment led to activation of HSF1 manifested by its increased phosphorylation on S326 and binding to chromatin in breast epithelial cells. Thus, we aimed to define whether HSF1 can contribute to estrogen-induced breast cell transformation and tumor progression. For this purpose, we have chosen non-tumorigenic MCF10a (basal-like), MCF12a (basal/luminal-like) cells, and ER-positive tumorigenic MCF7 cells. Using lentiviral vectors we modulated the level of HSF1 in these cells, either by its down- or up-regulation. HSF1 silencing affected the expression of epithelial-to-mesenchymal transition markers. It was connected with growth inhibition and the reduced ability of non-tumorigenic cells to form acini in three dimensional cultures (3D), while in tumorigenic cells – with the appearance of organized polar colonies characteristic for non-tumorigenic cells. These preliminary analyses suggest that HSF1 could support cell proliferation as well as non-polar, disorganized growth in (3D) characteristic for tumorigenic cells, whereas HSF1 silencing could partially reverse cancer phenotype of malignant breast cancer cells. The contribution of HSF1 to estrogen-induced transformation of mammary cells will be a part of further experiments.

Acknowledgements: This work was supported by grants no 2014/13/B/NZ7/02341 and 2015/17/B/NZ3/03760 from the Polish National Science Centre

. 9 HSPA2 - CYTOPROTECTIVE OR REGULATORY PROTEIN?

Dorota Ścieglińska, Agnieszka Gogler-Pigłowska, Katarzyna Klarzyńska, Damian Sojka, Zdzisław Krawczyk

Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, ul. Wybrzeze Armii Krajowej 15, 44-101 Gliwice, Poland

The human heat shock protein A2 (HSPA2), is a poorly characterized chaperone from the HSPA (HSP70) family. HSPA2 is abundantly expressed in the testis and it is involved in sperm cell differentiation, maturation, and activity. Several reports have pointed to HSPA2 as a cancer associated protein that promotes growth and invasion of malignant cells. Nevertheless, our recent studies have demonstrated that HSPA2 is also expressed in selected populations of somatic cells. Among others, we found high accumulation of HSPA2 in stratified and pseudostratified epithelia. These findings prompted us to get deeper insight into HSPA2 expression in the epidermis, a multilayered cornified epithelium which provides the outermost barrier between the organism and its environment. We found that HSPA2 is expressed in keratinocytes constituting the basal layer of epidermis, but not in the fraction displaying features of keratinocyte stem cells (KSC). These results suggest that HSPA2 can be attributed to undifferentiated keratinocytes which are responsible for maintaining epidermal homeostasis (e.g. committed progenitors and/or the daughter transit amplifying cells). In searching for a functional significance of HSPA2 expression in epidermis we choose a spontaneously immortalized epidermal keratinocyte HaCaT line, a suitable model for investigating proliferation and the differentiation of human keratinocytes. We found that a substantial reduction of the HSPA2 level, achieved with RNAi technology, had negligible effect on keratinocyte proliferation. Surprisingly, HSPA2 depletion exerted insignificant effect on cells’ sensitivity to heat shock. Instead, HSPA2-deficient cells showed a set of phenotypic changes typical for differentiated keratinocytes such us: lower clonogenic potential, reduced adhesiveness and increased expression of differentiation markers. Organotypic three-dimensional culture demonstrated that HSPA2-deficient cells formed terminally differentiated layer in reconstituted epidermal equivalents faster than the control ones. Collectively, our results implicate for the first time that HSPA2 is a unique member of the HSPA family, which is involved in regulation of keratinocytes differentiation. We presume that HSPA2 may prevent premature commitment of basal keratinocytes to terminal differentiation. The contribution of HSPA2 to regulation of epidermal homeostasis will be elucidated in further experiments.

Acknowledgements: This work has been supported by National Science Centre, research grant NCN no. DEC-2013/09/B/NZ5/01815

10 .

Session II

Molecular and Numerical Gears of Circadian Clocks

. 11 12 . CROSSTALK OF STRESS RESPONSE PATHWAYS AND THE CIRCADIAN CLOCK IN MAMMALS

Hans Reinke

Medical University, Dusseldorf, Germany

The circadian clock confers rhythmic expression to a large part of the mammalian genome, which leads to overt rhythmic changes in physiology and behaviour. All body clocks receive input from metabolic signals informing on physiological parameters such as energy and redox levels or body temperature. Circadian clock genes are in turn connected to a variety of output pathways, among them proteostatic mechanisms such as the heat shock response and autophagy pathways. However, the molecular basis of these interactions is still largely unknown. In a screen for novel circadian transcriptional regulators Heat Shock Factor 1 was found to be rhythmically active and to drive the expression of heat shock proteins at the onset of the dark phase in mouse liver. HSF1-deficient mice also have a longer free-running period than their wild type littermates, indicating a role for HSF1 in the master oscillator of the SCN. This effect might in part be mediated by the HSF1 target gene HSP90, which controls the stability of the core clock protein BMAL1. Autophagy is another protein degradation mechanism that exhibits crosstalk to the circadian clock, in particular in the context of aging. We found that the expression levels of transcriptional repressor components of the clock, most prominently the human Period homologue PER2, are strongly reduced in primary dermal fibroblasts from aged humans, while raising the expression of PER2 in the same cells partially restores diminished autophagy levels. This link is further corroborated by the finding that the circadian clock drives cell- autonomous, rhythmic autophagy levels in murine fibroblasts, and that knocking down PER2 decreases autophagy levels while leaving core clock oscillations intact. Finally, the Period homologue lin-42 regulates autophagy and life span in the nematode Caenorhabditis elegans, indicating an evolutionarily conserved role for the circadian clock in autophagy control and aging.

13 PERSONALIZING CANCER TREATMENTS THROUGH SYSTEMS CHRONOTHERAPEUTICS

Francis Lévi

Cancer Chronotherapy Unit, Warwick Medical School, Coventry, United Kingdom, and European Associated Warwick-INSERM Laboratory C2SysMed, INSERM UMR 935, Campus CNRS, and Assistance Publique-Hopitaux de Paris, Medical Oncology Dept, Paul Brousse Hospital, Villejuif, France

Objective: Chronotherapeutics aim at improving treatment outcomes through the delivery of medicines according to the Circadian Timing System (CTS), a complex hierarchical and dynamic network system involving molecular clocks located in all cells in the body. The approach is especially relevant in oncology, because of the severe toxicities of anticancer therapies and the frequent need for treatment durations over several years.

Rationale: The CTS controls most metabolic pathways, and cellular proliferation, resulting in up to 10-fold changes in tolerability according to dosing time for nearly 50 anticancer medications in mice or rats. The transcription/translation regulatory loops which constitute the molecular clocks are usually disrupted in cancer. The differences between clocks in healthy and cancer tissues account for up to several fold increase in antitumor efficacy following chemotherapy dosing at the time of best tolerability. Mathematical models help understand why chronotolerance and chronoefficacy can coincide. The clinical implementation of chronotherapy has translated into improved patient outcomes for 5-fluorouracil-leucovorin (FL), oxaliplatin (O), carboplatin, cisplatin, doxorubicin, theprubicin, vinorelbine and irinotecan (I) in cancer patients.

Results and discussion: However, both sex, and circadian robustness/disruption measured through rest-activity circadian rhythm monitoring, appeared as independent determinants of the optimal chronotherapeutic schedule, in meta-analyses of international randomized trials involving patients with metastatic colorectal cancer (mCRC). Proper chronomodulated delivery of anticancer medications indeed could jointly enhance treatment tolerability up to 5-fold as compared to conventional delivery, and achieve prolonged survival or cures from metastatic disease, through the integration of chronotherapy within curative intent medicosurgical strategies. Liver-directed chronotherapy has been further developed in GI cancers, based on improved knowledge regarding both (1) the circadian clocks in healthy liver and primary or secondary liver cancers in experimental models and (2) advances in the clinical chronotherapy in this disease. The delivery of chronomodulated irinotecan, 5-fluorouracil and oxaliplatin into the hepatic artery has indeed helped eradicate extensive metastatic disease in selected patients despite prior failure of conventional chemotherapy protocols. Recent findings have emphasized the critical relevance of the reciprocal regulation of Bmal1 and Rev-erbα for the determination of optimal circadian timing. The results support the need for coupling chronotherapy algorithms to circadian biomarkers, using e-health technologies, whose feasibility and clinical relevance have just been shown.

Conclusions and perspectives: The integration of experimental and clinical data into mathematical models is needed for the personalization of chronotherapy delivery including that of clock-directed therapies. The scaling up of data-based models from cells to whole organisms should help adjust chronomodulated treatment delivery algorithms to the CTS of individual patients remaining in their own environment.

14 . THE CIRCADIAN CLOCK PROTEINS, BMAL1 AND CLOCK, CONTROL G2/M CELL CYCLE TRANSITION

Elham Farshadi, Jie Yan, Pierre Leclere, Albert Goldbeter, Ines Chaves, Gijsbertus T.J. van der Horst

Erasmus Medical Center, Rotterdam, The Netherlands

Gating of cell division by the circadian clock has been observed in various studies. There is significant evidence that in mammals circadian rhythms affect the timing of cell division in vivo. Circadian clock disruption, either by light or genetic mutations, is linked to alterations in the rate of cell proliferation, apoptosis, DNA damage and metabolism, and to cancer predisposition. However, still little is known on how these two oscillators interact. In order to understand the mechanism behind the circadian gating of cell division, we have studied the impact of circadian clock deregulation on cell cycle progression. In this study we used a mouse fibroblast line with fluorescent reporter genes for clock and cell cycle phase (NIH3T33C). Using the NIH3T33C cells as a tool, we then analyzed timing of cell division when the circadian clock is disturbed, by knocking down Bmal1 or Clock, at the single cell level. Our data show that, in both cases, the length of the cell cycle is extended. We further showed the kinetics of S, G2, and M phases of the cell cycle in the NIH3T3 cell population in the absence of Bmal1 and Clock genes. The data so far indicate the specific effect of circadian clock on G2 phase by affecting an important G2/M regulator molecule. This observed effect is not specific for the Bmal1 or clock but for the whole Positive elements of the circadian clock. In agreement with this observation, a mathematical simulation of the effect of knockdown on the coupling between cell division and the circadian clock also revealed a lengthening of the cell cycle with decreasing Bmal1 or Clock mRNA . In conclusion, in the absence of the positive limb of the circadian clock, cell division takes longer and this observed effect is attributable to a delay in the G2 phase of the cell cycle. Ongoing experiments will delineate the mechanism behind the coupling between the two cycles.

. 15 FROM BENCH TO BEDSIDE: COUPLING BETWEEN CLOCK AND CELL CYCLE IN HEALTH AND DISEASE

Celine Feillet

Universite Nice Sophia Antipolis, Nice, CNRS, INSERM, Nice, France

Uncontrolled cell proliferation is one of the key features leading to cancer. Seminal works in chronobiology have revealed that surgical, genetic or environmental manipulation disrupting the circadian clock increase tumor development. In humans, circadian perturbation is now recognized as a risk factor for cancer. Based on these observations, the link between the circadian clock and cell cycle has become intuitive. But despite identification of molecular connections between the two processes, the influence of the clock on the dynamics of the cell cycle had never been formally observed. Recently, we combined single live cell imaging with computational methods to shed light on robust coupling between clock and cell cycle oscillators. The 2 oscillators are able to phase-lock each other so that in an expanding cell population the two oscillators are synchronized with a common frequency. In light of these results, we explore the impact of oscillators coupling in health and disease. We propose that the cell cycle may be synchronized or slowed down through coupling with the circadian clock, which results in reduced tumor growth. More than ever, systems biology has become instrumental to understand the dynamic interaction between the circadian clock and cell cycle, which is critical in cellular coordination and for diseases such as cancer.

16 . REACTIVE OXYGEN SPECIES AND CIRCADIAN RHYTHMS

Joanna Rzeszowska-Wolny

Institute of Automatic Control and Biotechnology Center, Silesian University of Technology, Gliwice, Poland

Reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, hydroxyl radical or singlet oxygen are mainly byproducts of cellular metabolism, but they are also purposely produced by cellular oxidases as participants of different signaling pathways. Their levels are increased radically by external factors such as ionizing and UV radiation as well as different chemical compounds and they were shown to increase in cells exposed to other stressing factors. Using fluorescent microscopy we studied the time-course changes of ROS levels in two different types of living cells. Cells were stained with CellRox Green and MitoSox, the low toxicity dyes which become fluorescent in the presence of ROS. Living Me45 human melanoma and HCT116 colon cancer cells were observed during 24 and 48 h with 0.5h intervals between image collection. ROS levels fluctuated in time and the dynamics and amplitude of changes differed for individual cells. Comparison of ROS level change profiles of different cells showed that at some time periods ROS changes became synchronized. In melanoma cells synchronized peak-like changes of ROS level appeared every 6 hours suggesting that some kind of molecular clock synchronizing the ROS levels in living cells may operate.

. 17 PLATFORM FOR REMOTE TESTING AND ANALYZING OF BIOMEDICAL DATA

Krzysztof Psiuk-Maksymowicz1,2, Damian Borys1,2, Roman Jaksik1,2, Sebastian Student1,2, Dariusz Mrozek3, Krzysztof Fujarewicz1,2, Andrzej Świerniak1,2

1Institute of Automatic Control, Silesian University of Technology, 44-100 Gliwice, ul. Akademicka 16, Poland; 2Biotechnology Centre, Silesian University of Technology, 44-100 Gliwice, ul. Krzywoustego 8, Poland; 3Intitute of Informatics, Silesian University of Technology, 44-100 Gliwice, ul. Akademicka 16, Poland

Testing biomedical hypotheses is performed based on advanced and usually many-step analysis of biomedical data. This requires sophisticated analytical methods and data structures that allow to store intermediate results, which are needed in the subsequent steps. However, biomedical data, especially reference data, often change in time and new analytical methods are created every year. This causes the necessity to repeat the iterative analyses with new methods and new reference data sets, which in turn causes frequent changes of the underlying data structures. The BioTest platform was developed as a support tool for biomedical data (coming from multiple experiments) management, and as a tool for remote verification of research hypotheses and effective analysis of new and archived data for cancer research. Multi-version data model provides access to research results of diverse origins (genomics, transcriptomics and proteomics) and dimensions, and allow their extensive use in diagnosis and treatment. The scalable platform built on the virtualized computational environment provides the possibility of executing in parallel multiple data workflows, as wells as configuring individual multi-step data analysis.

Acknowledgements: This work was supported by The National Centre for Research and Development grant No PBS3/B3/32/2015. Presented system was developed and installed on the infrastructure of the Ziemowit computer cluster (www.ziemowit.hpc.polsl.pl) in the Laboratory of Bioinformatics and Computational Biology, The Biotechnology, Bioengineering and Bioinformatics Centre Silesian BIO-FARMA, created in the POIG.02.01.00-00-166/08 and expanded in the POIG.02.03.01-00-040/13 projects.

18 . MICROSCALE THERMOPHORESIS: QUANTITATIVE INTERACTION ANALYSIS AND BEYOND

Marek Żurawski

NanoTemper Technologies, Cracow, Poland

MicroScale Thermophoresis (MST) is a powerful technique to quantify biomolecular interactions. It is based on thermophoresis, the directed movement of molecules in a temperature gradient, which strongly depends on a variety of molecular properties such as size, charge, hydration shell or conformation. Thus, this technique is highly sensitive to virtually any change in molecular properties, allowing for a precise quantification of molecular events independent of the size or nature of the investigated specimen. Microscale Thermophoresis allows quantification of binding affinities of proteins, nucleic acids and small molecules as well as measurement of enzymatic activities. In addition, functional studies of small molecule inhibitors are possible. When performing a MST experiment, a temperature gradient is induced by an infrared laser. The directed movement of molecules through the temperature gradient is detected and quantified using either attached or intrinsic fluorophores. By combining the precision of fluorescence detection with the variability and sensitivity of thermophoresis, MST provides a flexible, robust and fast way to dissect molecular interactions.

. 19 20 .

Session III

Chromatin Structure and Modulation of Transciption (session organized by EACR members)

. 21 22 . WWOX, THE FRA 16D CHROMOSOMAL FRAGILE SITE GENE, AT THE CROSSROADS OF CANCER AND CNS PATHOLOGY

C. Marcelo Aldaz

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas M.D. Anderson Cancer Center

WWOX, WW domain containing oxidoreductase, was cloned by our laboratory as a putative tumor suppressor gene mapping to the ch16q23.3 region. This genomic region, FRA16D, is the second most common constitutive chromosomal fragile site in the human genome. Thus, aberrations that cause loss of WWOX expression are common in various cancers including multiple myeloma (MM) and associate with poor prognosis. An example is the poor prognosis MM subset displaying chromosomal translocation t(14;16)(q32;q23) where breakpoints lie within intron 8 of WWOX, destroying a functional allele. Additionally, deletions affecting WWOX are very common in MM. Overall, WWOX expression is reported to be disrupted in ~40% of MM cases due to copy number loss or other aberrations. In spite of the high incidence of WWOX loss of function, no study to date has addressed the biological consequences of this common abnormality in MM initiation and/or progression. MM is characterized by the clonal expansion of neoplastic plasma cells (differentiated B cells). MM is the second most common hematological cancer in the USA with >30,000 new cases/yr. Despite treatment advances, remains a largely incurable disease with a median overall survival of 6yr. and only 2- 3yr. for high-risk MM. Treatment options often fail due to disease heterogeneity so there is an urgent need to identify common molecular targets across MM subtypes. We have now generated evidence indicating that: (1) Wwox deletion is tumorigenic in B cells and induces monoclonal gammopathies in mice; and (2) Wwox deletion results in defective DNA repair, increased microhomology use, spontaneous translocations and genomic instability in B cells. Constitutive genomic instability is central to MM initiation and progression. It is the main cause for the genomic and clonal heterogeneity characteristic of this neoplasia, and is a tool for MM cells to develop drug resistance. Although defects in DNA damage repair pathways have been implicated, the mechanisms underlying genomic instability in MM are largely unknown. We hypothesize that among other effects WWOX loss of function participates in MM progression by increasing genomic instability. Understanding the role of WWOX in DNA repair and genomic stability will provide insight into efficacy of DNA damaging chemotherapy treatment and sensitivity of WWOX deficient MM to DNA damage response inhibitors. We have also generated MM mouse models based on Wwox deletion and Myc activation that will be useful to investigate in vivo MM biology while providing novel preclinical testing tools for high-risk MM therapies. Studies on the role of WWOX in human disease are rapidly expanding from exclusively the field of cancer to metabolic diseases and CNS degenerative disorders. Recently the first human pedigrees with probands carrying homozygous germline loss of function WWOX mutations have been identified. These patients are characterized by severe CNS related pathology that includes epilepsy, ataxia and mental retardation (SCAR12, OMIM# 614322). Furthermore, we documented that Wwox full KO mice developed in our lab show an extremely similar phenotype to that observed in humans displaying spontaneous and audiogenic epileptic seizures. Since our original report multiple additional cases and phenotypic variants have been reported by others, including WWOX deletions and mutations found in multiple families with cases of recessive infantile epileptic encephalopathy (EIEE28, OMIM #616211). The described findings underscore the likelihood that WWOX plays a fundamental role in CNS development, physiology and neurodegeneration. In preliminary studies in collaboration with Dr. Ashok Shetty from TAMU, by using immunostaining markers of subclasses of GABA-ergic interneurons such as Parvalbumin (PV), Neuropeptide Y (NPY), we observed that brains from Wwox KO mice have significant reductions in PV and NPY expressing inhibitory interneurons in the hippocampus. These are major populations of interneurons critical for inhibition of principal neurons as well as synchronization of neural activity (critically needed for memory formation). We also observed an increase in IBA-1 and GFAP reactivity in the hippocampus that point to inflammation. These observations suggest on going neurodegeneration and indicate that the seizures seen in these mice are likely (at least partially) related to these abnormalities. In summary, WWOX is a highly conserved and tightly regulated gene throughout evolution and when defective or deregulated the consequences are important and deleterious as demonstrated by its association not only with poor prognosis in cancer but also with other important human pathologies such as metabolic syndrome and CNS related pathologic conditions.

. 23 ROLE OF TUMOUR SUPPRESSOR WWOX, THE GENE PRODUCT OF COMMON FRAGILE SITE

FRA16D, IN HUMAN DISEASES

Rami I. Aqeilan

Lautenberg Center for Immunology and Cancer Research, IMRIC, Hebrew University-Hadassah Medical School, Jerusalem 91120

The role of common fragile sites (CFSs) in cancer remains controversial. Two main views dominate the discussion: one proposes that CFSs are hotspots of genomic instability leading to inactivation of genes encoded within them while the opposing view suggests that CFSs are functional units and that loss of the genes they encode confers selective pressure, leading to cancer development. This later view is supported by emerging evidence showing that inactivation of CFS- resident tumor suppressor genes leads to dysregulation of the DNA damage response (DDR) and increased genomic instability. These two views of CFS function may not be mutually exclusive - both CFS sensitivity to breakage and selective advantage following loss of CFS tumor suppressor genes may contribute to cancer-related deletions. Our studies focus to explore the roles of CFS gene products with direct links to the DDR, genomic instability, cancer and other human diseases. One such gene is the WW domain-containing oxidoreductase (WWOX), product of FRA16D and a commonly deleted gene in breast cancer. We show that WWOX loss is associated with mammary tumor formation and that its deletion renders the genome less stable. Furthermore, brains of WWOX mutant mice display inc percentage of apoptosis perhaps contributing to neurodegeneration. Our findings suggest that gene products of CFSs, at least some, might have direct roles in DDR and that their inactivation might have driver roles in human pathogenesis.

24 . THE ROLE OF NOTCH SIGNALING PATHWAY IN REGULATION OF GENE EXPRESSION IN CANCER

Magdalena Orzechowska, Dorota Jędroszka, Elżbieta Płuciennik, Katarzyna Kośla, Magdalena Nowakowska, Izabela Baryła, Karolina Pospiech, Ewa Styczeń-Binkowska, Andrzej K. Bednarek

Department of Molecular Carcinogenesis, Medical University of Lodz, 90-752 Łódź, ul. Żeligowskiego 7/9, Poland

Cancer is the most common human genetic disease characterized by a transcriptional deregulation that leads to accumulation of various cellular abnormalities resulting in destructive effects on tissues and whole organism. Notch signaling is evolutionary conserved pathway regulating many essential cellular processes; and numerous data show that aberrant Notch signaling is associated with progression of tumorigenesis. In our study we examined gene expression data of over 5000 TCGA patient samples of breast, ovarian, prostate, kidney, lung, colon and brain tumors. We found that disease recurrence is strongly associated with aberrant Notch signaling, resulting from transcriptional deregulation of Notch receptors, ligands and effectors. Gene expression of different Notch members showed contrary effect on disease-free survival (DFS) in different types of cancer. In particular, lowered expression of NOTCH1 was associated with better DFS in all examined tumors. Lowered expression of other Notch receptors (NOTCH2-4) was favorable in majority of tumors, except colon adenocarcinoma and ER+ breast or ovarian cancers. Similar associations were established for other Notch members including regulators (i.e. ADAM17, PSEN2) and effectors (HES, HEY families) of pathway. To determine how aberrant Notch signaling influenced cellular and tissue biology we performed Gene Set Enrichment Analysis (GSEA) using molecular signatures for canonical pathways, transcription factor binding motifs and gene ontology groups. We also considered the dimensional grouping of patients according to particular set of variables (recurrence, stage, Notch pathway members) by employment of Multiple Factor Analysis (MFA) provided as FactoMineR and factoextra R packages. Our results suggest that in most cases, the recurrence of the disease correlate with higher tumor stages. The above may be explained by our further findings, that differential gene expression associated with aberrant Notch pathway is strongly associated with cell and tissue remodeling in carcinogenesis and tumor progression. Moreover, distinct Notch signatures present predictive potential to split patients into groups of good and bad prognosis regarding recurrence of the disease.

. 25 DYNAMICS AND SELF-ORGANIZATION OF DROSOPHILA CHROMATIN INTO TOPOLOGICALLY- ASSOCIATING DOMAINS

Sergey V. Ulianov1,2, Ekaterina E. Khrameeva3,4, Alexey A. Gavrilov1, Ilya M. Flyamer1,2, Pavel Kos5, Alexander Chertovich5, Mikhail S. Gelfand4,6, Yuri Y. Shevelyov7, Sergey V. Razin1,2

1Institute of Gene Biology, RAS, Moscow, Russia; 2Department of Molecular Biology, Lomonosov Moscow State University, Moscow, Russia; 3Skolkovo Institute of Science and Technology, Skolkovo, Russia; 4Institute for Information Transmission Problems (the Kharkevich Institute), RAS, Moscow, Russia; 5Physics Department, Lomonosov Moscow State University, Moscow, Russia; 6Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia; 7Department of Molecular Genetics of Cell, Institute of Molecular Genetics RAS, Moscow, Russia

Drosophila chromosomes are partitioned in large self-interacting domains (Topologically Associating Domains, TADs) separated by boundary regions or inter-TADs. How this spatial organization of chromatin is established and supported is not clear. We have performed Hi-C analysis on four Drosophila cell lines of different origins and annotated TADs using the Armatus software. Contrary to the previous studies, we did not observe a strong enrichment of TAD borders/inter-TADs with CTCF deposition sites, and instead another insulator protein Su(Hw) was preferentially present within the TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (house-keeping) genes. The tissue-specific genes reside preferentially in TADs and their transcription appears to correlate with a partial de-compaction of the TADs. Pairwise comparison of the cell lines demonstrated that, in some cases, activation of transcription within a TAD resulted in splitting of this TAD (i.e. in generation of a new boundary/inter-TAD). We proposed that integral features of active chromatin rather than the presence of any individual protein determine the TAD boundaries. Of particular importance may be the high level of histone acetylation that directly influences the ability of nucleosomes to interact with each other. Using computer simulations we demonstrated that polymers composed of long blocks of non-acetylated (capable to interact with each other) nucleosomes interspersed with shorter blocks of acetylated (incapable to interact with each other) nucleosomes adopts spatial configuration closely resembling organization of chromosomes in TADs. The important role of histone acetylation in determining TAD profiles was further confirmed by experiments with chemical inhibition of histone acetylases and histone deacetylases in S2 cells.

Acknowledgements: This work was supported by a Russian Science Foundation grant #14-24-00022.

26 .

Session IV

Regulatory RNA

. 27 28 . INTERPLAY OF TRANSCRIPTIONAL AND EPIGENETIC MECHANISMS IN NEURONAL ACTIVITY- DRIVEN GENE EXPRESSION

Angel Barco

Instituto de Neurociencias (Universidad Miguel Hernández – Consejo Superior de Investigaciones Científicas). Av. Santiago Ramón y Cajal. Sant Joan d’Alacant. 03550. Alicante, Spain.

Activity-driven transcription is a key event associated with long-lasting forms of neuronal plasticity. Although its deregulation has been linked to different neurological disorders, ranging from epilepsy to intellectual disability, our understanding of how activity-driven transcription operates at the genomic level is still limited. I will discuss the interplay between the posttranslational modification of histones, such as lysine methylation and acetylation, the production of putative regulatory RNAs and activity-driven transcription in the hippocampus, a brain region of great relevance in cognitive processes.

. 29 SMALL RNAS IN TRIPLET REPEAT DISEASES

Włodzimierz J. Krzyżosiak

Department of Molecular Biomedicine, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland

Triplet repeat expansion diseases include more than twenty human hereditary neurological disorders the molecular pathogenesis of which is poorly understood and effective treatments still do not exist. Our research is aimed at better understanding the role of RNA in molecular patomechanisms of these diseases and developing RNA-based treatment tools to either prevent their onset or slow- down their progression.

The first part of this presentation is focused on myotonic dystrophy type 1 (DM1) and more specifically on the role of microRNAs (miRNAs) in pathomechanisms of this disease. We demonstrated that the expression of the implicated gene DMPK is regulated by several miRNAs that bind either to specific sequence (miR-206, miR-148a) or repeated sequence (miR-15b/16) of DMPK transcript. We discuss the latter miRNA-target interaction in the context of competing endogenous RNA (ceRNA) hypothesis adapted to transcripts harboring simple repeat sequences.

The second part refers to our efforts to develop experimental treatment approaches for a group of polyglutamine diseases the Huntington’s disease is the best known example. We describe our attempts to achieve allele-selectivity in targeting mutant HTT transcript containing expanded CAG repeats in the presence of normal transcripts having shorter repeats. We succeeded in achieving these objectives using atypical siRNAs, and our next step is to explain molecular basis of selective mutant gene silencing.

30 . SHARPENING THE KNIVES OF CRISPR/CAS9

Krzysztof Chylinski

CRISPR Lab at Protein Technologies Facility, VBCF GmbH, Vienna, Austria

CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) systems constitute a family of prokaryotic adaptive defense mechanisms providing immunity against foreign genetic elements including bacteriophages and plasmids. To destroy foreign nucleic acids CRISPR-Cas systems utilize short RNAs, called crRNAs or CRISPR RNAs, that are in part complementary to the invader DNA. crRNAs are complexed with one or multiple Cas proteins, one of them being an executioner Cas nuclease that cleaves target DNA upon crRNA binding. So- called class II CRISPR-Cas systems use minimal DNA targeting machineries comprising only guiding RNA molecule(s) and a large single RNA-guided DNA endonuclease, namely Cas9 or Cpf1. Thus, class II systems were chosen to be adapted to programmable genome editing tools. CRISPR/Cas9 was the first CRISPR-based genome editing tool successfully used in a plethora of cell types and model organisms for gene deletion, insertion, mutation, transcription regulation, DNA visualization and epigenetic reprogramming. Although the tool quickly became the state-of-the-art in genome editing approaches, it still requires optimization in delivery, efficiency and specificity. Our work concentrates on optimization and development of CRISPR-based tools with a main focus on using purified protein and RNA components in a variety of mammalian cell lines and primary cells. This method provides superior delivery efficiency combined with high activity and very high specificity in both knock-out and knock-in approaches and can be used as well for transient regulation of gene expression. We further aim to expand a genome editing toolbox with a variety of orthologous CRISPR/Cas9 systems to widen the choice of specific target sites and allow for multi-task approaches with the use of orthogonal systems.

. 31 NEGATIVE REGULATORY LOOP BETWEEN MIRNA301 AND AKT – SIGNIFICANCE FOR TUMOR GROWTH

1Mayur V. Jain1, Ahmad Shareef1 Wirginia Likus2, Artur Cieślar-Pobuda3,4, Saeid Ghavami5, Marek J. Łos6,7

1Department of Clinical & Experimental Medicine, Division of Cell Biology Integrative Regenerative Med. Center (IGEN), Linköping University, Linköping, Sweden; 2The J. Kukuczka Academy of Physical Education in Katowice, Mikołowska 72a, 40-065 Katowice, Poland; 3Institute of Automatic Control, Silesian University of Technology, Akademicka 16, 44-100 Gliwice, Poland; 4Stem Cell Group, Nordic EMBL Partnership, Centre for Molecular Medicine Norway (NCMM); University of Oslo; Oslo, Norway; 5Department of Human Anatomy and Cell Science, College of Medicine, Faculty of Health Sciences, University of Manitoba, Winnipeg, Canada; 6ENT Department, School of Medicine in Katowice, Medical University of Silesia, 20-24 Francuska Street, 40-027 Katowice, Poland; 7 LinkoCare Life Sciences AB, 583 30 Linköping, Sweden

Micro-RNAs (miRs) are a new class of genes that have the capacity to regulate the expression of other genes, mainly at various levels of mRNA maturation, stability and translation. The aberrant expression of several miRs has recently been associated with different types of cancers. We have studied the interplay between selected miRs and major pro-survival signaling pathways. Here we show that miR301 inhibition influences PI3K-Akt pathway activity. Akt overexpression in MCF7 and MDAMB468 cells caused downregulation of miR301 expression. This effect was confirmed by co- transfection of miR301-modulators in the presence of Akt. Cells overexpressing miR301-inhibitor and Akt, exhibited increased migration and proliferation. Experimental results also confirmed PI3K, PTEN and FoxF2 as regulatory targets for miR301. Furthermore, Akt expression in conjunction with miR301- inhibitor increased nuclear accumulation of PTEN, thus preventing it from downregulating the PI3K- signalling. In summary, our data emphasize the importance of miR301 inhibition on PI3K-Akt pathway- mediated cellular functions. Hence, it opens new avenues for the development of new anti-cancer agents preferentially targeting PI3K-Akt pathway.

1 The presented data has recently been accepted for publication in Oncotarget, (2016) 7: 20953- 20965. doi: 10.18632/oncotarget.7996.

32 . A MODEL FOR THE EFFECTS OF IONIZING RADIATION ON MICRORNA – MEDIATED REGULATION OF MRNA LEVELS IN CELLS

Krzysztof Fujarewicz

Institute of Automatic Control, Silesian University of Technology, Akademicka 16, 44-100 Gliwice, Poland

Rapid changes in the level of many messenger RNAs (mRNAs) follow exposure of cells to ionizing radiation. Here we show that the irradiation-induced change of the level of mRNA from a transfected luciferase reporter gene which contains in its 3'- untranslated region sequences targeted by different microRNAs (miRNAs) depends on the miRNAs which interact with it. Based on this observation and our former microarray results, we created a mathematical model which predicts the mRNA changes caused by perturbation of microRNA-mRNA interactions due to radiation-induced modifications of RNAs. The model simulations suggest that globally, about half of the changes of mRNA levels in irradiated cells can be explained by perturbed miRNA-mRNA interactions. Only about 30 out of a few hundred miRNAs expressed in cells appear to be important for a good correlation between predictions of the model and experimental data, suggesting that these miRNAs play crucial roles in modulating the effect of radiation. In melanoma cells these miRNAs are characterized by a higher average content of GC and a higher number of targeted transcripts, and many are reported to play a role in development of cancer. The group of mRNAs whose level change as predicted by the model differ significantly in length, length of the 3'-end, number of miRNA-targeted sequence motifs, and structure of their gene's 5' regulatory region from those whose change do not fit to the model predictions. This mathematical modeling approach could be used to identify miRNAs which participate in responses of cells to ionizing radiation and other stressing factors or to drugs.

. 33 NEURONAL RNA PROCESSING IN MEMORY FORMATION

Witold Konopka

Laboratory of Animal Models, Neurobiology Center, Nencki Institute of Experimental Biology PAS, Warsaw, Poland

Learning and memory refer to an animal's ability to respond adequately to environmental signals. The extremely elaborate connectivity network of neurons in the brain is capable of governing animals' reactions (e.g., by enhancing or weakening single or multiple synapses). Such circuit plasticity is largely believed to be the very essence of memory formation. Gene expression understood as a transfer of information from DNA to proteins is mediated by a variety of RNA molecules both messanger and regulatory ones. Here I will present examples of animal models with modified expression of two proteins i.e. TDP-43 and Dicer involved in the regulation od RNA processing. TDP-43 (TAR DNA Binding Protein, 43 kDa), recognized so far mainly as a key pathological protein in a range of neurodegenerative disorders, is a potential candidate behind the processes regulating neuronal activity. Its neuronal depletion in transgenic rat model leads to enhanced memory of acquired fear and consistently, to more stable LTP (Long-Term Potentiation) in CA1 area. Under conditions of massive neuronal stimulation, two-fold reduction of total TDP-43 results in disruption of short-term plasticity mechanisms in hippocampal pyramidal cells. Furthermore, transgenic rats present alterations of dendritic spines, as well as altered AMPARs (α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid receptors) subunit assembly, regarding flop and flip splice variants. We consider that demonstrated modifications may underlie more general mechanism leading through disturbed TDP-43 to abnormal neuronal activity and in a longer run to degeneration of neurons. The local protein translation in dendrites allows neurons to selectively rebuild only those synapses that have been activated. However, substrates of protein synthesis (i.e., mRNA) have to be kept suppressed until they are needed. MicroRNAs -short, non-protein-coding RNA regulatory sequences that guide an RNA-induced silencing complex to target mRNAs seem to be perfect candidates in fulfilling this function in neurons. Removal of the Dicer1 gene (a key enzyme for microRNA production) in neurons leads to enhanced memory formation.

34 .

Session V

Biomaterials and Medical Biotechnology

. 35

36 . LINKCOR BIOENGINEERED CORNEA: A VIABLE ALTERNATIVE TO HUMAN CORNEA FOR KERATOCONUS AND CORNEAL DYSTROPHY PATIENTS

Mehrdad Rafat1,2, Ph.D.

1Department of Biomedical Engineering, Linköping University, Sweden; 2LinkoCare Life Sciences AB (Ltd.), Sweden

According to most recent global estimates from the WHO in 2010, corneal diseases and abnormalities are the second largest cause of unilateral and bilateral blindness worldwide accounting for 23 million people globally. Diseased or damaged corneas are typically replaced by donor human corneas from deceased patients. However, a significant shortfall in donor numbers exists, and less than 1% of those in need may receive a therapeutic option that has been unchanged for more than 50 years and is limited by the lack of suitable donor tissue and rejection. Although prosthetic artificial corneas are developed as a substitute for donor corneas and, in some cases, appear to have short- term success in patients, their post-surgical complications and high cost make them a last resort. These issues have prompted the advancement of bioengineered tissue alternatives. Bioengineered corneas with favorable biological properties, which are additionally amenable to low-cost mass production, are in urgent need. LinkCor bioengineered cornea is a transparent cell-free but cell- interactive tissue-mimetic scaffold developed based on our patented Regenerative Medicine and Tissue Engineering platform technology. It mimics and functions as an alternative to human cornea. It can partially or fully replace damaged or diseased human cornea helping corneal blind or low vision patients by stopping the progression of the diseases and helping them regain their sight. LinkCor® is primarily designed and tested for keratoconus and corneal dystrophy patients though its suitability for other corneal indications are being evaluated. LinkCor® can be prefabricated and customized as per specific needs of each patient. LinkCor® has been successfully tested in vitro and in vivo for its physical and biological properties the results of which will be presented.

. 37 LINEAGE SPECIFIC REGULATION OF LAMIN B1 ASSOCIATED CHROMATIN DOMAINS IN THE HEMATOPOIETIC TISSUE

Marie Rogne1,*, Adnan Hashim1*, Oksana Svaerd1, Vincent Oei2, Håvard A Kristiansen3, Philippe Collas4,5, Karl Johan Malmberg2,6, Judith Staerk1,3,5

1Centre for Molecular Medicine Norway (NCMM), Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, Oslo, Norway; 2Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway; 3Department of Haematology, Oslo University Hospital, Oslo, Norway; 4Department of Molecular Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway; 5Norwegian Center for Stem Cell Research, University of Oslo, Oslo, Norway; 6Department of Hematology, Karolinska University Hospital, Stockholm, Sweden; Center for Infectious Medicine, Karolinska Institutet, Stockholm, Sweden; K.G. Jebsen Center for Cancer Immunotherapy, Institute of Clinical Medicine, University of Oslo, Oslo, Norway * These authors contributed equally to this work

The nuclear lamina is composed of lamins, which are intermediate filaments and lamin- associated proteins. Lamins have been implicated in regulation of gene expression by tethering chromatin regions to the nuclear periphery. In hematopoietic cells little is known about how chromatin regions are configured with respect to the nuclear lamina, and how those interactions correlate with transcriptional activity and epigenetic signatures. We therefore generated a global high resolution map of genomic regions that dynamically and constantly reside within LaminB1-Lamin Associated Domains (LMNB1-LADs) for CD34+ progenitor and mature blood cell types, and show for the first time that LMNB1 has a differentiation-specific LAD signature. In CD34+ cells and monocytes, facultative and constitutive LADs are silent for gene expression, as generally described for LADs. Strikingly, in lymphoid cell types, LADs contain transcriptionally active regions correlating with a distinct enrichment of epigenetic marks around transcription start sites. Moreover, we demonstrate that epigenetic signatures marking LMNB1-LADs are not only distinct between different blood cell types, but also between facultative and constitutive LADs of the same cell type. Taken together, our data identify a novel layer of gene regulation during hematopoiesis represented by different degrees of transcriptional activation and epigenetic markings of facultative and constitutive LADs.

38 . THE RATE-LIMITING ENZYME IN CARBOHYDRATE METABOLISM, PFKFB3 – DIFFERENTIAL EXPRESSION IN IPS AND CANCER STEM CELLS

Artur Cieślar-Pobuda1,2, Mayur Vilas Jain3, Gunnar Kratz4,5,6, Joanna Rzeszowska-Wolny2, Saeid Ghavami7, Emilia Wiechec3,4

1Centre for Molecular Medicine Norway, Oslo, Norway; 2Biosystems Group, Institute of Automatic Control, Silesian University of Technology, Akademicka 16, 44-100 Gliwice, Poland; 3Department of Clinical and Experimental Medicine, Division of Cell Biology, Linköping University, 581 85 Linköping, Sweden; 4Integrative Regenerative Medicine Center (IGEN), Linköping University, 581 85 Linköping, Sweden; 5Experimental Plastic Surgery, IKE, Linköping University, 581 85 Linköping, Sweden; 6Department of Plastic Surgery, County of Östergötland, Linköping, Sweden; 7Department of Human Anatomy and Cell Science, University of Manitoba, Manitoba, Canada

Various studies have revealed fenotypic similarity between the induced pluripotent stem cells (iPSc) and cancer stem cells (CSCs). Both iPS and CSCs are fast proliferating cells with high propensity to form tumors, suggesting that molecular mechanisms of cell cycle regulation may be similar. In this work we compare the restriction (R) point-governed regulation of the cell cycle progression in different cellular systems (iPSc, cancer, CSCs and normal cells). We focus on glycolysis related enzymes: PFKFB3 and PFK1, and show differences in their expression in various cells. Moreover, our work reveals that cancer and iPS cells when cultured in hypoxic conditions alter expressions of PFKFB3 and PFK1, such that they more resemble CSCs (high PFKFB3, low PFK1 gene expressions). Results show also cell type related differences in response to inhibition of PFKB3. Presented study provides new insight into molecular mechanism of cell cycle regulation in various types of cells, especially between CSC and cancer cells. It also shows that iPS cells, cancer and CSC can be easily distinguished a basis of PFKB3 and PFK1 expression, which may bring some new promises in a stem cell biology and cancer research.

. 39 PREVENTIVE MEASURES AGAINST CHRONIC REJECTION OF HEART TRANSPLANTS

Jacek Z. Kubiak1, Malgorzata Kloc2, Rafik M. Ghobrial2

1UMR 6290 CNRS/University Rennes 1, Institute of Genetics & Development of Rennes, Rennes, France; 2The Houston Methodist Research Institute, Department of Surgery, The Houston Methodist Hospital, Houston, Texas, USA

The chronic rejection is the major threat for survival of transplanted organs. The cellular and molecular mechanisms of chronic rejection of transplanted organs remain obscure. However, macrophages are known to play a critical role in the injury and repair of allografts. Among multiple factors influencing macrophage infiltration of allografts, the actin cytoskeleton, which is regulated by a triphosphatase Ras homolog gene family member A (RhoA), is of the utmost-׳small guanosine-5 importance. To define the role of macrophage-specific RhoA pathway involvement in chronic rejection, we generated mice with monocyte/macrophage-specific deletion of RhoA. Hearts from BALB/c (H-2d) donors were transplanted into RhoAflox/flox (no Cre) and heterozygous Lyz2Cre+/-RhoAflox/flox recipients treated with cytotoxic T-lymphocyte-associated protein 4 immunoglobulin to inhibit early T-cell response. Allografts were assessed for chronic rejection and monocyte/macrophage functions. The deletion of RhoA clearly inhibited macrophage infiltration, hyperplasia of vasculature, and abrogated chronic rejection of the allografts. Our finding of the dependence of chronic rejection on monocyte/macrophage RhoA signaling pathways may lead to the development of novel anti-chronic rejection therapies.

40 . POLYMETHACRYLATES AS DRUG CARRIERS - STRUCTURE VS CELL VIABILITY IN MCF-7 CELL LINES

Anna Mielańczyk1, Magdalena Skonieczna2, Łukasz Mielańczyk3, Dorota Neugebauer1

1Department of Physical Chemistry and Technology of Polymers, Faculty of Chemistry, Silesian University of Technology, ul. M. Strzody 9, 44-100 Gliwice, Poland; 2Biosystems Group, Institute of Automatic Control, Silesian University of Technology, ul. Akademicka 16, 44-100 Gliwice, Poland; 3School of Medicine with the Division of Dentistry in Zabrze, Department of Histology and Embryology, Medical University of Silesia, ul. Jordana 19, 41-808 Zabrze, Poland

Doxorubicin (DOX) is an anthracycline-based, commonly used chemotherapeutic drug, which causes severe side effects: heart failure, cardiomyopathy, myelosuppression, stomatitis, and leukemia. Therefore, application of DOX in the form of polymer conjugate might reduce side effects and improve the therapeutic effect by increasing the bioavailability of DOX, reducing the renal and hepatic clearance, and protecting from premature degradation of the active compound. Two series of sugar-centered polymethacrylates, namely V-shaped and 3- or 4-arm star- shaped polybases with pending hydroxylamine groups and 4- or 6-arm star-shaped polyacids with pending carboxylic groups, were designed and evaluated in vitro toward their potential usage as drug delivery systems in breast cancer (MCF-7) treatment [1,2]. Further, in case of polybases cytostatic properties of both free-DOX polymers and polymer-DOX conjugates with different number of DOX molecules bonded via ketimine groups, were examined using cytotoxicity test, apoptosis assay and cell cycle experiments on breast cancer cell lines - DOX resistant (MCF-7/R) and wild type (MCF-7/W) [3]. The main goal of our research was to find correlation between the structure of polymeric matrix (directly influencing on their charge and size) and their cytostatic activity. Among all tested conjugates, the most promising results with induction of apoptosis without inducing necrosis in both MCF-7 cell lines were obtained for conjugate based on 4-arm stars with low content of DOX (4 µg/mL). The apoptosis assay on MCF-7/R revealed that the efficacy of the drug after conjugation with a carrier tripled while its dose was six time lower.

References: 1. Mielańczyk A, Neugebauer D (2014): J. Polym. Sci. Part: A. Polym. Chem., 17: 2399-2411. 2. Mielańczyk A, Neugebauer D (2015): Bioconjug. Chem., 26: 2303-2310. 3. Mielańczyk A, Skonieczna M, Mielańczyk Ł, Neugebauer D (2016) Bioconjug. Chem., 27: 893-904.

Acknowledgements: The authors gratefully acknowledge financial support from the National Science Center, Decision No DEC-2012/07/N/ST5/ 708 01875 (A.M. and D.N.),grant KNW-2-043/D/5/N from Medical University of Silesia in Katowice (Ł.M.) and SUT BK-277/Rau1/2015 t.3 711 from Silesian University of Technology in Gliwice (M.S.). The biological experiments were performed in the Biotechnology Center of the Silesian University of Technology in Gliwice and Silesian Medical University in Katowice using equipment financed by the Silesian Biofarma program supported by POIG.02.03.01-24-099/13 grant

. 41 42 .

Poster abstracts

1. Mathematical modeling of biological processes and structures (1-19) 2. Regulation of cellular processes in normal and pathological cells (20-49) 3. Transcriptome and proteome (50-63) 4. Molecular markers and targets (64-69) 5. New methods, new molecules and new approaches (70-81) 6. New molecules and new therapies (82-101)

. 43

44 .

Mathematical modeling of biological processes and structures (1-19)

. 45

46 . 1. SENSITIVITY ANALYSIS OF A MODEL OF TUMOR GROWTH WITH STRONG ALLEE EFFECT

Krzysztof Łakomiec, Krzysztof Fujarewicz

Silesian University of Technology, Gliwice, Poland

In our previous work [1] we proposed a novel method of spatiotemporal radiotherapy optimization. We used a spatial tumor growth model to simulate behavior of the tumor and adjoint sensitivity analysis to optimize in-silico the radiotherapy plan. However, the model of tumor growth and response to radiotherapy [2] that we used has one big disadvantage. Due to lack of degradation term in the model the simulated tumor cannot be completely killed by radiation. Even if the radiation reduces the tumor cell density to value nearly zero the tumor will grow back after the irradiation. In this work we proposed a simply model of tumor growth and response to radiation therapy with added degradation term based on so-called strong Allee effect. The Allee effect term is commonly used in populations modeling where the strong Allee effect means that the growth function of the model is negative for a low population density (the model has a growth threshold which below the population goes to extinction). Recent work [3] suggests that the strong Allee effect can be important in spreading of malignant tumors. We use the proposed model and adjoint sensitivity analysis to calculate the spatiotemporal gradient of predefined scalar objective function characterizing the model's solution. The calculated gradient can be used to more precisely predict behavior of the tumor during radiotherapy.

References: 1. Fujarewicz K, Łakomiec K (2016): Adjoint sensitivity analysis of a tumor growth model and its application to spatiotemporal radiotherapy optimization. Math.Biosci.Engineer. 13: 1131-1142. 2. Rockne R, Alvord Jr. EC, Rockhill JK, Swanson KR (2009): A mathematical model for brain tumor response to radiation therapy. JMathBiol 58: 561-578. 3. Sewalt L, Harley K, van Heijster P, Balasuriya S (2016): Influences of Allee effects in the spreading of malignant tumours. JTheorBiol 394: 77-92.

. 47 2. MODEL OF GROWTH AND DECLINE OF A SINGLE CELL POPULATION

Karolina Kurasz, Aleksandra Krzywoń, Krzysztof Fujarewicz

Silesian University of Technology, Gliwice, Poland

Dynamics of growth and decline of single cell population were analysed based on Juska's model [1]. Analysis of the dynamics based on general considerations concerning the main properties of microorganisms and their interactions with the environment which supposed to be affected by the activity of the population. The model is described by differential equations containing minimal sets of parameters characterizing activity of the population. Juska's model assumes that the factors leading to the decline of the population have to be considered separately and they are: accumulation of metabolites -toxins in the medium and the exhaustion of resources of energy. This work is based on biological experiments and computer simulations. A series of biological experiments was made in order to designate the number of cells at specific time points. For this purpose, cells were seeded on 6-well dishes with different initial amounts of cells and various medium volume. For numerical estimation of the parameters MATLAB software was used.

References: 1. Juska A (2010): Minimal models of growth and decline of microbial populations. MathemJTheoretBiol 269: 195200.

48 . 3. MATHEMATICAL MODELING OF BYSTANDER EFFECT

Aleksandra Krzywoń, Maria Wideł, Krzysztof Fujarewicz

Institute of Automatic Control, Silesian University of Technology, Akademicka 16 Street, 44-100 Gliwice, Poland

Bystander effect is the phenomenon, where unirriadiated cells exhibit some biological effects in response to signals received from nearby irradiated cells. These responses are manifested in neighboring unirradiated cells as decrease of survival, cytogenetic damage, increase in the level of apoptosis, biochemical changes, gene expression and epigenetic changes. Bystander effect has been observed after ionizing and non-ionizing radiation and even other stressors. Our experiments performed on human malignant melanoma cells irradiated with UVA, UVB and UVC bands and co-cultured with normal human dermal fibroblasts revealed, that despite of classical damaging bystander effect in neighbor cells, the non-exposed fibroblasts exerted protective bystander effect towards irradiated melanoma cells. This effect was particularly seen in the case of UVA exposure. The aim of modeling the bystander effect induced by ultraviolet radiation was to distinguish appropriate bystander effect from effects resulting from the interaction of cells residing in a common environment, and eventually dying because of life processes associated with decrease of nutrients and accumulation of toxic metabolites. The results of experimental studies designed to create a mathematical model involved in counting the number of cells in different time points (24, 48 and 72 h). Modeling bystander effect was divided into three stages: modeling of a single population of cells, modeling of mutual influence of life processes and modeling of bystander effect caused by ultraviolet radiation. Each model was created by adding new equations and parameters to the initial model and estimating only new parameters. The parameters estimated in the previous steps was unchanged. Mathematical modeling demonstrated that it is impossible to explain the appearance of bystander effect only on the base of life processes of the two co-existing cell populations, one of which is irradiated, and clearly indicates that specific bystander signaling has to be engaged.

Acknowledgements: This research was supported by the grant DEC2012/05/B/ST6/03472 from the Polish Ministry of Science and Higher Education

. 49 4. A SIMPLIFIED MODEL OF INTERACTIONS BEETWEN HSF AND NF-κB SIGNALING PATHWAYS

Małgorzata Kardyńska, Jarosław Śmieja

Institute of Automatic Control, Silesian University of Technology, Akademicka 16, 44-100 Gliwice, Poland

NF-κB is a family of transcription factors that regulate the transcription of numerous of genes among which there are anti-apoptotic genes (Cataldi et al., 2003). Therefore, inhibition of NF-B pathway may constitute one of the goals in anticancer therapies. Experimental results show that heat shock induces such inhibition in cancer cells (Janus et al., 2011). The ultimate goal of the study is to develop a model reflecting the transcription factor NF-κB reduction through heat shock pre-treatment. So far, numerous models of NF-κB pathway have been developed, both, stochastic and deterministic, whereas much fewer models of HSF pathway have been published. In deterministic models, the output of the model is fully determined by the parameter values and the initial conditions. Such models are useful approximation used for description of fast reaction channels of processes involving a large number of molecules. However, in the case of NF-κB response, observation of single cells show high heterogeneity of the cell population. The cells exhibit large variability due to stochasticity in gene transcription and therefore stochastic models better reflects the behavior of individual cells. Unfortunately, stochastic models are usually more complicated and require more computational power. Therefore, combined stochastic-deterministic modeling has gained increasing popularity, using ordinary differential equations for description of fast reactions involving a large number of molecules and stochastic switches to account for changing activity of the genes involved. Here we present a simplified, deterministic model of NF-κB signaling pathway, based on West et al., 2015, which we modified by adding a stochastic gene transcription. In addition, the model has been modified to take into account interactions with HSF signaling pathway by adding a simple attenuation function, which affect processes of IKK activation and IκBα phosphorylation (the most probable interaction of these two signaling pathways). The proposed model reflects both: (i) NF-κB response damping through heat shock pre-treatment and (ii) heterogeneity of NF-κB response in the cell population.

References: 1. Cataldi A, Rapino M, Centurione L, Sabatini N, Grifone G, Garaci F, Rana R (2003): NF-κB activation plays an antiapoptotic role in human leukemic K562 cells exposed to ionizing radiation. JCellBiochem 89: 956–963. 2. Janus P, Pakuła-Cis M, Kalinowska-Herok M, Kashchak N, Szołtysek K, Pigłowski W, Widlak W, Kimmel M, Widlak P (2011): NF-κB signaling pathway is inhibited by heat shock independently of active transcription factor HSF1 and increased levels of inducible heat shock proteins. GenCellDevMolCellMech 16: 1168–1175. 3. West S, Bridge L, White M, Paszek P, Biktashev V (2015): A method of ‘speed coefficients’ for biochemical model reduction applied to the NF- κB system. JMathemBiol 70: 591-620.

This work is supported by the NCN grant DEC-2013/11/B/ST7/01713 (JŚ) and BKM/506/RAU1/2016/5 (MK). Calculations were performed on the Ziemowit computational cluster (http://www.ziemowit.hpc.polsl.pl) created in the POIG.02.01.00-00-166/08 project (BIO-FARMA) and expanded in the POIG.02.03.01-00-040/13 project (Syscancer).

50 . 5. SIMULATION STUDY OF THE CONNECTION BETWEEN EPITHELIAL - MESENCHYMAL TRANSITION AND NF-ΚB PATHWAY

Monika Kurpas1, Bing Tian2,3, Allan R. Brasier2,3,4, Marek Kimmel1,5

1Faculty of Automatic Control, Electronics and Computer Science, Silesian University of Technology, 44-100 Gliwice, ul. Akademicka 16, Poland; 2Department of Internal Medicine, University of Texas Medical Branch (UTMB), Galveston, TX, USA; 3Sealy Center for Molecular Medicine, UTMB, Galveston, TX, USA; 4Department of Biochemistry and Molecular Biology, UTMB, Galveston, TX, USA; 5Department of Statistics, Rice University, 6100 Main St. MS-138, Houston, TX 77005, USA

Epithelial-mesenchymal transition (EMT) is an important process in which epithelial cells lose features such as polarity and cell-cell adhesion, and acquire a fibroblast-like phenotype, and motility and invasiveness, characteristic of mesenchymal cells. EMT is involved in normal physiological processes, such as embryonic development or wound healing, but also in cancer progression and metastasis. The transforming growth factor beta (TGF-β), a cytokine that induces growth arrest, tissue fibrosis, and EMT, activates NF-κB-dependent inflammatory gene program via a mechanism that is still not fully elucidated. Moreover, NF-κB/Rel-A Ser276 phosphorylation has been reported as required for activation of type II EMT. In this work, we inferred the topology and dynamics of the biological pathway connecting EMT circuit with NF-κB module, based on the results of biological experiments. We also developed a deterministic mathematical model, which we use to investigate the dependencies between these two important cellular processes. Our preliminary results indicate that TGF-β stimulation itself does not cause increased NF-κB nuclear import and activation of the proinflammatory genes, such as the well-described effects of treatment with tumor necrosis factor α, but it promotes the NF-κB/Rel-A Ser-276 phosphorylation and the onset of EMT. Moreover, the increased expression of EMT regulatory genes seems to induce an increase of the expression of an unknown paracrine factor, which then activates the canonical NF-κB pathway. This hypothetical mechanism seems to be a logical explanation of the observed delay in activation of the inflammatory gene program.

. 51 6. USING STOCHASTIC MODELING FOR DESIGN AND ANALYSIS OF COMPETITIVE MIRNA BINDING SITES

Marzena Dołbniak, Jarosław Smieja

Systems Engineering Group, Silesian University of Technology, Akademicka 16, 44-100 Gliwice, Poland

Understanding sources and consequences of stochasticity is important for designing successful experiments. Large variability in experimental results makes it hard or even impossible in interpretation. In this work we analyzed two alternative models of miRNA-based regulatory mechanisms. The first one describes behavior of wild-type cells, i. e. only natural intracellular regulatory mechanisms. In the second one, competitive miRNA binding site for miR-21 was introduced, representing plasmid- coded mRNA. In both cases a simplified component of NFκB pathway was considered [1]. While NFκB is a target of a large number of miRNAs, precise mechanisms and roles of miRNA-regulated NFκB and NFκB-regulated miRNAs are still unknown [2]. The problem lies also with lack of mathematical models including miRNA. Our model includes NFκB, Interleukin 6 and two miRNA species: miR-21 and miR - 149. Plasmid with binding sites for miR-21 was introduced to imitate experimental procedures using siCHECK™ Vectors. Following plasmid transfer, the cells are assayed for the presence of the reporter by directly measuring the reporter protein itself or the enzymatic activity of the reporter protein. The computational analysis was performed to verify if miRNA dynamics can be observed using this experimental system and how introduction of the second competitive binding site affects the observed pathway. Cellular heterogeneity (variability among cells) was also included. Intrinsic stochasticity resulting from the finite numbers of molecules interacting within the system was modeled using stochastic gene activation. Additionally, unequal distribution of molecules during cell division was modelled by changing the initial conditions of simulations. This simple example shows that introducing competitive miRNA binding site can change not only expression of targeted mRNA but affects the whole pathway. In consequence, response of wild type and transfected cells may differ. Important conclusion is that to compare simulation results with experimental data, one must consider the experimental setup and experiments cannot focus on mRNA measurement only, since conclusions drawn from them may be misleading.

References: 1. Xue X, Xia W Wenzhong H (2013): A modeled dynamic regulatory network of NF-kB and IL-6 mediated by miRNA, BioSystems, 114: 214-218. 2. Ma X, Becker Buscaglia LE, Barker JR, Li Y (2011): MicroRNAs in NF-kB signaling, JMolCellBiol 3: 1-8.

The authors were supported by grant nr BKM/506/RAU1/2016/t.6 (MD). Additionally, MD was holder of scholarship DoktoRiS-Scholarship program for Innovative Silesia.

52 . 7. ANALYSIS OF SYSTEM WITH SWITCH-LIKE INTERACTION IN THE P53-REGULATORY MODULE

Magdalena Ochab, Krzysztof Puszyński, Jerzy Klamka

Institute of Automatic Control, Faculty of Automatic Control, Electronics and Computer Science, Silesian University of Technology, 44-100 Gliwice, ul. Akademicka 16

Dynamics of biological processes is very complicated, because the rates depend on different regulatory molecules concentrations. Moreover the maximum rate of the process is limited by natural cells capabilities. Generally, when described by mathematical formulas, biological processes are represented by a functions, which tends to form as sigmoid functions, as a Hill or Michaelis-Menten functions. Due to the strong nonlinearities in such models, analysis of these systems is very difficult and the results can be obtained only by the numerical simulations. In contrary to this method we propose to use piece-wise linear, differential equations model. Systems with switch-like interactions are quite well examined problem in automatic control. In such systems the phase space is divided into domains by thresholds for given variables. Within each domain, depending on the state, the system is described by different set of linear differential equations. Analysis of the piece-wise linear system can be divided into three parts. Firstly, behaviour in each domain should be analyzed by calculation of its focal points. If the focal point calculated from the condition dxi/dt = 0 lies inside the domain, which is considered, this point is the steady-state and is called Regular Steady Point (RSP). If the focal point lies outside the considered domain, the system will leave it and enter a new domain. The main problem with analysis of the systems with switches is discontinuity on the boundary of the domains, so we made the basic assumption to replace the step functions by the continuous, steep sigmoid functions. In consequence we can localize the Singular Steady Points (SSP). SSP is calculated by comparison derivatives to zero, in the case when at least one variable lies on its threshold, for example on the boundary of the domain. Finally we localize the cycles, which are the closed temporal sequences of states in the phase space. In this work we are focused on the p53 regulatory core. The model consists of four variables p53 (P), cytoplasmic MDM2 (C), nuclear MDM2 (N) and PTEN (T) and includes 2 feedback loops. We create piece-wise linear model with 4 threshold, one for p53 level, two for nuclear MDM2 level and one for PTEN level. We analyze the behaviour of such system, especially in the case of different IR dose, which induces increased degradation MDM2 rate. We calculate the RSP, SSP and localize the cycles in this model for different IR dose and determine cell behaviour in this cases. Existence of the cycle for small IR dose results in proteins oscillations in cells with cell cycle arrest and DNA repair. Model with high IR dose contains a cycle, however the system reach the RSP in the domain with high p53 level. Moreover we examined how different initial conditions influence the final state of the system.

Acknowledgements: The research presented here was partially funded by the Polish National Centre for Science granted by decision number DEC-2012/05/D/ST7/02072 (KP) and DEC- 2014/13/B/ST7/00755 (JK) and by BKM/506/RAU1/2016/15 (MO).

. 53 8. BLOOD FLOW MODELLING - COMPARISON OF TWO CASES: WITH COARCTATION OF THE AORTA AND AFTER THE VASCULAR MALFORMATION REMOVAL

Bartłomiej Melka

Institute of Thermal Technology, Silesian University of Technology, Konarskiego 22, 44-100 Gliwice, Poland e-mail: [email protected], www.itc.polsl.pl/bmelka

Computational Fluid Dynamics (CFD) became one of the tools based on a noninvasive technique used for the blood flow visualization. Unfortunately, CFD has still limiting disadvantages. Some of them are: time demanding calculations, multistep process of geometry preparation, troubles with this process automatization etc. Nevertheless, the CFD implementation on a wider scale is urgently awaited in the angiology field. In the future, it could be used for the flow field visualization as a part of the angiography procedure. In the research, the use of CFD modelling was presented for two geometries. The first one consisted of the aorta with a coarctation of the aorta (CoA). The second geometry was unaffected by this vessel malformation. In both cases, the geometry contained five outflows. Hence, the studied case covered the flow through the main body bifurcation. In the research, the flow simulation results coming from these two models were compared. It was noticeable, that in the case without CoA the flow during systole in the descending aorta was higher by 4pp (percentage points). The local pressure drop caused by CoA decreased and the distribution of the flows into smaller arteries also has changed positively. The rheological properties of blood were treated with the averaged values for the blood of a healthy man. At the inlet, the pulsatile flow was implemented to simulate the heart work. Additionally, a lumped model was used to simulate response of the systemic vascular bed. It was built on the base of electrical analogy. Capacitors in this model mimicked the accumulation behavior of the system due to arteries elasticity and resistors represented the circulatory system resistivity. Moreover, the non- Newtonian behavior of the blood flow was modelled using two approaches. The first one was in accordance with the Power Law model and the second one was set as the Carreau model. In the future investigations, the analysis could be enriched with Fluid Structure Interaction (FSI). Additionally, the blood could be modelled according to multiphase approach which seems more accurate than model with averaged properties of the plasma and morphological elements. Nevertheless, in the literature, the most often way of the blood flow modelling is similar to presented study.

Acknowledgements: This research is supported by National Science Centre, Poland within project No 2014/13/B/ST8/04225. This help is gratefully acknowledged herewith.

54 . 9. HEMODYNAMIC SIMULATION OF PULSATILE FLOW IN A CORONARY ARTERY MODEL USING OPEN SOURCE MFIX SOFTWARE SUITE

Piotr Krajewski1, Wojciech Adamczyk1, Maria Gracka1, Bartłomiej Melka1, Marek Rojczyk1, Andrzej J. Nowak1, Adam Golda2, Ziemowit Ostrowski1*

1Biomedical Engineering Laboratory, Institute of Thermal Technology, Silesian University of Technology, Poland; 2Department of Cardiology, Gliwice Medical Center, Poland *Corresponding author: ul. Konarskiego 22, 44-100 Gliwice, Poland, [email protected]

The aim of this study is to perform calculations of the multiphase flow in simplified geometry (section of the human coronary artery) using open-source computational fluid dynamics (CFD) software – MFiX Software Suite [1]. MFiX (Multiphase Flow with Interphase eXchanges) is a general- purpose computer code which was developed at the National Energy Technology Laboratory (NETL, USA) for describing the hydrodynamics, heat transfer and chemical reactions in the fluid-solids systems. This open-source software has over three decades of development history and more than 3,500 registered users worldwide. For current research the simplified and idealized curved geometry with the inflexible walls and constant diameter of the human blood vessel were assumed. Numerical blood model assumes two- phase and non-Newtonian approach. The blood is modelled as dispersed phase – red blood cells (RBCs) suspended within the continuous phase – plasma [3]. The main outcome of the research is to show possibilities of the open source code usage for hemodynamics multiphase CFD modelling. The comparison of the results obtained within current research (i.e. using MFiX code) against commercial CFD package results (ANSYS Fluent, ANSYS Inc., USA) is presented.

References 1. MFiX Software Suite, https://mfix.netl.doe.gov (accessed 26.10.2016) 2. ANSYS Fluent User’s guide, Rel. 15, ANSYS, Inc, 2013. 3. Jung J, Lyczkowski RW, Panchal CB, Hassanein A (2006): Multiphase hemodynamic simulation of pulsatile flow in a coronary artery. JBiomech 39: 2064–2073.

Acknowledgements: This research is supported by National Science Centre, Poland within project No 2014/13/B/ST8/04225. This help is gratefully acknowledged herewith.

. 55 10. NUMERICAL MODELLING OF PULSATILE BLOOD FLOW IN COARCTATED AORTA USING MULTIPHASE KINETIC THEORY

Maria Gracka

Institute of Thermal Technology, Silesian University of Technology, Gliwice, Poland

In recent years Computational Fluid Dynamics (CFD) methods proved their aplicability in bio- engineering. The cardiovascular diseases presently have became the leading cause of death in the world mainly in the developed and industrialized societies. Therefore understanding of basic mechanisms and phenomena occurring in the cardiovascular system could be useful in early detection of the development of lesions in blood vessels, where pharmacological treatment can be used instead of expensive and complicated surgical procedures. The scope of this work was numerical analysis of the blood flow within aorta. In presented project two numerical models are presented. Eulerian multiphase approach is used in model of the blood flow which assumes blood as a mixture of three blood components plasma, red blood cells (RBC) and white blood cells (WBC) [2]. Multiphase model was compared to single-phase model that considered blood as a homogenous, non-Newtonian fluid with average rheological properties of viscosity and density. Geometry used in the numerical model was created based on data generated during Gadolinium-enhanced Magnetic Resonance Angiography (MRA). The real geometry of a female 8-year old patient with moderate thoracic aortic coarctation (approximately 65% area reduction) [4] was adopted for present study. Model of the geometry included: ascending aorta, arch, descending aorta und upper branches such as innominate artery, left common artery and left subclavian artery. Numerical model assumed rigid walls of the blood vessel. To develop numerical models of the blood flow within human aorta the commercial software ANSYS Fluent (ANSYS Inc., USA) has been used [3]. Using User Defined Functions (UDFs) the pulsatile boundary condition has been implemented to mimic periodic cardiac cycle [1]. As an inlet bounady condition the velocity profile has been set. As the outlet boundary condition the outflow condition has been used. Volumetric share of blood flow through all of the branches of the aortic model was based on [4].

References: 1. Huang J, Lyczkowski RW, Gidaspow D (2009): Pulsatile flow in a coronary artery using multiphase kinetic theory. JBiomech 42: 743-754. 2. Laurent F, Massot M, Villedieu P (2004): Eulerian multi-fluid modeling for the numerical simulation of coalescence in polydisperse dense liquid sprays. JComputPhysics 197: 505-543. 3. ANSYS Academic Research, Release 17.0, Help System, UDF Manual, ANSYS, Inc. http://www.vascularmodel.com/sandbox/doku.php?id=repository_aorta, repository ID OSMSC0111 (accessed April 30th, 2016).

Acknowledgements: This research is supported by National Science Centre, Poland within project No 2014/13/B/ST8/04225. This help is gratefully acknowledged herewith.

56 . 11. LUMPED PARAMETER MODEL OF HUMAN CIRCULATORY SYSTEM

Dominika Bandoła

Institute of Thermal Technology, Silesian University of Technology, ul. Konarskiego 22, 44-100 Gliwice, Poland

The design and development of the medical equipment requires a precise and accurate numerical model reproducing the physical phenomena inside the vessel. The complex computational fluid dynamics (CFD) simulation of the pulsatile blood flow requires proper outflow boundary conditions (BCs) that take into account characteristic impedance, peripheral resistance and capacitance in a systemic peripheral vascular bed [1]. In addition, inlet BC must be capable to describe the pulsatile inlet blood flow. The work presents a lumped parameter model (LPM) of the blood flow in the systemic cardiovascular system that is based on electrical analogy. Proposed LPM is able to mimic response of the vascular bed to the flow changes analyzed using CFD model of aorta [2]. In this approach the blood flow rate and corresponding pressure drop is computed using electrical analogy. All features of the vascular system are described by their electrical analogues, such as: resistors, capacitors, coils and diodes. Additionally the conservation of the mass principle is applied converted into the Kirchhoff’s law [3]. The case of 8-years old girl with coarctation of the descending aorta was analyzed; the obtained results include the time-dependent blood flow and the pressure of the blood ejected from the heart. The resistances and compliances of vessels were estimated [4]. The proposed model was tested using MATLAB software (MathWorks Inc., USA). The heart rate, duration of the systolic and diastolic phases and cardiac output can be adopted according to patient specific data. The work shows that LPM based on the Windkessel model is sufficient tool, give the reliable results and make investigations of the cardiovascular system precise.

References: 1. Simaan MA, Faragallah G, Wang Y, Divo E, Left Ventricular Assist Devices: Engineering Design Considerations, In: New Aspects of Ventricular Assist Device, Dr. Guillermo Reyes (ed.), InTech, DOI: 10.5772/24485. Available from: http://www.intechopen.com/books/new-aspects-of-ventricular-assist-devices/left-ventricular-assist-devices-engineering-design- considerations. 2. Rojczyk M, Ostrowski Z, Adamczyk W, Melka B, Bandoła D, Gracka M, Knopek A, Nowak AJ, Golda A (2016): CFD analysis of blood flow within aorta of patient with coarctation of aorta, Studia Informatica 37: 43-55. 3. Mossa HAL (2008): Engineering Modeling of Human Cardiovascular System, The 1st Regional Conference of Eng. Sci. NUCEJ 11: 307- 314. 4. Bandoła D (2016): Identification and modeling the pulsatile blood flow in the cardiovascular system using a zero-dimensional model in an electrical analogy. In: Arch Inst Tech Ciepl Ostrowski Z, Rojczyk M, Ryfa A, Stanek W (eds), Gliwice

Acknowledgements: This research was supported by National Science Centre (Poland) within project No 2014/13/B/ST8/04225. This help is gratefully acknowledged herewith.

. 57 12. MODELLING BRAIN VASCULAR NETWORK IN SILICO

Krzysztof Psiuk-Maksymowicz1,2

1Institute of Automatic Control, Silesian University of Technology, 44-100 Gliwice, ul. Akademicka 16, Poland; 2Biotechnology Centre, Silesian University of Technology, 44-100 Gliwice, ul. Krzywoustego 8, Poland

The aim of this work was creation of the vascular network of brain relying on magnetic resonance angiograms of the brain and statistical informations about micro-vasculature. Geometrical arrangement of arterial and venous trees is possible to observe for example by means of the magnetic resonance imaging techniques with or without the use of contrast enhancing the image. We used data obtained from two types of non-contrast enhanced magnetic resonans angiographies: phase contrast and time-of-flight sequences. Processing these images we obtained structure (skeleton) of vascular network of the major vessels of the brain. Vessels not visible in angiograms were generated in silico by means of Monte Carlo simulations. The latter were based on the statistics for arterioles, capillaries and venules which differ from each other in diameters and lengths. Developed algorithm generating vascular structures exploits physiological parameters of a brain. Implementation additionally allows to simulate pressures and blood flow for selected segment of the network. We analyzed the model in terms of physiological correctness of generated vascular network structure. Application of particular model parameters resulted in creation of incorrect vascular tree, for example structure which does not cover whole computational space. The potential application of the model are studies of brain perfusion depending on the correct vascular structure as well as comparison of the vasculature between different brains and attempt to find pathological or distinct lesions.

Acknowledgements: This work was financially supported by internal grant of Silesian University of Technology for young researchers BKM-514/RaU1/2015/31.

58 . 13. IMPACT OF CORONARY ARTERY MYOCARDIAL BRIDGE ON THE BLOOD FLOW PROFILE. NUMERICAL SIMULATIONS IN THE SIMPLIFIED GEOMETRY

Marek Rojczyk1*, Jarosław Wasilewski2, Tadeusz Osadnik2, Ireneusz Szczygieł1, Wojciech Adamczyk1, Dominika Rojczyk1, Mariusz Gąsior2, Ziemowit Ostrowski1

1Biomedical Engineering Laboratory, Institute of Thermal Technology, Silesian Univ. of Technology, Gliwice, Poland; 23rd Department of Cardiology, SMDZ in Zabrze, Medical University of Silesia, Katowice, Poland *Correspondence: Konarskiego 22, 44-100 Gliwice, Poland, [email protected]

The purpose of this research is to evaluate blood flow profile by means of Computational Fluid Dynamics (CFD) in the relation to atherosclerotic plaque localization which is typically located proximally to the overlying artery muscle band what is named as Myocardial bridge (MB) [1]. Myocardial bridge is an innate anomaly in which a segment of a coronary artery is surrounded by the myocardium. Therefore systolic compressions by MB on artery wall may induce retrograde blood flow in the adjacent proximal segment. In the middle part of the left anterior descending artery MB can be found most commonly [2]. The assumption of the study was to prove the hypothesis that compression of artery at systole (“milking” effect of MB) generate bidirectional blood flow proximally to the MB with pro-atherogenic low-oscillatory endothelial shear stress (ESS), whereas in the bridged segments, normal or even high ESS protect tunneled artery against atherosclerosis [3,4]. In the presented study simplified geometry model of the coronary artery with bifurcation and MB is analyzed using CFD. The 2D geometry is based on data from coronary computed tomography angiography. Numerical simulations were performed using ANSYS Fluent code (ANSYS Inc. USA). Inflow boundary conditions being blood flow velocity function of time is applied [1]. Blood is considered as Newtonian fluid. The contraction of the vessel lumen due to presence of MB is applied to mimic the muscle performance during cardiac cycle. Lumped Parameter Model (LPM) is used for outlet boundary condition to account for changes in vascular resistance and impedances. The obtained CFD results revealed the presence of low and oscillatory velocities of blood in proximal to MB region, resulting in low values of ESS. Our study indirectly confirm the dependency between disturbed blood flow and plaque localization in the presence of MB.

References: 1. Wasilewski J, Mirota K, Peryt-Stawiarska S, Nowakowski A, Poloński L, Zembala M(2012): Introduction to the computational fluid dynamics based on computer simulation of flow in the left coronary artery. KardiochirTorakochirPol 9: 366-374 (in Polish). 2. Teofilovski-Parapid G, Jankovic R, Kanjuh V, Virmani R, Danchin N, Prates N, Simic D, Parapid B (2016): Myocardial bridges, neither rare nor isolated − Autopsy Study. AnnAnat in press (DOI: 10.1016/j.aanat.2016.09.007). 3. Chatzizisis YS, Giannoglou GD (2009): Myocardial bridges spared from atherosclerosis: overview of the underlying mechanisms. CanJCardiol 25: 219–222. 4. Wasilewski J, Roleder M, Niedziela J, Nowakowski A, Osadnik T, Głowacki J, Mirota K, Poloński L (2015): The role of septal perforators and "myocardial bridging effect" in atherosclerotic plaque distribution in the coronary artery disease. PolJRadiol 16: 195-201.

Acknowledgements: The research was supported by Faculty of Energy and Environmental Engineering within Ministry of Science and Higher Education (Poland) statutory research funding scheme. This help is gratefully acknowledged herewith.

. 59 14. TWO APPROACHES OF SIMPLIFIED BREAST DEFORMATION MODELLING

Marta Danch-Wierzchowska, Damian Borys, Andrzej Świerniak

Institute of Automatic Control, Silesian University of Technology, ul. Akademicka 16, 44-100 Gliwice, Poland

Breast cancer is the most frequent women’s cancer around the world. Diagnosis is mainly done using Magnetic Resonance Imaging (MRI) which is the most sensitive imaging technique for soft tissue parameters changes. This basic diagnostic information can be supported by metabolic imaging from Positron Emission Tomography (PET), which is usually done in hybrid device with Computer Tomography (CT). Fused prone PET and MR images allow to observe both tissue structure and it’s metabolic activity simultaneously, which increase significantly diagnostic confidence, far more than side-by-side review. Unfortunately, for many reasons both studies are performed in different patient position. During MRI examination patient lays in prone position while PET/CT and, what is the most important, surgery are performed in supine position. Since changing position from prone to supine leads to breast deformation, we propose a Matlab algorithm based on finite element method (FEM) to model it. We present also results obtained with model created in ANSYS. Physical based deformation algorithms use Finite Element Methods (FEM) to perform any deformation. Our preliminary study consisted of creating a model based directly on breast medical images. The novelty of our Matlab approach is implementing large breast deformations, with mesh created directly on medical images, without using any third-party software. The same deformation is performed in ANSYS software. Both simulations are based on Lagrange equation of motion and deformations are performed according to gravitational forces. For both simulations (Matlab, ANSYS) chestwall shape is not-identical, probably caused by different muscle tension in prone and supine position. However, after deformation main breast shape is preserved. Presented models use the simplest constitutive tissue description (isotropic, linear elastic, homogeneous), which gives acceptable results. Results are obtained in less than 1 min per image on average portable computer, with fully automated algorithm. This allows us to believe that our algorithm, extended and improved, would be useful in clinical practice.

Acknowledgements: This work was supported by the Polish National Science Centre (NCN) under Grant No. 2011/03/B/ST6/04384 (AS), the Polish National Center of Research and Development grant no. STRATEGMED2/267398/4/NCBR/2015 (MILESTONE - Molecular diagnostics and imaging in individualized therapy for breast, thyroid and prostate cancer) (DB) and the Institute of Automatic Control, Silesian University of Technology under Grant No. BKM-514/RAU1/2016/t.7 (MDW). Calculations were performed using a ANSYS licence purchased in the GeCONiI, POIG.02.03.01-24- 099/13 project, on the Ziemowit computer cluster in the Laboratory of Bioinformatics and Computational Biology, created in the EU Innovative Economy Programme POIG.02.01.00-00-166/08 and expanded in the POIG.02.03.01-00-040/13 project.

60 . 15. QUALITY EVALUATION OF CHOSEN SEGMENTATION TECHNIQUE IN MAGNETIC RESONANCE IMAGING DATA ANALYSIS

Katarzyna Bednarczyk, Franciszek Binczyk, Joanna Polańska

Silesian University of Technology, Institute of Automatic Control, Gliwice

Introduction: Despite of the recent advances in the biological sciences and technological innovation, brain tumour remains one of the most debilitating and deadly disease. Magnetic resonance imaging (MRI) is a common test used to diagnose different subtypes of brain tumour. One MRI session usually produces a large series of images of different modalities (at least the T1, T2, and FLAIR). Detailed analysis of those is very time consuming and specialists are looking for new tools that would enable accurate image analysis to detect even small dangerous changes in the brain. Aim: This work is a comprehensive study on Naive Bayes Classifier as a tool for the automatic detection of brain tumour using co-registered different MRI modalities. The goal was to evaluate its properties as the algorithm for automated tumour delineation. Material and methods: Seventeen patients with three basic MRI sequences (T1, T2, FAIR) were considered. Each sequence included 53 images of cross-sections of the brain manually segmented by the experienced clinician. The first stage of the work involved data pre-processing and normalization. Different methods of data normalization were examined, and their impact on the further classification procedure was investigated. Then the various classifiers were constructed. Quality assessment of data classification was made by calculating relevant indicators, including Dice index, accuracy, FPR, NPR, sensitivity, specificity in Monte Carlo random validation experiment. In the final stage of work, a comparison of all the proposed methods was conducted in order to indicate the best solution and identify possible causes of errors. Results: The basic simplest classifier, assuming future independence and univariate normal distributions of the signal, gave significantly better results than those applying more sophisticated multidimensional probability distribution functions. The automatic detection of the tumour area for this classifier was obtained with mean accuracy of 71%, according to the Dice index. The median of Dice index equal to 76% demonstrates the very skewed distribution of the Dice index across images. It was observed that lower classification quality was associated with varying discrepancies of the data among which the anomalies in tissues (eg. necrosis) and poor image quality (low signal and/or high noise level) played a crucial role. Conclusions: It was proved that fast Naive Bayes classifier based on T1, T2 and FLAIR MRI sequences is able to detect tumour area with very good accuracy. Its results depend strongly on tumour grade/type. Assuming future development and tuning of the classification algorithm, it is possible to support oncological diagnostics by such modern bioinformatics tools.

Acknowledgments: The work was financially supported by SUT grant 02/010/BK_16/3015, FB was financed by BKM/514/RAU1/2015/18

. 61 16. AUTOMATED ANALYSIS OF CELL CYCLE PHASE-SPECIFIC DNA DAMAGE

Artem V. Luzhin, Artem K. Velichko, Sergey V. Razin, Omar L. Kantidze

Laboratory of Structural and Functional Organization of Chromosomes, Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia.

The comet assay is one of the most widely-used approaches for detecting DNA damage; generally, it provides information on the cell population-averaged level of DNA damage. Here, we present a technique for easy measurement of standard comet characteristics and an annotation of the cell cycle phase of each comet. The approach includes the modified neutral comet assay and a pipeline for CellProfiler software designed to analyze DNA damage-related characteristics and annotate the cell cycle phase of each comet. The approach allows to reduce the time of comet assay procedure, because there is no need to synchronize the cell population, and users can analyze more than thousands comets per experiment. Using this technique, we performed cell cycle phase-specific analysis of DNA damage induced by etoposide and showed that the proposed protocol provides important additional information that often remains hidden in a standard comet analysis of an asynchronous cell population.

62 . 17. METHODS FOR FILTERING 2D GEL ELECTROPHORESIS IMAGES

Michał Marczyk

Data Mining group, Institute of Automatic Control, Silesian University of Technology, 44-100 Gliwice, ul. Akademicka 16, Poland

Two-dimensional gel electrophoresis (2DGE) is a powerful tool for separation and fractionation of complex protein mixtures from tissues, cells or other biological samples. A result of the experiment on a single gel is a grey scale image with even thousands black spots, where each correspond to single protein. On the analyzed image, beside composition of the sample we observe some amount of noise as high-intensity spikes with sharp boundaries covering a few pixels, white noise and larger artefacts resulting from dust and other pollutions acquired during the experimental setup. Also, proteins with similar molecular properties can form streaks in which spots are hardly identifiable and quantifiable. The properly designed smoother reduce the noise, keeping the true signal not too much distorted. Omission of the denoising step may for example lead to an increased detection of false spots. The comparison of different filtering methods is made on selected fragments of manually annotated real gels and multiple synthetic images. In order to imitate the real 2DGE image more accurately and retain its characteristics, the information about distributions of background signal, noise and spots was estimated using real data. Two background correction methods were tested: the one based on iterative polynomial fits (in both image axis, line by line) and using rolling ball algorithm (a circular disc with size larger than the largest spot feature in the image is used as a structural element in the morphological opening). Three noise filtering methods were compared: standard median filtering (window of predefined size is placed at each pixel in the image and the signal value is averaged), Median Modified Wiener filter (preserves the morphology of close spots, and avoid spot and spike fuzzyfication) and 2D matched filtering (divide the range of image intensity into overlapping fragments and in each perform Gaussian filtering). Comparison of different methods was provided by means of the sensitivity and false discovery rate (FDR) of spot detection. Overall quality of detection was measured by harmonic mean of sensitivity and 1-FDR termed as F1 score. Additionally, the ability to reproduce the true spot intensity on synthetic images was checked. In this study different algorithms for background correction and noise filtering of 2DGE images were compared. Using real images and artificially created datasets it was shown that background correction and noise filtering are necessary to improve the quality of detection and quantification of protein spots. However, most of the methods used require to set up properly their parameters to get better results than when no image cleaning was performed. Thus it is needed to find an adaptive way of choosing proper settings, based on properties of analyzed images.

Acknowledgments: This work was financially supported by internal grant of Silesian University of Technology for young researchers 02/010/BKM16/0047/33. All calculations were carried out using GeCONiI infrastructure funded by project number POIG.02.03.01-24-099/13.

. 63 18. REACTION ENERGY PROFILES FOR OXIDATIVE ALIPHATIC CARBON-CARBON BOND CLEAVAGE REACTIONS OF CU(II) CHLORODIKETONATE COMPLEXES. DOUBLE-Ζ VS. TRIPLE- Ζ BASIS SET FOR GEOMETRY OPTIMIZATION

Agnieszka Stańczak1, Anna Miłaczewska2, Tomasz Borowski2, Artur Góra1

1Tunneling Group, Biotechnology Centre, Silesian University of Technology, ul. Krzywoustego 8, 44-100 Gliwice, Poland; 2Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Science,Krakow 30-239, Poland

Oxidative aliphatic carbon−carbon (C−C) bond cleavage reactions catalyzed by first row transition metal salts that involve O2 as the terminal oxidant are of significant current interest for the preparation of bulk and fine chemicals. The understanding of the role of anions on Cu(II)/O2 chemistry may give a more rational approach for economical and environmentally friendly syntheses of important precursor compounds (e.g., α-keto esters and 1,2-diketones) for the pharmaceutical industry[1]. In this studies, density functional theory (DFT) methods have been used to examine the + chloride ion binding properties of [(6-Ph2TPA)Cu(PhC-(O)C(Cl)C(O)Ph)] under anaerobic conditions. Geometry optimization was performed with the spin-unrestricted formalism using Gaussian 09 suite of program with B3LYP-D2 exchange-correlation functional and a triple-ζ quality that combined the lacv3p+ basis set for copper and 6-31G for hydrogen and carbon and 6-311G* for chlorine, nitrogen, oxygen and the three aliphatic carbon atoms of the chlorodiketonate[2]. Computed reaction enthalpy profiles were compared with those from the previous study[3], in which a double-ζ basis set (LANL2DZ for copper and 6-31G for other elements) was used for geometry optimization. Geometry optimization with triple-ζ basis set for the key atoms of the system is feasible. There are some significant differences between barrier heights computed for geometries optimized with the double-ζ and the effective triple-ζ basis sets. However, the reaction mechanism suggested by both calculations is basically the same. The binding of the chloride ion to copper very significantly lowers the barrier.

References: 1. Malkhasian AYS. et al. (2007): InorgChem 46: 2950−2952. 2. FrischMJ et al. (2009): Gaussian 09, revision D.1; Gaussian, Inc.: Wallingford, CT. 3. Saraf S et al. (2016): InorgChem 55: 6916-6928.

Acknowledgements: This work was supported by the National Science Centre, Poland (DEC- 2015/18/M/NZ1/00427).

64 . 19. THERMODYNAMIC DESCRIPTION OF ANTICANCER DRUGS REACTIONS WITH POLYMERS FOR DRUG DELIVERY SYSTEM

Katarzyna Bernaczek, Anna Mielańczyk, Zbigniew Grzywna, Dorota Neugebauer

Department of Physical Chemistry and Technology of Polymers, Faculty of Chemistry, Silesian University of Technology, 44-100 Gliwice, Strzody 9, Poland

Doxorubicin [1] (DOX) is one of the anthracycline antitumor antibiotics, which is used in the treatment of a wide range of cancers including breast cancer, lung cancer, sarcoma, and other. Despite the efficacy of DOX, its clinical application is limited by a dose-dependent cardiotoxicity [2]. Strict control of DOX dosage is problematic and limits its use. Methotrexate [3] (MTX) is an antagonist of folic acid, also used as anticancer drug (malignant lymphoma, osteosarcoma, and breast cancer). Its mechanism of action bases on the inhibition of enzyme dihydrofolate reductase involved in folic acid metabolism. However, MTX therapy is limited by side effects such myelosuppression, nephrotoxicity, and gastrointestinal mucositis. In recent years, there has been increasing amount of research associated with drug delivery systems. The use of nanocarriers can improve drug solubility and the therapeutic index for example, by targeted delivery and longer blood circulation times. Nowadays, the most extensively analyzed are polymeric drug carriers [4]. Apart from the widely studied block copolymers, star-shaped architectures have also gained considerable attention. Bond formation between the drug and its carrier is of interest for its importance to fully characterize such interactions to help optimize their bioavailability for the patient. A challenge is the prediction of drug-polymer affinity. This property is described by the equilibrium constant (Ka), and standard Gibbs free energy change (ΔG°). Another important thermodynamic parameters are: energy of interaction between the binding partners (the standard enthalpy change ΔH°), and a measure of the systemorder (standard entropy change ΔS°). Isothermal titration calorimetry (ITC) is a powerful method to study a wide range of interactions both physical and chemical. ITC method allows directing measurement of the enthalpy, the stoichiometry and equilibrium constant associated with the binding. The results can help explore the interaction mechanisms and allow comparative analysis. Novel, pH-sensitive star-shaped copolymers with sugar core were studied in the reaction with DOX and MTX to improve their properties and minimize drugs side effects. The calorimetric results provide quantitative information on the binding of drugs to star-shaped nanoparticles and suggest that these carriers possess properties justifying their consideration as future drug carriers.

References: 1. Agudelo D, Bourassa P, Bérubé G, Tajmir-Riahi H.A (2016): JPhotochemPhotobiolB: Biology 158: 274-279. 2. Jenkins GR, Lee T, Moland CL, Vijay V, Herman EH, Lewis SM, Davis KJ, Muskhelishvili L, Kerr S, Fuscoe JC, Desai VG (2016): ToxicolAppliedPharmacol 310: 159-174. 3. Nicum S, Midgley R, Kerr D. (2000): JRSocMed 93: 416. 4. Liechty WB, Kryscio DR, Slaughter BV, Peppas NA (2010): AnnuRevChemBiomolEng 1: 149–173.

. 65 66 .

Regulation of cellular processes in normal and pathological cells (20-49)

. 67 68 . 20. NHEJ AND HRR INHIBITORS POTENTIATES AN EFFECT OF X-RAY RADIATION N CISPLATIN-SENSITIVE AND CISPLATIN-RESISTANT HUMAN OVARIAN CARCINOMA CELLS

Anna Macieja1, Aleksandra Rodacka2, Mieczysław Puchała2, Tomasz Popławski1

1Department of Molecular Genetics, University of Łódź, 90-236 Łódź, Pomorska 141/143, Poland; 2Department of Molecular Biophysics, University of Łódź, 90-236 Łódź, Pomorska 141/143, Poland

Radiation therapy is widely used as the part of cancer treatment. In incurable cancers (e.g. ovarian cancer at advanced stage), it is an effective way of killing cancer cells. Ionizing radiation causes awide spectrum of DNA lesions, including the most severe DNA double strand breaks (DSBs). The aim of our study was to determine the influence of DSBs repair pathways inhibitors on X- ray effects in human ovarian carcinoma cells. We used inhibitors of NHEJ and HRR - two main DSBs repair pathways. A12B4C3 and DDRI-18 are known as non-homologous end joining (NHEJ) inhibitors, whereas YU238259 is a newly described inhibitor of homologous recombination repair (HRR). To determine an effect of DSBs inhibitors on radiation-treated cancer cells, we used two human ovarian carcinoma cell lines with different sensitivity to cisplatin: A2780 and A2780cis, which are sensitive or resistant to cisplatin, respectively. We analyzed the cytotoxic and genotoxic activity of X-rays alone and X-rays combined with DSBs inhibitors. Viability of cells was assessed using Cell Counting Kit-8 (Sigma). The level of DSBs was assessed by SCGE (single cell gel electrophoresis, the comet assay). Viability of the cancer cells and the level of DSBs were evaluated after 24 and 48 h after irradiation. We observed that DSBs inhibitors are able to enhance the cytotoxic activity of X-ray and to increase the level of X-ray induced DSBs in human ovarian carcinoma cells. We also observed that all of the tested inhibitors decreased viability of radiation-treated cells, and this effect was more pronounced after 48 h. All of the tested inhibitors are able to sensitize human ovarian carcinoma cells to X-rays. The most potent of them– DDRI-18 – sensitizes A2780 and A2780cis cells to X-rays 2- and 4-fold, respectively. Remaining two inhibitors sensitizes human ovarian carcinoma cell lines to X-rays about 1.5 fold. Similar effect was observed for DSBs level. The results of our study suggest, that DSBs inhibitors can potentiate the effect of radiation therapy in human ovarian carcinoma cells.

Acknowledgements: This work was financially supported by the National Science Centre (Poland), according to the decision No. DEC-2013/11/B/NZ7/01340.

. 69 21. MODULATION OF CYTOTOXIC ACTIVITY OF ONCOSTATIN M MUTANTS THROUGH DIFFERENTIAL INTERACTIONS WITH GP130 AND OSM RECEPTORS

Beata Chęcińska, Sebastian D. Pawlak, Radosław Kwapiszewski, Anna Molga, Albert Jaworski, Adrian Jasiński, Piotr Rózga, Małgorzata Teska-Kamińska, Michał Szymanik, Bartłomiej Żerek

Drug Discovery Department, Adamed Group, 05-152 Czosnów, Pieńków 149, Poland

Oncostatin M is a member of interleukin-6 family. Differentiation of its pleiotropic function is associated with the formation of active complexes with two types of receptors. Among these, we can specify gp130 receptor recognized by IL-6 family ligands. It is involved in the activation of multiple signaling pathways of MAPK, JAK/STAT3, PI3-K, playing important roles in the immune response, inflammation, proliferation, carcinogenesis and cell differentiation. Gp130 protein heterodimerizes with LIFR and OSMR, creating two types of receptor complexes, type I - gp130/LIFR and type II - gp130/OSMR. We developed a set of fusion proteins consisting of modified variants of human cytokine Oncostatin M and strongly cytotoxic, active domain of Exotoxin A from Pseudomonas aeruginosa. The OSM mutants were characterized by differential affinity for OSMR and gp130 receptors. The effect of the mutations on ligands affinity was proposed for modulation of cytotoxic activity towards cancer cell lines. Fusion proteins were expressed in Escherichia coli using pET expression system and purified by affinity chromatography using histidine-tag on Ni-NTA resin. Obtained proteins were characterized biophysically using circular dichroism (CD) spectroscopy. Interaction with the Oncostatin M specific receptors, gp130 and OSMR, was confirmed with SPR. Cytotoxicity was evaluated with MTT assay on panel of human cancer and normal cell lines. For in vivo potential we examined the efficacy of one of the most promising agent on mice xenograft model of human lung carcinoma (A549). The calculated secondary structures of fusion molecules were correlated with their component parts. Receptor binding kinetics parameters confirmed that linking the cytokines to toxin domains does not disrupt their biologic properties. In MTT cell cytotoxicity assays proteins displayed specific cytotoxic effects on various cancer cell lines and very low activity on normal cells (IC50 >4000 ng/ml). These effects were related to inserted mutations aimed at modulating affinities to corresponding receptors. Cytotoxic activity of single components was significantly lower on most cancer cell lines in comparison to fusion molecules. The most cytotoxic protein displayed in vivo tumor growth inhibition activity at the dose 0.1 mg/kg therefore it can be very promising molecule used in the treatment of cancer.

70 . 22. FLUCTUATION OF REACTIVE OXYGEN AND NITROGEN SPECIES LEVEL IN HUMAN COLON CARCINOMA CELLS

Tomasz Hejmo1,2, Magdalena Skonieczna1, Dorota Hudy1, Aleksandra Poterała-Hejmo1, Joanna Rzeszowska-Wolny1

1Silesian University of Technology, Institute of Automatic Control, Gliwice, Poland, 2Medical University of Silesia, Zabrze, Poland

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are groups of highly reactive chemical molecules such as free radicals (molecules with unpaired electron). ROS and RNS are involved in cell signaling, immune responses, proliferation, differentiation and apoptosis thus playing an important role in normal cell functioning. Production of ROS and RNS occurs during cellular respiration (for example, during the activation of NADPH oxidase membrane in the respiratory chain), in cyclooxygenase or lipooxygenase pathways. They are known to be important regulatory elements: it has been reported that reactive oxygen species can induce phosphorylation and activation of signaling proteins such as transcription factors, cytokines, tyrosine kinases and growth factors. However, overproduction of ROS and RNS results in oxidative stress and DNA damage which can lead to mutation and/or apoptosis. Elevated levels of reactive oxygen species play a role in the pathogenesis of diseases such as cancer, Parkinson's disease, Alzheimer's disease, diabetes and atherosclerosis. Here we present results of analysis of total reactive oxygen species, DNA-associated reactive oxygen species, nitric oxide and superoxide anion level in HCT116 (human colon carcinoma) cell line cultured in standard condition. Experiments were performed using flow cytometry and fluorescent dyes (DHCF-DA, CellROX, DAF-FM and MitoSox respectively) at six time-points followingmedium change. We observed that the level of reactive oxygen species – both total and DNA-associated – is fluctuating, with the statistically significant increase at 6 and 24 hours after medium change. The level of selected free radicals - superoxide anion and nitric oxide – also changed significantly 6 hours following medium change. There is no correlation with cell cycle or feedback between nitric oxide and superoxide.

Acknowledgments: This work was supported by BKM/506/RAU1/2016/ (A.P-H.), BK-213/Rau1/2016, t.3. (T.H), NCN 2012/07/B/NZ1/00008 (J.R.W.), NCBiR PBS3/B3/32/2015 (M.S).

. 71 23. CHANGES OF REACTIVE OXYGEN SPECIES IN LIVING CHO CELLS

Sylwia Kała, Patryk Bil, Anna Daniłowicz, Karolina Gajda, Aleksandra Poterała-Hejmo, Joanna Rzeszowska-Wolny

Institute of Automatic Control, Silesian University of Technology, Gliwice.; Center of Biotechnology, Silesian University of Technology

Reactive Oxygen Species (ROS) are involved in many cellular processes and their increased levels were found in tumor and other pathological cells. There are also experiments suggesting that the ROS levels may change and be important for the cell cycle progression. We developed procedure for monitoring the levels of ROS in living cells by fluorescent microscopy during many hour experiment with read-out every 30 minutes. The global levels of ROS and superoxide were measured in Chinese Hamster Ovary cells (CHO) after staining with CellRox Green or MitoSox fluorescent dyes respectively, and analyzed with ImageJ program (plugin: Lineage Tracker). In series of experiments we observed changes of global ROS and superoxide levels in unsynchronized and synchronized with Nocodazol cell populations and in single cells. During 48 h observation we observed subpopulations of cells that divided with different rates even in population which was synchronized. Some cells divided regularly and their daughter cells divided at the same time during second division but in some small population daughter cells after first division showed asynchronous divisions and differed in time of the second division. Individual cells differed in the levels of reactive oxygen species and nearly all cells showed increase in the global content but not concentration of ROS short time before division. Our results show that CHO cells strictly regulate concentration of reactive oxygen species but not the total amount of cellular ROS and suggest that ROS are important for proper progress of the cell cycle.

References: Hancock JT, Desikan R, Neill SJ (2001): Role of Reactive Oxygen Species in Cell signaling pathways. BiochemBiomedAspOxidatModific 29: 345- 350

Acknowledgements: This work was supported by the Polish National Science Center Grant 2015/19/B/ST7/02984 (J.RW, P.B), BKM/506/Rau1/2016/ (S.K., A.D., K.G., A.PH)

72 . 24. HEAT SHOCK MODULATES BINDING OF SPEN TO CHROMATIN IN MOUSE SPERMATOGENIC CELLS

Joanna Korfanty1, Tomasz Stokowy2, Wiesława Widłak1

1Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, ul. Wybrzeze Armii Krajowej 15, 44-101 Gliwice, Poland; 2Department of Clinical Science, University of Bergen, Norway

SPEN protein belongs to a protein family characterized by a SPOC (Spen Paralog and Ortholog C-terminal) domain at the C-terminus, and four RNA recognition motifs at the N-terminus. This large (~400 kDa) transcriptional repressor may bind to DNA and RNA and plays an important role in the regulation of transcription. Therefore, we postulate that altered expression of SPEN could be a reason of general transcriptional repression observed during the heat shock in heat-sensitive cells. Using antibodies to different parts of the protein we determined the intracellular distribution of SPEN by immunohistochemistry in control and heat shocked testes. The protein preferentially accumulated in the nuclei of spermatogenic cells and revealed a significant increase of expression after heat shock. Western blot analysis of fractionated testes extracts showed the presence of the full- length protein (which potentially represents a nascent polypeptide precursor) in the cytoplasm, and a shorter mature forms (created after proteolytic digestion) detected only in the nuclear fractions. Moreover, Western blot analysis of whole testes and nuclear and cytoplasmic fractions revealed an increase of SPEN protein level already 15 min after heat shock, both in testes shocked at 38°C and at 43°C. Looking for SPEN binding to chromatin in mouse spermatogenic cells we performed ChIP-seq. We found that N-terminal as well as internal part of SPEN can bind to chromatin while C-terminal SPEN fragment cannot. Using antibody to N-terminal or internal part of SPEN we observed remodeling of the protein binding to chromatin induced by heat shock both at 38°C and at 43°C. SPEN binding was found preferentially in intergenic and intronic sequences. Among annotation to promoters predominate non-coding genes. We noticed enrichment of SPEN binding after heat shock around genes coding for small nuclear RNAs.

Acknowledgements: This work was supported by the Polish National Science Centre (Grant 2011/03/N/NZ3/03926).

. 73 25 CHANGES OF NF-κB SYSTEM DYNAMICS DURING THE HEAT SHOCK RESPONSE

Anna Naumowicz1,2, James Bagnall3, Paweł Paszek3, Michael White3, Wiesława Widłak1, Marek Kimmel 2

1Center for Translational Research and Molecular Biology of Cancer, Maria Skołodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch; 2Institute of Automatic Control, Faculty of Automatic Control, Electronics and Computer Science, Silesian University of Technology; 3Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road, Manchester M13 9PT, UK

The NF-κB signaling system is an essential component of cellular response to stress as the main ragulator of numerous genes important for inflammation, immune response and cell survival. The cellular response to hyperthermia includes the transcriptional HSF1-dependent activation of genes encoding heat shock proteins (HSPs) as a part of an internal repair mechanism. These two types of stress response interfere with each other. Heat shocked cells do not exhibit typical NF-κB induction in response to TNFα stimulation. To investigate cellular responses to cytokine input after heat shock we visualized NF-κB system dynamics using time-lapse fluorescent microscopy. We produced MCF7 cells stably expressing human p65 EGFP-tagged. Resting cells showed a cytoplasmic localization of p65-EGFP. Continuous treatment with 10 ng/mL of TNFα led to rapid nuclear translocation of the fusion protein. Cells responded heterogeneously. Over a long time-course (up to 10 h) some of single cells exhibited oscillations in p65-EGFP translocations. Cytokine treatment directly after 1 h of heat stress (HS) at 43°C led to the attenuation of NF-κB system activation manifested by the lack or time delay in p65- EGFP translocation, or reduction of the first peak amplitude. Stimulation after 1 h of recovery time at 37°C showed more responding cells than directly after HS but with the lower first peak amplitude and lower frequency than non-heated cells. However when the cytokine stimulation was carried out after 2 h of recovery time at 37°C cells responded with an increased frequency of p65-EGFP translocations. Amplitude of the first peak was lower and amplitudes of the next peaks were higher. Possible mechanism responsible for changes in NF-κB dynamics after heat shock may be related with an inhibition of IKK activity. 1 h of HS at 43°C led to IKKα and IKKβ insolubility. Using Western blotting we observed IKKα and IKKβ in the insoluble protein fraction directly after HS and until 2 h of recovery time at 37°C. Heat shock can cause conformational changes in IKK leading to exposition of hydrophobic amino acids. However, after longer recovery period (≥2 h) NF-κB signaling was no longer inhibited. The mechanism responsible for IKKs repair may be related to increased level of heat shock proteins (HSP) synthesized as a result of HSF1 transcriptional activity. Thermotolerance, which was acquired due to HSPs accumulation protects cells and as a result we did not observe NF-κB system attenuation when cells were subjected to second HS at 43°C after 4 h of recovery period at 37°C.

Acknowledgements: This work was supported by NCN grant DEC-2012/04/A/ST7/00353 and White’s lab, Faculty of Biology, Medicine and Health, University of Manchester, Oxford Road, Manchester M13 9PT, UK. AN was supported by Fundacja im. Jakuba hr. Potockiego

74 . 26. HEAT SHOCK PROTEIN HSPA2 PARTICIPATES IN MAINTENANCE OF REDOX HOMEOSTASIS AND MITOCHONDRIAL DYNAMICS IN NON-SMALL CELL LUNG CARCINOMA CELLS

Damian Sojka1, Damian Matyśniak1, Magdalena Głowala-Kosińska2, Małgorzata Łysek-Gładysińska3, Krystyna Klyszcz1, Dorota Ścieglińska1

1Center For Translational Research and Molecular Biology of Cancer, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch, Poland; 2Department of Bone Marrow Transplantation and Oncohematology, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland; 3 Department of Cell Biology and Electron Microscopy, The Jan Kochanowski University in Kielce, Kielce, Poland

The HSPA2 gene is a poorly characterized members of the human heat shock protein HSPA (HSP70) family. This gene is abudantly expressed in the testis and also in several types of somatic tissues. Recently, it was also revealed that HSPA2 is expressed in different types of tumors. Available evidence suggest that HSPA2 promotes growth and invasion of several types of cancer cells. In our previous works we showed that HSPA2 protein is present in non-small cell lung carcinoma (NSCLC) cell lines. We also found that high HSPA2 expression in NSCLC tumors correlates negatively with patients survival. Our preliminar results suggest that the level of HSPA2 expression can be modulated by oxidative stress. These observations allowed us to hypothesize that HSPA2 could be involved in the maintenance of cellular redox balance in normal and cancer cells. In this study we used RNAi technology to knockdown HSPA2 expression in normal bronchial epithelial cells and in several types of NSCLC lines that differed by endogenous expression of the HSPA2 gene. The effect of HSPA2 knockdown on the level of reactive oxygen species (ROS) was evaluated by several types of molecular ROS probes. We found that HSPA2 depletion resulted in changes in ROS level in NSCLC cells, but not in normal bronchial epithelial cells. We also detected that the alterations in mitochondrial system could be the main source of ROS disbalance in HSPA2-depleted cells. HSPA2 knockdown caused decrease mitochondrial membrane potential, reduced number of active mitochondria accompanied by changes in mitochondrial ultrastructure in cancer cells. Mitochondria in HSPA2- depleted cells also produced superoxide at significantly lower rates than the control cells. To conclude, in this work we showed for the first time that depletion of the HSPA2 protein from NSCLC cells can affect the redox homeostasis and mitochondrial dynamics. At present, the potential targets (direct and indirect) of HSPA2 in regulation on mitochondria dynamics and/or structure are unknown. It is even more important that besides mortalin (HSPA9) any other member of the HSPA family has been directly associated with the mitochondrial dynamics. Therefore further research on HSPA2 involvement in regulation of redox balance and mitochondrial dynamics are needed.

Acknowledgements: This work has been supported by National Science Centre, research grant NCN no. DEC-2013/09/B/NZ5/01815

. 75 27. ESTABLISHMENT OF IN VITRO MODEL SYSTEM DESIGNED FOR FUNCTIONAL STUDIES OF SELECTED GENES IN OVARIAN CANCER BIOLOGY

Alexander J. Cortez, Katarzyna A. Kujawa, Patrycja Tudrej, Krystyna Klyszcz, Katarzyna M. Lisowska

Center for Translational Research and Molecular Biology of Cancer, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology Gliwice Branch, 44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland

In our previous microarray study we identified a set of genes related with different survival of the patients with high-grade serous ovarian cancer [1]. Among these were genes coding for, e.g., Cartilage Oligomeric Matrix Protein (COMP), Fibroblast activation protein (FAP), Microfibrillar associated protein 5 (MFAP5), Secreted frizzled protein 2 (SFRP2) and Integrin-beta like 1 (ITGBL1). We checked for expression of these genes in five commercially available ovarian cancer cell lines (A2780, ES2, OAW42, OVCAR3 and SKOV3) and one cell line (OVPA8) established from cells derived from ascitic fluid of high-grade serous ovarian cancer patient. The cell lines that were not expressing a given gene were used to obtain stable overexpression using retroviral transfer system. The coding sequence of a gene of interest was cloned into PLNCX vector and transduced into chosen cell line. Control cell line was obtained by transduction of an empty PLNCX vector. Variant clones were also produced using constructs with Histidine tags, designed for alternative detection of an overexpressed genes. So far, the described model system was used to study an effect of selected genes on the cell proliferation, migration rate, adhesion and sensitivity/resistance to platinum compounds and paclitaxel, the drugs used in the first-line treatment of ovarian cancer patients. Further studies will help to understand the role of the selected genes in ovarian cancer progression and treatment.

References: 1. Lisowska KM et al (2016): JCancerResClinOncol DOI 10.1007/s00432-016-2147-y. Published online: 30 March 2016

Acknowledgments: This study was supported from grant 2012/04/M/NZ2/00133 from National Science Center to K.M. Lisowska.

76 . 28. ACTIVATION OF PMAIP1/NOXA DURING HEAT STRESS

Marek Chadalski1, Joanna Korfanty1, Agnieszka Toma-Jonik1, Anna Naumowicz1,2, Natalia Vydra1, Wiesława Widłak1

1Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland; 2Institute of Automatic Control, The Silesian University of Technology, 44-100 Gliwice, Poland

PMAIP1/NOXA is the primary p53-responsive gene, a member of the BCL-2 protein family, the overexpression of which leads to apoptosis. We found that also HSF1 (heat shock transcription factor 1) could be involved in Pmaip1 regulation. We determined HSF1 binding to its consensus binding site (interestingly, located in the Pmaip1 intron) and Pmaip1 up-regulation following heat shock in mouse spermatocytes, which are extremely heat sensitive. HSF1 is the main mediator of the heat shock response typically inducing cytoprotective Heat Shock Proteins (HSPs) expression what enables cells to survive. We proposed that the balance between HSPs and PMAIP1/NOXA expression, both regulated by HSF1, could be the main molecular mechanism involved in switching from pro-survival to pro-death signaling in cells subjected to elevated temperatures. Thus, we screened mouse tissues to compare their heat-sensitivity with expression of cytoprotective HSPs and apoptotic PMAIP1/NOXA proteins. Using RT-PCR, Western blot and immunohistochemistry (IHC) we characterized the Pmaip1 and Hspa1 expression pattern in different mouse organs up to 24h post-recovery from in vivo performed heat shock. Then, heat-induced cell death was estimated using TUNEL test. At the mRNA level we found the highest heat-induced activation of Pmaip1 in thymus, spleen, stomach, intestine, brain, and testes, while Pmaip1 was not expressed or activated e.g. in salivary gland, pituitary, kidney, urinary bladder, liver, skeletal muscle. TUNEL test demonstrated that apoptosis was rapidly induced by heat shock in testes, thymus and spleen, but not in stomach, heart and pancreas. By Western blot we detected PMAIP1/NOXA activation following heat shock e.g. in stomach and thymus. Interestingly, in stomach we found two protein isoforms: 11-12 kDa (corresponding to calculated MW of the mouse protein) and ~6 kDa (corresponding to human protein MW). This suggests that alternative transcription or translation start sites could be utilized in the mouse Pmaip1. We noticed also a very strong additional specific anti-PMAIP1 staining at the level of ~18-19 kDa (reduced by blocking peptide) suggesting that PMAIP1 could be modified (possibly ubiquitinated). This form is preferentially detected by IHC making impossible to detect unmodified isoforms and estimation how expression of PMAIP1 overlaps with HSPs expression in individual cells. Further studies are needed to confirm above observations and to elucidate which PMAIP1 isoform is acting as proapoptotic protein.

Acknowledgements: This work was supported by grant no 2014/13/B/NZ3/04650 from the Polish National Science Centre.

. 77 29. HSF1-DEPENDENT CHANGES IN THE TRANSCRIPTOME OF THE MCF7 CELLS AFTER HEAT SHOCK OR ESTROGEN EXPOSURE

Agnieszka Toma-Jonik1, Wiesława Widłak1, Tomasz Stokowy2,3,4, Joanna Tobiasz2, Natalia Vydra1

1Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, ul. Wybrzeze Armii Krajowej 15, 44-101 Gliwice, Poland; 2Faculty of Automatic Control, Electronics and Computer Science of Silesian University of Technology, ul. Akademicka 16, 44-100 Gliwice, Poland; 3Department of Clinical Science, University of Bergen, Norway; 4Gerstein Lab, Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, USA

Estrogen (E2) is an important mitogenic factor which enhances cancer cells proliferation. We found that E2 treatment leads to activation of HSF1 (Heat Shock transcription Factor 1) in human breast adenocarcinoma MCF7 cells. HSF1 acts as the cytoprotective factor and activator of the heat shock response. It is the main regulator of the heat shock proteins (HSPs) expression, which general function is to assist protein folding either during de novo synthesis or under stress conditions associated with protein misfolding. Additionally, HSF1 plays a key role in tumor biology supporting the malignant transformation as well as tumor progression. We aimed to study whether HSF1 can support genomic action of the estrogen receptor. We silenced HSF1 in MCF7 cells using specific lentiviral shRNA. To find out the changes in the whole transcriptome after E2 or heat shock treatment we performed the RNA-Seq. The Gene to GO BP (Gene Ontology Biological Process) analysis revealed that silencing of HSF1 resulted not only in downregulation of genes involved in response to stress and protein folding, but also in deregulation of genes involved in signal transduction. Looking for differences in response to estrogen treatment we found that among activated genes, those involved in cell adhesion were over-represented in cells with silenced HSF1, but not in control ones. This findings suggest that in the presence of estrogen HSF1 could change the ability of cells to contact with extracellular matrix and other cells influencing their motility as well as their metastatic potential.

Acknowledgements: This work was supported by grants no 2014/13/B/NZ7/02341 and 2015/17/B/NZ3/03760 from the Polish National Science Centre.

78 . 30. INITIAL CHARATCTERIZATION OF CELLULAR RESPONSIVENES TO THE NOVEL FGFR INHIBITOR

Aleksandra Rusin, Katarzyna A. Kujawa, Magdalena Olbryt, Patrycja A. Tudrej, Aleksander J. Cortez, Katarzyna M. Lisowska

Center for Translational Research and Molecular Biology of Cancer, Maria Skołodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch

No selective inhibitor targeting the family of fibroblast growth factor (FGF) receptor tyrosine kinases has been approved up to date. A potent and selective FGFR inhibitor, CPL-304-110, was discovered in Celon Pharma S.A. The aim of the current project is preclinical and clinical development of this novel anti-cancer drug that is intended to be used in the treatment of squamous lung, stomach, bladder, and other FGFR-driven types of cancer. Herein, we report preliminary screening results f of antiproliferative activity of the FGFR inhibitor in selected cell lines. Four cell lines were initially selected for screening: bladder SW780, lung NCI H1703, breast CAL-120 and colon HCT116. CPL-304-110 was tested at 0.01, 0.1, 1 and 10 µM concentrations for 72 hours in 8 technical replicates. After incubation with FGFR inhibitor, Alamar Blue was added to the cell culture for 1 hour, and then fluorescence was measured using TECAN Infinite 200Pro microplate reader (excitation wavelength - 560nm; emission wavelength - 590 nm). Data was analyzed with GraphPad Prism. IC50 values were between 1 µM and 23 µM. Most responsive was SW780 line that is known to contain FGFR3 exon18::BAIAP2L1 exon 2 fusion. CAL-120 cell line showed lover responsiveness, despite FGFR1 amplification.

Acknowledgements: The research was co-financed by the National Center of Research and Development and pharmaceutical company Celon Pharma S.A., project "CELONKO", grant number STRATEGMED2/266776/17/NCBR/2015.

. 79 31. MITOCHONDRIAL IMPAIRMENT IN HELA CELLS TREATED WITH AMINOALKANOL DERIVATIVES OF XANTHONE

Daniel Sypniewski1, Natalia Szkaradek2, Anna M. Waszkielewicz2, Tomasz Loch1, Michał Szapski1, Henryk Marona2, Ilona Bednarek1

1Department of Biotechnology and Genetic Engineering, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Katowice, Poland; 2Department of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Krakow, Poland

Xanthone derivatives display a broad spectrum of pharmacological activities including antitumor potential, however, the molecular mechanism of their action has not been fully elucidated. Reactive oxygen species (ROS) are one of the most important mediators in signaling pathways in tumor cells. Additionally, ROS mediate significant proportion of anticancer action of agents used in chemo-, radio- and photodynamic therapy; while excessive ROS levels are considered to have deleterious influence on cells, they may also contribute to mechanisms underlying the efficiency of many anticancer drugs. The aim of the study was to examine whether ROS production in HeLa cells under the xanthone treatment leads to mitochondrial impairment and apoptosis enhancement. Four aminoalkanol xanthone derivatives, synthesized at the Dept. of Bioorganic Chemistry (CMUJ, Kraków, Poland), were included in the study due to their previously designated significant antitumor activity. Natural xanthone gambogic acid and anticancer drugs (doxorubicin, bleomycin) were used as reference compounds. ROS levels were measured by the detection of oxidized form of carboxy- H2DCFDA by fluorescent microscopy. Mitochondrial morphology and transmembrane potential was estimated by rhodamine 123 staining. Apoptosis was evaluated microscopically and by caspase-3 activity measurements. mtDNA damage was estimated by Real-Time PCR according to earlier papers [Rothfuss et al. Nucl. Acid. Res. 2010, 38: e24], genomic DNA damage was analyzed by comet assay. Results indicate that all aminoalkanol xanthones used in our study led to significant increase in ROS levels, deterioration of mitochondrial morphology (symptoms of mitochondrial swelling and appearence of megamitochondria was observed), decrease in mitochondrial transmembrane potential. We also proved that mitochondrial dysfunction and cellular apoptosis enhancement correlated with xanthone-induced oxidative stress. All xanthones also induced damage of mtDNA while genomic DNA remained intact. Finally, we indicate that the use of antioxidant N-acetylcysteine partly reversed the effects of xanthone treatment. Altogether these results suggest that our new aminoalkanol xanthones induce oxidative stress by ROS generation in HeLa cell cultures leading to mitochondrial impairment and apoptosis which can be neutralized by using antioxidants. Therefore, we propose that oxidative stress in xanthone-treated HeLa cells plays an important role in the mechanism of their anticancer activity, and this mechanism is similar to the activity displayed by one of the most active anticancer xanthones: gambogic acid.

Acknowledgements: The study was supported by Medical University of Silesia funding (KNW-1- 028/N/5/0, KNW-1-043/N/6/B).

80 . 32. THE TRANSCRIPTION OF CK19 IN ADIPOSE DERIVED STEM CELLS AFTER COCULTURE WITH PORCINE LIMBAL EPITHELIAL CELLS

Bartosz Sikora1, Aleksandra Skubis1, Bartosz Różanowski2, Urszula Mazurek1

1Department of Molecular Biology, School of Pharmacy with the Division of Laboratory Medicine, Medical University of Silesia in Katowice, 41-200 Sosnowiec,ul. Jedności 8, Poland; 2Department of Genetics and Cell Biology, Institute of Biology, Pedagogical Academy of Kraków, Poland

Adipose tissue is a reservoir of mesenchymal stem cells. It was proven that MSCs are able to differentiate into cells derived from all germ layers. The pluripotency of stem cells from adipose tissue (ADSC) is still under investigation. Until now it was shown that ADSC can differentiate into mesodermal tissues however more and more publications confirm that ADSC possibly differentiate also into ectodermal tissues. ADSC may be differentiated by the influence of extracellular environment. The cultivation of this kind of stem cells in a co-culture provides the direct influence of compounds and cytokines that are secreted by cell from differentiating layer, which influences expression changes of characteristic surface proteins. This means that the process of differentiation appears. One of relevant proteins which take part in such differentiation is cytokeratin 19 (CK19) encoded by KRT19 gene. This protein was localized in epithelial tissues including limbal epithelium. It was shown that limbal stem cells derived from basal layer express KRT19, but the partially differentiated cells from limbal and corneal epithelial layer do not. It could be assumed that the expression of KRT19 in differentiated mesenchymal stem cells should be downregulated. This research presents the results of ADSC differentiation into corneal epithelium by co- culturing with porcine limbal epithelial stem cells based on the number of copies of mRNA encoding characteristic membrane proteins of ADSC and gene encoding cytokeratin 19. This research is based on real-time RTqPCR method.

. 81 33. INFLUENCE OF TIME ON SPP1 GENE EXPRESSION DURING OSTEOBLAST DIFFERENTIATION OF ADIPOSE DERIVED STEM CELLS

Aleksandra Skubis1, Bartosz Sikora1, Małgorzata Kimsa2, Bartosz Różanowski3, Agnieszka Witkowska4, Urszula Mazurek1

1Department of Molecular Biology, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Poland; 2Department of Biochemistry, School of Medicine in Katowice, Medical University of Silesia, Poland; 3Department of Genetics and Cell Biology, Institute of Biology, Pedagogical Academy of Kraków, Poland; 4Department of Internal Diseases, Diabetology and Nephrology in Zabrze, Medical University of Silesia, Poland

Adipose tissue is a rich source of mesenchymal stem cells (MSCs). Mesenchymal stem cells have the ability to differentiate into mesoderm-lineage cells, which hold great promise for use in regenerative medicine. They can be obtained by the less invasive method of lipoaspiration and a greater number of cells than bone marrow stem cells. In this study, MSCs were isolated from adipose tissue and differentiate into osteoblasts. Cell surface antigens for ADSC cells were analyzed by fluorescence-activated cell sorting (FACS) with used of Flow Cytometry Kit (R&D Systems, Minneapolis, MN). Normal human adipose derived stem cells (ADSCs) were routinely maintained in the DMEM medium (Dulbecco's Modified Eagle Medium, Lonza, Basel, Switzerland), that was supplemented with fetal bovine serum (FBS), amphotericin B and a penicillin-streptomycin complex at 37˚C in a 5% CO2 incubator. In our experiment we used standard components of medium: dexamethasone, L-ascorbic acid 2-phosphate and beta-glycerophosphate. Medium were changed every 2–3 days. After osteogenic differentiation, the presence of extracellular calcium was confirmed using an Alizarin red stain. Total RNA was isolated from ADSCs by using Trizol reagent (Invitrogen). Quantitative reverse transcription- polymerase chain reaction analysis was used to evaluate the expression of gene SPP1 (Secreted Phosphoprotein 1) coding osteopontin. The protein encoded by this gene is involved in the attachment of osteoclasts to the mineralized bone matrix. The results show that time of differentiation affects gene expression of osteoblast marker.

82 . 34. ARGONAUTE 2 IMMUNOPRECIPITATION REVEALED LATS1 AS A NOVEL PRO-APOPTOTIC TARGET OF MIR-21 IN T CELLS

Nato Teteloshvili1,2†, Katarzyna Smigielska-Czepiel1,2,5†, Ye Yuan1, Annika Seitz1, Debora de Jong1, Bea Rutgers1, Pytrick Jellema1, Roelof Jan van der Lei1, Izabella Slezak-Prochazka1, Elisabeth Brouwer2,3, Annemieke M.H. Boots2,3, Bart-Jan Kroesen2,4, Anke van den Berg1,2, Joost Kluiver1

1Department of Pathology and Medical Biology; 2Groningen Research initiative on healthy Ageing and Immune Longevity (GRAIL); 3Department of Rheumatology and Clinical Immunology; 4Department of Laboratory Medicine, University of Groningen, University Medical Center Groningen, The Netherlands 5Current address: Medical University of Silesia, Katowice, Poland †These authors contributed equally

MiR-21 is an important suppressor of T-cell apoptosis that is also overexpressed in many cancer types. The exact mechanisms underlying the anti-apoptotic effects of miR-21 are not well understood. In the present study, we set out to investigate T-cell specific miR-21 target genes related to its anti-apoptotic signaling. To that end we applied AGO2 RNA Immuno Precipitation followed by gene expression microarray (RIP-Chip) in Jurkat cells. We showed that expression of miR-21 rapidly increases upon αCD3/αCD28 activation of Jurkat cells. Inhibition of miR-21 reduced cell growth and resulted in pronounced apoptosis induction. MicroRNA target gene identification by AGO2 RNA- Immuno Precipitation (IP) resulted in the identification of 72 predicted miR-21 target genes that were 2-fold enriched in the AGO2-IP fraction of miR-21 overexpressing cells. Of these, 71 were enriched at least 2-fold more in the miR-21 overexpressing as compared to AGO2-IP fraction of control cells. Among these, we found several genes involved in the regulation of apoptosis, cell cycle and immune function. The target gene for which the enrichment was most prominently increased upon miR-21 overexpression was the pro-apoptotic protein LATS1. Luciferase reporter assays and Western blot analysis confirmed targeting of LATS1 by miR-21. Finally, LATS1 knock down partially rescued the impaired growth induced by miR-21 inhibition. Collectively, these data identify LATS1 as a miR-21 target important for the anti-apoptotic function of miR-21 in T-cells and likely in many types of cancers.

. 83 35. THE ROLE OF MIRNA-3613-3P IN NEUROBLASTOMA

Iwona Nowak, Elżbieta Boratyn, Małgorzata Durbas, Irena Horwacik, Hanna Rokita

Laboratory of Molecular Genetics and Virology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University,

Neuroblastoma is the most common extracranial solid tumour of childhood. Its clinical presentation varies from spontaneous regression to rapid progression, despite aggressive therapy, and depends on many genetic and biological factors. MiRNAs comprise a class of small noncoding RNAs responsible for post-transcriptional regulation of gene expression and were found to have a significant impact on neuroblastoma pathogenesis. The deregulation of many miRNAs in neuroblastoma and its impact on prognosis and therapy has been described, however the role of many other microRNAs is yet to be fully elucidated. We used miRNA microarrays to investigate changes in miRNA expression in BE(2)C neuroblastoma cells exhibiting MCPIP1 protein overexpression, characterized by potent decrease in proliferation and cellular ATP content. MiRNA-3613-3p was one of the most significantly changed microRNAs and its deregulated expression was also found in other pathological states. Next, we confirmed the downregulation of miRNA-3613-3p in BE(2)C cells under the conditions of MCPIP1 overexpression, as well as found the differential expression of this miRNA in a range of other neuroblastoma cell lines: SKNSH, L-AN-1, L-AN-5, IMR-32, Kelly, CHP-134. Our bioinformatic analysis of the function of predicted targets for the miRNA-3613-3p, using Gene Onthology and Panther algorithms, revealed its potential involvement in tumorigenesis and regulation of mitogenic pathways. Individual target genes for the investigated miRNA were sought in several databases using different algorhitms. Visualisation of the complementarity of selected transcripts (such as DFFB, APAF1, DICER, RORA, NF1, MAP3K1, KIF3A and VHL) and miRNA-3613-3p was done in, among others, RNA hybrid programme. In order to investigate the link between the level of miRNA-3613-3p and its putative targets we measured the expression of selected targets at both mRNA and protein level in cells overexpressing MCPIP1 protein. Our obtained results showed that potential targets of miRNA-3613p are DFFB, APAF1, DICER and RORA. Our data shows, that recently described miRNA-3613-3p could have a role in neuroblastoma tumorigenesis and heterogeneity of its clinical presentation. The suggested targets for the miRNA should be further validated experimentally.

Acknowledgements: This research was supported by: Polish National Science Center; Grant number: 2011/03/B/NZ1/00024 and Research Project Competition for Young Researchers and PhD Students of the Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University; Grant numbers: 1/2014 and 12/2016.

84 . 36. DUSP13 GENE – THE NOVEL POTENTIAL P53 TARGET

Małgorzata Krześniak, Agnieszka Gdowicz-Kłosok, Artur Zajkowicz, Iwona Matuszczyk, Marek Rusin

Center for Translational Research and Molecular Biology of Cancer, M. Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, ul. Wybrzeże Armii Krajowej 15, Poland

The p53 is a transcription regulator modulating the expression of a wide array of genes. Activation of p53 by phosphorylation induces the cell cycle arrest or apoptosis. We observed that in A549 cells, in spite of strong p53 phosphorylation on Ser46 induced by actinomycin D and nutlin-3a co-treatment, apoptosis did not take place. By comparison of transcriptome of A549 cells treated with various combinations of actinomycin D, nutlin-3a or camptothecin we observed strong upregulation of the alternative isoform of DUSP13. DUSP13 gene codes for two major isoforms of dual specificity phosphatases: DUSP13A (MDSP) and DUSP13B (TMDP). These isoforms show strong tissue specific expression. We observed strong, synergistic upregulation of DUSP13 mRNA using next generation sequencing and quantitative RT-PCR methods. This isoform is transcribed from an alternative promoter in intron 3. This alternative promoter of DUSP13 with the potential p53 binding site was cloned into pGL3-Basic reporter vector coding for firefly luciferase gene. We found that p53 from expression vector activated the alternative DUSP13 promoter in various cell lines. The p53-activated isoform of DUSP13 protein contains, at the N-terminal end, a signal of transport into endoplasmic reticulum. Our hypothesis is that the novel DUSP13 isoform may be a component of exosomes and/or it may be directly secreted outside of the cell. Based on our results we conclude that DUSP13 is a novel, p53-regulated gene identified by transcriptome analysis of cells with synergistically activated p53.

Acknowledgements: This work was supported by grants no. 2013/11/B/NZ5 and 2014/15/D/NZ5/03410 from the Polish National Science Centre (NCN)

. 85 37. NATURAL COMPOUND PARTHENOLIDE INDUCES MITF-M DOWNREGULATION AND SENESCENCE IN PATIENT-DERIVED MITF-MHIGH MELANOMA CELL POPULATIONS

Mariusz L. Hartman1, Małgorzata Sztiller-Sikorska1, Aleksandra Mielczarek1, Beata Talar1, Dariusz Nejc2, Małgorzata Czyż1

1Department of Molecular Biology of Cancer, Medical University of Lodz, 92-215 Lodz, Mazowiecka Street 6/8, Poland; 2Department of Surgical Oncology, Medical University of Lodz, 93-509 Lodz, Paderewskiego 4, Poland

Parthenolide (PN), a sesquiterpene lactone from the herbal medicine feverfew (Tanacetum parthenium), exerts multiple anti-cancer activities, including inhibition of nuclear factor κB (NF-κB). The M isoform of microphthalmia-associated transcription factor (MITF-M) is a central regulator of melanoma cell phenotype involved in the regulation of differentiation, proliferation and survival, and it has not been yet reported to be affected by PN. In the present study, patient-derived wild-type BRAF or BRAFV600E melanoma cell populations exerting diverse basal expression and activity of MITF-M and NF-κB were used. Cellular and molecular effects of PN activity were measured by flow cytometry, microscopy, Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). The results confirm PN-mediated NF-κB inhibition and show that in MITF-Mhigh melanoma cell populations, PN rapidly reduces the percentages of MITF-positive cells and down-regulates MITF-M at the protein level, both in the nucleus and cytosol. Mechanistically, PN-induced MITF-M depletion is independent of lysosomal enzymes, MEK/ERK1/2 signaling, proteasome and caspase activity as confirmed by the usage of selective inhibitors of these pathways: chloroquine, trametinib, bortezomib and z-VAD-fmk, respectively. Interestingly, PN induces phosphorylation of ERK1/2. PN inhibits histone deacetylase 1 (HDAC1) concomitantly with the reduction of MITF-M mRNA level, which suggests PN- controlled regulation of MITF-M at the transcriptional level. PN induces cell growth arrest and apoptosis in melanoma cell populations with low basal MITF-M expression, whereas in those with MITF-Mhigh PN induces senescence shown as enlargement of cells, increase in cell granularity and activity of senescence-associated β–galactosidase (SA- β-gal). In addition, MITF-Mhigh melanoma cell populations were characterized by low expression of MCL-1, which might create the conditions suitable for PN-induced senescence. Our findings that PN efficiently reduces the levels of MITF-M and HDAC1 in melanoma cells irrespective of BRAF mutation status, especially when combined with previous results of PN inhibitory effect on NF-κB activity, might have important clinical implications. In addition, our results highlight possible multifactorial effects of PN limited by the molecular context such as basal MITF-M expression level and other cell-autonomous differences such as NF-κB activity and MCL-1 level.

Acknowledgements: This work was financially supported by 2014/15/B/NZ7/00947 from National Science Centre (Poland), and 502/1-156-01/502-14-302 from Medical University of Lodz.

86 . 38. AUTOPHAGY REGULATION IN COLON ADENOCARCINOMA

Martyna Bednarczyk1, Katarzyna Walkiewicz1, Paweł Kozieł1, Małgorzata Muc-Wierzgoń1, Urszula Mazurek2

1Department of Internal Medicine, School of Public Health in Bytom, Medical University of Silesia in Katowice; 2Department of Molecular Biology, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia in Katowice

Beclin1 is located on chromosome 17q21 and codes for a protein that binds to the apoptosis- controlling protein Bcl-2. It is the first identified mammalian autophagy effector, that was reported to be deleted or significantly decreased in ovarian, breast and prostate cancers, suggesting that autophagy may be a tumor-suppressive mechanism. Among the autophagy-related genes, beclin 1 established the first connection between autophagy and cancer, and is regarded as a haplo-insufficient tumor suppressor gene. Beclin 1 participates in autophagosome nucleation at the early step of autophagy. In addition, it interacts with many positive and negative regulators that affect autophagic activity and tumorigenesis. It acts as a crucial mediator in autophagy regulation. The aim of this study was to compare the expression profile of BECN1 in colon cancer specimens from different clinical stages of cancer. The analysis of the expression profile of BECN1 gene was performed using oligonucleotide microarrays of HG-U133A (Affymetrix, Santa Clara, CA). Appointment of differentiating genes was performed with the use of PL-Grid Infrastructure (http://www.plgrid.pl/). The results showed that the expression of BECN1 mRNA is lower in colon adenocarcinoma than in the control, depending on the of the clinical stage of cancer: p = 6.866942E-4. The value of FC, which shows log2 fluorescence signal difference between the treatment groups were CSI vs C = - 1.3238523, CSII vs C = -1.4172313, and CSIII vs C = -1.631746 and SCIV vs C = -1.7723753. Conclusions: Reduction expression of BECN1 mRNA depends on the clinical stage of adenocarcinoma.

. 87 39. ENFORCED MCPIP1 EXPRESSION AND ITS INHIBITORY EFFECT ON MYCN ONCOGENE, PI3K/AKT/MTOR PATHWAY AND CELL CYCLE IN NEUROBLASTOMA

Elżbieta Boratyn, Iwona Nowak, Małgorzata Durbas, Irena Horwacik, Hanna Rokita

Laboratory of Molecular Genetics and Virology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland

MCPIP1 (monocyte chemoattractant protein-1-induced protein1) also called regnase-1 is a multidomain protein encoded by the ZC3H12A gene and one of important negative regulators of inflammation. Neuroblastoma (NB) is the most common extracranial solid tumour of childhood. One of the most important neuroblastoma markers is MYCN oncogene which the high expression of which causes poor patient outcome. Additionally, MYCN is a significant regulator of genes involved in important processes of the described neoplasm, like cell cycle progression and escape from immune system. Our recent studies showed an inverse correlation between MYCN and MCPIP1 expression levels in neuroblastoma cell lines and tumours. In the present study we investigated impact of the enforced wild type or mutant MCPIP1 expression in neuroblastoma cell lines (BE(2)-C, Kelly). We found significant decrease in MYCN expression at the gene and protein levels and we propose that MCPIP1-driven MYCN downregulation relies on a known mechanism of MYCN destabilization engaging Akt/mTOR pathway. Further, we performed measurements of expression levels of cell cycle regulators like cyclins, cyclin-dependent kinases and their inhibitors (i.e. p21, p27). The obtained data show that MCPIP1 overexpression could lead to cell cycle arrest of NB cells. Based on these results we can draw two important conclusions: (i) in BE(2)-C neuroblastoma cells overexpressing MCPIP1 the inhibition of Akt/mTOR leads to indirect MYCN oncoprotein destabilization; (ii) cell cycle arrest of neuroblastoma cells could be caused by MCPIP1 overexpression and MYCN oncogene downregulation.

Acknowledgements: The study was supported by grant no. 2011/03/B/NZ1/00024 from the Polish National Science Center and grants from Research Project Competition for Young Researchers and PhD Students of the Faculty of Biochemistry, Biophysics and Biotechnology Jagiellonian University awarded to Elżbieta Boratyn in 2014, 2015, 2016. Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University is a partner of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education.

88 . 40. EXPRESSION OF MIRNAS WITH PROBABLE INFLUENCE ON WNT4 GENE EXPRESSION IN ENDOMETRIAL CANCER PATIENTS

Tomasz Janikowski1, Nikola Zmarzły1, Emilia Wojdas3, Andrzej Witek2, Agnieszka Jęda-Golonka2, Urszula Mazurek1

1Department of Molecular Biology School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec; 2Department of Gynecology, Obstetrics and Oncologic Gynecology, School of Medicine in Katowice; 3Department of Instrumental Analysis, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia in Katowice

Introduction: Endometrial cancer is the most common gynecological malignancy in developed countries. The Wnt/β -catenin signaling pathway is crucial in all types of cells influencing such processes like: proliferation, inflammatory response, differentiation. The role of WNT4 in endometrioid endometrial adenocarcinoma (EEC) is associated with changes in the Wnt/β-catenin pathway. Thus it is important to recognize miRNAs that influence the down regulation of WNT4 in endometrial cancer. The aim of this study was to analyze the expression profile of miRNAs that can silence Wnt4 expression in endometrial cancer. Patient and methods: Gene expression analysis was completed for 19 endometrial endometrioid adenocarcinomas and 5 normal specimens (obtained from women with diagnosed uterine fibroids, benign ovarian tumors and a prolapsed uterus with histopathologically confirmed endometrium in the proliferative phase) using Affymetrix HG-U133A oligonucleotide microarrays. The miRNA expression experiment was done for 6 endometrial cancer patients and 4 normal specimens with the use of miRNA 2.0 (Affymetrix) The statistical analysis was performed using the GeneSpring13.0, Affymetrix TAC and Panther Gene onthology software. Results: The expression of Wnt4 was down-regulated in all endometrial cancer grades in comparison to the control. The fold change were: in Grade 1 -1.75, in grade 2 -3.36 and in grade 3 - 2.85. Among 448 differentially expressed miRNA only mir-15b was statistically significant with a fold change for G1 1.01 and for G2 – 3.6 in comparison to the control. Conclusion: Changes in expression of miR-15b in grade 2 endometrial cancer suggest a possible correlation between the miRNA and Wnt4 expression.

Acknowledgements: This research was financed by the Medical University of Sillesia nr KNW-1- 170/N/6/O

. 89 41. NOVEL ROLE OF WWOX TUMOR SUPPRESSOR GENE IN REGULATION OF BRAIN CELLS DIFFERENTIATION

Katarzyna Kośla, Ewa Styczeń-Binkowska, Magdalena Nowakowska, Elżbieta Płuciennik, Karolina Pospiech, Izabela Baryła, Dorota Jędroszka, Magdalena Orzechowska, Andrzej K. Bednarek

Department of Molecular Carcinogenesis, Medical University of Lodz, Poland

The WWOX gene has been shown to be crucial for proper central nervous system development as well as important for brain cancerogenesis. According to a numerous studies conducted on various tumor tissues and cell lines it is not a classical tumor suppressor. Its role is not limited only to cell cycle control or genome integrity maintenance, but its influence on cell functioning is more global. The aim of our study was to elucidate the WWOX protein role in the biology of human neural progenitors (hNSC). We silenced the WWOX gene by lentiviral shRNA delivery and investigate what consequences it causes for hNSC biological functions and differentiating potential. Inactivation of WWOX resulted in reduced cells mitochondrial redox activity. It also strongly affected cells adhesion. The hNSC cells with silenced WWOX showed considerably stronger adhesion to ECM protein mixture as well as to fibronectin alone. Moreover cells lacking WWOX expression secrete considerably less MMP2 and MMP9. The most striking was the cells behavior in 3D culture. While differentiation of the cells cultured as a monolayer appeared to be unaffected by WWOX deficiency, 3D cultured hNSC with silenced WWOX remained as isolated, single cells that did neither proliferate nor differentiate, whereas control cells differentiated and exhibited extensive network formation. Although the 2D cultured cells did not exhibit morphological changes during spontaneous differentiation to neurons, the CAGE gene expression analysis revealed that in fact gene regulation in the absence of WWOX protein is much altered. The presented study suggests that proper WWOX expression is important for hNSC maintenance and differentiation.

Acknowledgements: The study was funded by Medical University of Lodz grant 502-03/0-078-02/502- 04-020 and by National Science Centre, Poland grant 2015/17/D/NZ2/01989.

90 . 42. WWOX TUMOR SUPPRESSOR GENE IN THE REGULATION OF BASIC ENERGY METABOLISM IN GLIOBLASTOMA CELL LINES

Ewa Styczeń-Binkowska, Katarzyna Kośla, Izabela Baryła, Elżbieta Płuciennik, Karolina Pospiech, Magdalena Nowakowska, Dorota Jędroszka, Magdalena Orzechowska, Andrzej K. Bednarek

Department of Molecular Cancerogenesis, Medical University of Lodz, 90-752 Lodz, ul. Żeligowskiego 7/9, Poland

WWOX gene is considered a tumor suppressor. Much of previous research showed that genetic alteration in WWOX gene and the decrease of its expression are involved in cancerogenesis. Moreover, recent publications have demonstrated participation of WWOX gene in regulation of cell metabolism. Scientists reached a conclusion that cancer cells can switch their metabolism in order to adjust the mechanisms responsible for energy production to changes in the tumor microenvironment. Recent studies have shown that among the glioblastoma (GBM) cell lines scientists distinguished the lines which are typically glycolytic, glycolytic with moderately functional oxidative phosphorylation (OXPHOS) and typically OXPHOS. The aim of the current study was to determine how WWOX tumor suppressor is involved in glucose metabolism in GBM cell lines presenting different metabolic phenotype. As a research model we used T98G and U87MG GBM cell lines. T98G cell line is recognized as an OXPHOS type, which efficiently carry out process of oxidative phosphorylation and U87MG is considered to be glycolytic type, and is characterized by intensified glycolysis. To determine whether the WWOX gene is involved in the regulation of GBM cell glycolysis we upregulated its expression in U87MG and silenced it in T98G. The WWOX gene cDNA was introduced into U87MG GBM cells by a pLNCX2 retroviral transduction. To obtain WWOX silencing, the T98G cells were transduced by lentiviral vector harboring WWOX shRNA. Our experiments were carried out on GBM cells grown for 48 hour in an atmosphere of 20% or 0.1% O2 concentration. We then assessed the relative expression of WWOX and HIF1 genes, as well as that of genes related to glycolysis: SLC2A1, HK2, PFK1, LDHA, PKM2. The relative expression of these genes was estimated using real-time RT-PCR technique. The results of the experiments show decrease of examined genes’ expression as a result of WWOX overexpression in glycolytic U87MG cells. Consistently, silencing of WWOX expression in OXPHOS T98G cells led to increase of the examined genes expression. We conclude that WWOX overexpression in U87MG cells caused modification of their phenotype to less glycolytic. In turn, T98G cells as a result of silencing WWOX expression changes their OXPHOS phenotype to more glycolytic. In our study we demonstrated differences between cell lines in terms of expression of glycolysis genes that can depend on WWOX expression. Therefore, we conclude that WWOX gene is able to switch glucose metabolism of GBM cells.

. 91 43. THE ROLE OF WWOX GENE IN BIOLOGY OF ENDOMETRIAL CELLS

Elżbieta Płuciennik1, Magdalena Nowakowska1, Marta Popęda2, Małgorzata Gałdyszyńska2, Katarzyna Kośla1, Karolina Pospiech1, Izabela Baryła1, Ewa Styczeń-Binkowska1, Magdalena Orzechowska1, Dorota Jędroszka1, Andrzej K. Bednarek 1

1Department of Molecular Carcinogenesis, Medical University of Lodz, Poland; 2Faculty of Biomedical Sciences and Postgraduate Training, Medical University of Lodz, Poland

In Poland (2013), endometrial cancer was ranked in the 4th place in terms of morbidity and 11th in terms of mortality in women. In the 1980s, Bokhman et al. proposed to differentiate two types of endometrial carcinogenesis process based on histopathological characteristics and molecular pathways (endometroid endometrial carcinoma, EEC and non-endometrioid endometrial carcinoma, NEEC). The newest classification from the Cancer Genome Atlas (TCGA) identified four prognostically significant subgroups (according to alternation such as POLE mutations, microsatellite instability, copy-number low, copy-number high ). However, some of patients with endometrial cancer do not show the above-mentioned molecular alterations. Our previous in vitro and patients study revealed the potential role of WWOX gene in modulation of endometrial cells bahaviour and features. The aim of the present study was to analyze the impact of differential WWOX expression on biological cancer-related processes in endometrial cell lines, both non-cancerous and cancerous with various differentiation status. We constructed an in vitro model in normal endometrial cell line THESC, and two endometrial cancer cell lines Ishikawa (well-differentiated) and the MFE296 (moderately differentiated) cells, in which the WWOX tumor suppressor gene was silenced by Gipz lentiviral shRNA. We examined the changes in invasiveness via biological assays, such as zymography, migration through a basement membrane, the adhesion of cells to extracellular matrix proteins, anchorage-independent growth and colony formation assay. We also evaluated the correlation between the mRNA expression of the WWOX gene and genes involved in the processes of carcinogenesis (CTNNB1, ZEB1, CDH1, EZR, VIM, PTEN, SPARC) by RT-qPCR. Our results revealed that WWOX regulated changes in adhesion potential to ECM protein in both the normal and cancer cell lines. Downregulation of the WWOX gene reduced the ability of endometrial cells to invade through the basement membrane but only for the poorly differentiated cell line, MFE296. Moreover, this was associated with a reduction by 36% of the activity of metalloproteinase 2 (MMP-2). Furthermore, WWOX gene altered the ability of endometrial tumor cells to grow in suspension, but only in poorly differentiated cells (an increase by 27.7% for MFE296/shWWOX), while in well-differentiated Ishikawa cells it affected the ability to form colonies (an increase by 68% for Ishikawa/shWWOX). Analysis of expression at the mRNA level showed that silencing WWOX gene in the poorly differentiated MFE296 cell line causes more than 2-fold increase in the expression of the two genes essential for cell adhesion: CDH1 and EZR, as well as SPARC gene which protein product regulates cell growth through interactions with the extracellular matrix (ECM) and cytokines. In conclusion, these results for the first time provide a new information on the participation of WWOX gene in the process of adhesion of normal endometrial cells, and endometrial cancer cells, to ECM proteins. Moreover, WWOX gene affects metastasis processes (migratory capacity, activities of matrix metalloproteinase-2 and in the ability to grow in suspension) as well as the expression of CDH1, EZR, and SPARC in poorly differentiated endometrial cancer cells.

Acknowledgements: This study was funded by the National Center of Sciences N N407 168940

92 . 44. THE ROLE OF WWOX GENE IN REGULATING GLYCOLYSIS IN WOMEN DIAGNOSED WITH GESTATIONAL DIABETES MELLITUS

Izabela Baryła1, Elżbieta Płuciennik1, Ewa Styczeń-Binkowska1, Dorota Jędroszka1, Katarzyna Kośla1, Magdalena Nowakowska1, Magdalena Orzechowska1, Karolina Pospiech1, Katarzyna Mac-Marcjanek2, Andrzej Zieleniak2, Monika Żurawska-Kliś3, Andrzej Bednarek1

1Department of Molecular Carcinogenesis; 2Department of Structural Biology, Faculty of Biomedical Sciences and Postgraduate Education, Medical University of Lodz, 90-752 Lodz, ul. Zeligowskiego 7/9, Poland; 3Diabetology and Metabolic Diseases Department, Medical University of Lodz, 92-213 Lodz, ul. Pomorska 251, Poland

WWOX gene is known as tumor suppressor gene, the expression of which is greatly reduced in many kinds of cancer. However, in addition to neoplastic changes in mice lacking WWOX expression, researchers have demonstrate metabolic abnormalities, including hypoglycemia. WWOX protein possess two N-terminal WW domains and a central short dehydrogenase domain. WW domain is responsible for protein-protein interactions. Several reports have showed that WWOX protein interacts with a number of transcription factors involved in cellular metabolism, cell cycle regulation and cellular differentiation like AP-2α and γ, NFKB, p73 and DVL2, which play a role in the WNT pathway, that denotes WWOX as a global modulator of gene expression. Using immunoprecipitation method, more than 200 proteins was identified as putative WWOX partners. In this group, there are present proteins involved in the metabolism of glucose (enolase ENO1, pyruvate kinase PKM and ATP-citrate lyase ACLY), gestational diabetes genetic risk factors (enolase ENO1, pyruvate kinase PKM and ATP-citrate lyase ACLY) and risk factors associated with type 2 diabetes (pyruvate kinase PKM and transferase and transferase N-acetylglucosamine (GlcNAc) OGT). At the same time, WWOX acts as a negative regulator of hypoxia-induced factor HIF1A, which is responsible for directing cell metabolism towards glycolysis rather than tricarboxylic acid (TCA) cycle. The reason is fact that HIF1A is a transcription factor and HIF1A is required to induce transcription of glycolytic genes. This allows suggesting that WWOX may be responsible for regulation of sugar metabolism processes, and thus disturbances in its expression level can cause metabolic disorders, including gestational diabetes. In our study the expression level of WWOX, HIF1A and several genes involved in glucose metabolism (PFK1, LDHA, PKM2, HK2) and glucose transporters (SLC2A1, SLC2A4) was measured using RT-qPCR in peripheral blood leukocytes in the case of patients with gestational diabetes (GDM) vs normal glucose tolerance (NGT) patients. The study was conducted in a group of 93 subjects, 78 patients with GDM and 15 controls. The results show that GDM patients, as compared to the control group, have nearly 2.5 times lower WWOX expression level while HIF1A expression level is more than twice elevated. According to HIF1A, the expression level of genes encoding glycolytic enzymes of are higher in GDM patients than NGT, which suggests that gestational diabetes patients are in hypoxic conditions and anaerobic glycolysis is a main route of metabolism. What is more, the significant negative correlation between WWOX and lactate dehydrogenase (LDHA) and glucose transporter (SLC2A4) was observed in case of gestational diabetes patients. These results demonstrate a potential role of WWOX gene as a negative regulator of HIF1A and by this way WWOX is involved in regulation of glucose metabolism.

Acknowledgements: This work was supported by Polish National Center for Science (NCN) grant No. 2015/17/N/NZ4/02805.

. 93 45. CHARACTERISTICS OF THE METHYLATION PATTERNS OF GENE PROMOTERS AND THE EXPRESSION PROFILES OF LGALS1 & LGALS3 GENES IN T24 BLADDER CANCER CELL LINE

Dominik Bieg1, Marta Poczęta1, Ewa Nowak1, Daniel Sypniewski1, Aleksandra Mielczarek-Palacz2, Zdzisława Kondera-Anasz2, Ilona Bednarek1

1Department of Biotechnology and Genetic Engineering; 2Department of Immunology and Serology; School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, ul. Jedności 8, 41-200 Sosnowiec, Poland

Bladder cancer is one of the most frequent malignancies of the urinary system in the world. Except for mutations occurring in the genome, alterations underlying this disease are epigenetic, e.g. disorders of DNA methylation affecting genes transcription and expression. Aberrant methylation of gene promoters is a crucial factor in molecular pathogenesis of many cancers and can be potentially useful as a biomarker. There is growing evidence that galectin-1 (LGALS1) and galectin-3 (LGALS3) are involved in bladder cancer tumorigenesis, nonetheless there are still no sufficient data regarding the contribution of DNA methylation alterations. It should be noticed that galectins are implicated in essential biological processes such as cellular communication, apoptosis, inflammation and immune response. The current study attempted to investigate the methylation status and expression profile of LGALS1 and LGALS3 genes in the T24 cancer cell line. The T24 cell line culture was performed under standard conditions (37˚C; 5% CO2; RPMI medium supplemented with 10% FBS and 50µg/ml gentamycin). DNA and RNA were isolated by spin- column technology. MS-PCR (methylation specific PCR) was used to determine methylation status of LGALS1 and LGALS3 gene promoters by distinguishing 5-methylcytosine from unmethylated cytosine in bisulfate converted DNA. Expression profiles of genes of interest were examined by RT-PCR (reverse transcription PCR). Specific primers were designed using MethPrimer (for MS-PCR) and Primer-BLAST (for RT-PCR) tools. As a result of in silico analysis, TBP (TATA-binding protein) gene was chosen as a control for both PCR reactions. The results reveal that in T24 cancer cell line, the lack of LGALS1 and LGALS3 gene promoters methylation is accompanied by the expression of galectin-1 and galectin-3 proteins. The collected data might suggest that evaluation of methylation pattern of LGALS1 and LGALS3 gene promoters can possibly provide some information on expression of galectin-1 and galectin-3.

Acknowledgements: The research was supported by Medical University of Silesia funding: KNW-1- 028/N/5/0, KNW-1-043/N/6/B, KNW-2-014/N/5/N, KNW-2-B10/N/6/K

94 . 46. EPIGENETIC REGULATION OF MDR1 GENE ACTIVITY IN BLADDER CANCER

Marta Poczęta1, Ewa Nowak1, Dominik Bieg1, Daniel Sypniewski1, Justyna Sikora2, Zdzisława Kondera-Anasz2, Ilona Bednarek1

1Department of Biotechnology and Genetic Engineering; 2Department of Immunology and Serology; School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, ul. Jedności 8, 41-200 Sosnowiec, Poland

Bladder cancer is one of the most commonly diagnosed cancersin the world. The activation of oncogenes or tumor suppressor gene mutations may contribute to the development of this cancer. Epigenetic changes, including alterations in DNA methylation of gene promoter regions, may play an important role in initiation as well as progression of cancer. Generally, DNA methylation is associated with loss of gene expression. In addition, DNA hypomethylation may be a potential biomarker for bladder cancer. The human MDR1 (multidrug resistance 1 gene) encodes an integral membrane protein, P-glycoprotein (Pgp). The MDR1 gene/P-gp is expressed not only in various normal human tissues but also in various malignant tumors. The aim of our studies was to seek the link between the MDR1 gene expression and methylation pattern of its promoter region in human bladder cancer cell line T24 (ATCC® HTB-4™) and bladder cancer tissue microsection samples. Extraction of DNA and RNA from examined samples was performed by using ZR-Duet™ DNA/RNA MiniPrep (Zymo Research). Bisulphite-modified DNA was subject to MSP-PCR (Methylation-Specific PCR). Additionally, the changes in expression level of analyzed gene was examined by Real-Time™ RT-PCR. TBP and β-actin housekeeping genes were used as a control. The results show that in 80% of analysed tumor tissue samples demethylation of the promoter region MDR1 accompanied by the expression of this gene. In human cancer cell line T24 and 20% of analysed samples revealed the presence of both methylated, as well as, unmethylated variant of MDR1 gene. Additionally, Real-Time™ RT-PCR revealed overexpression of analyzed gene in 47% of examined bladder cancer tissue samples. The presented data suggest that study of MDR1 gene expression and methylation pattern could be an important aspect of understanding the epigenetic regulation and development of bladder cancer.

Acknowledgements: The study was supported by the funds of Medical University of Silesia: KNW-1- 028/N/5/0, KNW-1-043/N/6/B.

. 95 47. THE BIOLOGICAL IMPLICATIONS OF ESTROGEN RECEPTOR STATUS AND WWOX GENE IN BREAST CANCER

Karolina Pospiech, Magdalena Nowakowska, Elżbieta Płuciennik, Katarzyna Kośla, Izabela Baryła, Ewa Styczeń-Binkowska, Dorota Jędroszka, Magdalena Orzechowska, Andrzej K. Bednarek

Department of Molecular Cancerogenesis, Medical University of Lodz, 90-752 Lodz, Zeligowskiego 7/9, Poland

Tumour suppressive character of the WWOX gene was confirmed in numerous studies on various experimental models. As high as 80% incidence of deletions in breast cancer was observed, within the 16q region, where this gene is located. Significant reduction in expression of the WWOX gene was observed not only in breast cancer, but also in other tumour types including ovary, lung, colon and liver. In vitro studies of the soft agar growth after WWOX gene expression restoration in breast cancer cell lines MDA-MB-453, T47D, BT-474, MDA-MB-231 and HCC1937 significantly reduced their ability to form colonies, whereas morphogenesis test in Matrigel revealed branched structures of MDA- MB-231 cells transfected with WWOX, suggesting that WWOX alone has the ability to reverse 3D growth toward normal cell phenotype. WWOX protein possesses enzymatic short-chain dehydrogenase/ reductase domain. It is assumed that in steroid hormone-regulated tissues (where WWOX expression is the highest) this protein acts as a steroid dehydrogenase. The relationship between WWOX and hormone receptors was shown both on animal models, as well as in breast cancer patients. Herein, we have tried to assess the relationship between the presence of estrogen receptor and WWOX gene in breast cancer cell lines. The study was based on estrogen-responsive (MCF7, T47D) and estrogen-unresponsive (MDA-MB-231, BT20) cell lines, for which depending on native expression, WWOX gene was either induced by retroviral transfection (MDA-MB-231, T47D) or silenced with shRNA (MCF7, BT20), and cells were estrogen stimulated. Under the influence of 17β- estradiol presence, adhesion to ECM proteins, invasion, redox activity, proliferation and apoptosis were assessed. For the examined cell lines we have noticed alterations in biological characteristics of the cells, after estradiol treatment depending on WWOX gene level. Most significant changes were observed in adhesion to ECM proteins after estrogen treatment for estrogen-responsive cell lines, however also other biological parameters were changed. The results obtained point at a complex role of WWOX gene in breast cancerogenesis, which seems to be in tight relationship with estrogen receptor presence.

96 . 48. THE INFLUENCE OF TEMPERATURE ON THE ACTIVITY OF POTASSIUM CHANNELS

Agata Wawrzkiewicz-Jałowiecka1, Beata Dworakowska2, Zbigniew Grzywna1

1Department of Physical Chemistry and Technology of Polymers, Faculty of Chemistry, Silesian University of Technology, 44-100 Gliwice, Strzody 9, Poland; 2Department of Physics, Division of Biophysics, Warsaw University of Life Sciences – SGGW, 02-787 Warszawa, Nowoursynowska 166, Poland

Potassium channels are pore-forming transmembrane proteins that regulate many biological processes by controlling potassium flow across cell membranes. Alterations in potassium channel functions or expressions contribute to different diseases such as epilepsy, cardiac arrhythmia, and neuromuscular symptoms.There are strong evidences that cancer is also associated with dysregulated channel expression. Moreover, potassium channels regulate cancer cell behavior such as proliferation and migration. Thus, investigation of the factors influencing the activity of K+ channels, and mechanisms standing behind that, is a meaningful challenge. In this work, we analyze the impact of temperature on the activity of large conductance voltage 2+ and Ca -activated potassium channel (BKCa). In this aim, we have carried out the patch-clamp experiments at different temperatures in the range of 17-37C with ΔT = 5C step. We compare such characteristics of the obtained data as: channel’s open state probability, mean dwelling-times of open and closed states, dwell-time distributions of open and closed states, and the long-range memory (measured by the Hurst coefficient). Results of the current research allow us to infer about the possible mechanism of how the changes in temperature may affect the channel’s behaviour. It turns out that the most promising hypothesis suggests prominent indirect influence of that factor by altering properties of the cell membrane and, in consequence, the magnitude of its fluctuations, which are important for the channel gate dynamics according to our previous research.

. 97 49. FADU-DERIVED RADIOCHEMOTHERAPY-RESISTANT CLONAL CELL LINES AS AN EXPERIMENTAL MODEL SYSTEM IN EXOSOME RESEARCH: PRELIMINARY STUDY

Agata Abramowicz1, Ingeborg Tinhofer-Keilholz2, Piotr Widlak1, Monika Pietrowska1

1Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland; 2Department of Radiooncology and Radiotherapy, Charite University Hospital, Berlin, Germany

Exosomes are small nanovesicles secreted into extracellular milieu by fusion of late endosomes with plasma membrane. They can be found in various body fluids like blood, urea, cerebrospinal fluid or saliva. However their function is still intensively studied, their impact on intercellular communication and immune response modulation is increasingly studied making them a promising object for biomarkers investigation as well as for disease development and therapy response comprehension. Indeed, variable sensitivity of cancer cells within a tumor to treatment is commonly observed and the cause of this phenomenon is certainly multi-faceted and as desirable as elusive. Here in our study we examined FaDu cells, differing in terms of sensitivity to radiation and cisplatin treatment, as an experimental model for investigng potential role of exosomes in response to genotoxic agents. We did this by characterizing proteome components of their cargo. The four clonal cell lines ( C78, C5, C46 and C54) derived from human HNCC cell line FaDu (ATCC HTB-43), established in the late sixties from a punch biopsy of a hypopharyngeal tumor, were characterized according to their radiochemosensitivity. The clonogenic assay was performed using 2, 4, 6 and 8 Gy doses of ionizing radiation and 10, 100, 500, 1000 ng/mL doses of cisplatin. Cell viability and proliferation ability were assessed using Crystal Violet assays. For immnodetection of stress-induced proteins like p53 the Western blot analysis was performed. Exosomes were isolated using ultrafiltration method and purified by size-exclusion chromatography with Sepharose 2B. They were evaluated by Western blot and electron microscopy techniques. Our studies indicated that the selected clonal cell lines varied in their sensitivity to genotoxic agents. The C78 clonal cell line was clearly resistant to both ionizing radiation and cisplatin. The low treatment doses (2 Gy or 10 ng/mL of cisplatin) had no significant influence on clonal ability of this cell line, whereas for other more sensitive cell lines (C54, C46), a two-fold decrease in the number of colonies was observed. Cisplatin dose of 100 ng/mL completely inhibited growth of C54 and C46 cells with about one fifth of C78 and C5 cells still able to form colonies. Moreover, sensitivity of individual clones was correlated with p53 status: resistant cells express p53 protein at the same level as FaDu wild type cell line, whereas p53 was not detected for sensitive clones. Western blot analysis and electron microscopy confirmed the presence of exosomes in samples derived from the examined cell lines. Based on the presented results we conclude that the FaDu-derived radiochemotherapy- resistant clonal cell lines have a great potential in proteomic studies of exosomes secreted by cells of varying sensitivity to genotoxic agents.

98 .

Transcriptome and proteome (50-63)

. 99 100 . 50. LEUKEMIA SUBTYPE BIOMARKER VALIDATION IN A LARGE GENE EXPRESSION STUDY

Wojciech Łabaj1, Anna Papież2, Joanna Polańska2

1Institute of Informatics, Silesian University of Technology, ul. Akademicka 16, 44-100 Gliwice, Poland; 2Institute of Automatic Control, Silesian University of Technology, ul. Akademicka 16, 44-100 Gliwice, Poland

Biomarker discovery is an important field of study in the development of novel prognostic and diagnostic techniques in medicine. Leukemia in its various subtypes poses a significant threat to the contemporary society and enhancing the performance of treatment remains a significant problem. With the ever growing time and cost efficiency of microarray gene expression techniques they are a valuable source of providing answers to issues such as leukemia biomarker identification. The Microarray Innovations in LEukemia (MILE) project has been designed in order to test gene expression with the aim of searching for patterns in acute and chronic leukemia subtypes and their relation with the Myelodysplastic Syndrome. In previous studied we have shown that a deep data analysis with adequate procedures leads to potential biomarker discovery. In the course of this research we demonstrate the results of in silico performed validation using Stage II MILE data produced on a custom Amplichip Leukemia platform. Inference carried out in the domain of gene differentiation as well as functional analysis is verified with an independent data set. Eventually, we present the similarity between our candidate biomarker lists, in terms of their qualitative as well as functional features, and their counterparts determined in the course of two stages of the MILE study. Our work validates the suitability of deep data analysis techniques for tasks such as investigating a complex scientific project performed on a large collection of samples with the aim of examining molecular mechanisms of multiple disease subtypes.

Acknowledgements: This work was supported by SUT grants no. BK/213/Rau1/2016/10 (JP), BKM/506/RAU1/2016 (AP) and BKM/507/RAU2/2016 (WL). The calculations were carried out using GeCONiI infrastructure (POIG.02.03.01-24-099/13).

. 101 51. DISCOVERING NEW BIOLOGICALLY RELEVANT PATHWAYS BY GENE SET ENRICHMENT ANALYSIS

Joanna Żyła, Michał Marczyk, Joanna Polańska

Data Mining Group, Faculty of Automatic Control, Electronics and Computer Science, Silesian University of Technology, 44-100 Gliwice, ul. Akademicka 16 Poland

Aim: The study is concentrated on investigation of the results of Gene Set Enrichment Analysis (GSEA) [1] under different ranking metrics and their ability to detect highly relevant pathways or potentially newly discovered gene sets. The GSEA algorithm was chosen as it is the most commonly used method in enrichment analysis. Materials and methods: Six microarray datasets of clear cell Renal Cell Carcinoma (ccRCC) were used: GSE14762, GSE781, GSE6344, GSE15641, GSE14994 and GSE11024. For each dataset Gene Set Enrichment Analysis with phenotype permutation was performed based on the KEGG pathway database resource (273 pathways). The GSEA procedure was run with three different ranking metrics i.e. absolute value of signal-to-noise ratio (|S2N|), absolute value of Moderated Welch statistic (|MWT|) and Baumgartner-Weiss-Schindler statistic (BWS). The presented metrics were chosen according to our previous findings, which prove that these metrics give sensitive and specific results of functional analysis [2]. For each metric and microarray dataset, significant pathways were established at 0.01 significance level. The coverage between results given by different ranking metrics was checked and unique pathways from the literature were investigated. Results: In a group of significant pathways there are 132, 119 and 207 gene sets (for |MWT|, |S2N| and BWS respectively). |MWT| shows the biggest coverage across six datasets (about 13%). BWS gives the smallest number of pathways enriched only in single dataset (nearly 23%). Further, the coverage between results, when we apply different ranking metrics, was checked for pathways detected in all datasets. All gene sets found by |S2N| were also enriched for |MWT| and BWS metrics. BWS shows 7 distinctive pathways, while |MWT| 2. Out of unique BWS gene sets 5 were previously reported to ccRCC, whereas for |MWT| all 2 were known in literature. Conclusions: The presented work shows that out of 3 metrics tested, the most commonly used |S2N| has the lowest potential for finding relevant gene sets or discovering new ones. |MWT| gives a large number of common pathways across datasets but BWS shows ability to give invariable results. BWS also shows higher potential in finding new gene sets or with proved connection to disease under investigation.

References: 1. Subramanian A, et al. (2005): Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. PNAS 102: 15545-15550. 2. Zyla J, Marczyk M, Polanska J (2016):. Sensitivity, Specificity and Prioritization of Gene Set Analysis When Applying Different Ranking Metrics. In: 10th International Conference on Practical Applications of Computational Biology & Bioinformatics (pp. 61-69). Springer International Publishing.

Acknowledgements: The work was financially supported by SUT grant BK/213/Rau1/2016/10 (02/10/BK_16/3015) for MM and NCN grant no. 2013/08/M/ST6/00924 (JZ and JP). All calculations were carried out using infrastructure of GeCONiI (POIG.02.03.01-24-099/13).

102 . 52. MOLECULAR PROFILING OF TUMOR-DERIVED EXOSOMES IN PLASMA OF CANCER PATIENTS

Monika Pietrowska1, Sonja Funk2, Marta Gawin1, Łukasz Marczak3, Piotr Widłak1, Theresa Whiteside2

1Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska - Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland; 2 University of Pittsburgh Cancer Institute, Pittsburgh PA, USA; 3Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland

Tumor-derived exosomes (TEX) present in body fluids of cancer patients are thought to be responsible for delivering suppressive signals to immune cells, interfere with anti-tumor immunity and thus have impact on response to therapy or even outcome. We propose that the unique molecular and proteomic profiles of TEX in cancer patients’ plasma, which are similar if not identical to the profiles of cancer cells from which TEX originate, can serve as an equivalent of a “liquid biopsy.” If so, then they would allow for serial, real-time, non-invasive monitoring of tumor presence, progression/regression during a therapy and predicting outcome for cancer patients. The methods for exosome isolation from plasma were described in a recently published report [1]. Proteins derived from exosomes were subjected to qualitative and quantitative analysis using mass spectrometry (LC-MS analysis with MS/MS-based identification). Proteins were consecutively digested with two enzymes: endoproteinase Lys-C and trypsin. Exosomal proteins enzymatically fragmented were fractionated using SAX homemade tip-columns, and peptides were separated chromatographically using nanoLC system equipped with C18 column, analyzed and identified by tandem mass spectrometry (MS/MS). We have preliminary data for immunocapture of CSPG4+ MTEX (melanoma derived exosomes) from plasma of melanoma patients. Having first performed titrations to determine the optimal ratio of exosomes vs. biotinylated CSPG4-specific mAb 225.28, we established that 7μg of mAb 225.28-coated beads was optimal for capture of 2.1x109 exosomes present in combined fractions #3 and #4 (pre-capture) separated by mini-SEC from 1 mL of patients’ plasma. In this experiment, captured MTEX represented 33% of recovered exosomes. Western blot analysis showed that captured MTEX were enriched in VLA-4 and TYRP, while these markers were not or barely detected in non-captured exosomes or in the bulk exosome fraction. Our preliminary mass spectrometry data obtained with proteins from (1) pre-capture (MeP input), (2) captured (MeP cap) and non-captured (MeP uncap) melanoma exosomes indicated that we could discriminate between these exosome preparations. The immunocaptured MTEX (2) contained 55 proteins that were not shared with the other two fractions. Quantitative analysis could be implemented to monitor the presence and concentration of exosomal proteins in TEX that are relevant to melanoma and its progression, and to incorporate these proteins as components of the targeted assay.

References: 1. Hong CS, Funk S, Muller L, Boyiadzis M, Whiteside TL (2016): Isolation of biologically active and morphologically intact exosomes from plasma of patients with cancer. JExtracellVesicles 5: 29289.

. 103 53. DEMETHYLATION LEVEL DIVERSIFICATION AMONG DIFFERENT GENOME REGIONS N HUMAN AML

Agnieszka Cecotka1, Grainne Manning2, Christophe Badie2, Simon Bouffler2, Joanna Polańska1

1Data Mining Group, Faculty Of Automatic Control, Electronics and Computer Science, Silesian University of Technology, 44-100 Gliwice, Akademicka 16 Poland; 2Biological Effects Department, CRCE, Public Health England, United Kingdom Corresponding author: [email protected]

Introduction: DNA methylation is one of epigenetic mechanisms as well as one of gene expression control processes. Hypermethylation of the gene promoter region can inactivate its transcription, which occurs very often in cancer diseases. Differantially methylated genome regions are specific for various types of cancer. The result of this phenomenon can be an inactivation of tumour suppressor genes. Global hypomethylation of a cancer genome leads to chromosomal instability. In this study we concentrated on diversification of changes in methylation level in AML patients, comparing it to healthy control across the whole genome. Materials and methods: An experiment was performed for human acute myeloid leukaemia. For 5 patients, DNA methylation profile was checked, using Infinium 450k microarrays. 485512 sites of the whole genome (all chromosomes) were examined for methylation level. For each site we obtained results as β-value, which represents methylated to the sum of methylated and unmethylated ratio. As a control, the set of data was downloaded from GEO database. It contained information about methylation level in bone marrow cells collected from 20 healthy donors. The aim of this study was to detect differentially methylated regions in genome in AML patients compared to healthy control. Classical statistical methods were used to detect demethylated sites of the genome. Then, integration methods were applied according to Illumina genome region annotation, in order to find the demethylated region of a genome. For the regions found, e.g. gene promoters, a functional analysis was performed using Gene Ontology database. Results: The analysis shows that there are significant differences in demethylation level between several regions of a genome. Hypermethylation (in AML vs control) occurs mostly in gene promoter regions. Hypomethylation is very strong at the intergenic sites of genome. The analysis shows that different numbers of demethylated regions are located on particular chromosomes. The functional analysis of the genes with hypermethylated promoter region, thus the genes with inactivated expression, shows which molecular processes change during the investigation. Conclusion: DNA methylation is a gene expression control process. It can be modified in cancer disease, in the present case in AML, but not in the same way across the whole genome. The gene promoter regions become hypermethylated, which causes inhibition of expression of a part of genes. Methylation level at intergenic sites goes down. The changes in methylation level can have an influence on functionality of cells.

Acknowledgements: The work was financially supported by SUT grant BKM/506/RAU1/2016/t.33 (AC) and SUT grant BK/213/Rau1/2016/10 (JP).Calculations were carried out using infrastructure of GeCONiI (POIG.02.03.01-24-099/13).

104 . 54. IN VITRO CULTURED OVARIAN CANCER CELLS EXPRESS EXTRACELLULAR MATRIX- ASSOCIATED GENE SIGNATURE INDICATIVE OF POOR PROGNOSIS

Patrycja A. Tudrej1, Katarzyna A. Kujawa1, Magdalena Olbryt1, Barbara Nikiel2, Ewa Zembala-Nożyńska2, Alexander J. Cortez1, Katarzyna M. Lisowska1

1Center for Translational Research and Molecular Biology of Cancer; 2Tumor Pathology Department, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland E-mail: [email protected]

In our previous microarray study performed on 101 ovarian cancer samples, we identified a gene signature related to shorter patient survival [1]. Majority of those genes were related to extracellular matrix structure and function and were presumed to be expressed mainly by stromal fibroblasts. Now, we have analyzed whether these genes could be expressed by ovarian cancer cells from five commercially available lines and from our newly established line OVPA8 derived from high- grade serous ovarian cancer. Gene expression was assayed by reverse transcription-PCR and Western blot analysis. Using RT-PCR, we analyzed OVPA8 cells, together with SKOV3, OAW42, OVCAR3, ES2 and A2780, with respect to the expression of genes from our prognostic signature. Out of 13 genes which were assayed, four were expressed in all six cell lines (these were LOX, VCAN, FN1 and PLAU), three were expressed in five lines (COL11A1, POSTN, SFRP2), and next three in three lines (DSPG3, MFAP5, CONP). Other genes (ITGBL1, HNT, FAP, THBS2) were expressed in at least two lines. These observations were partially confirmed at the protein level by Western blot analysis. We show that multiple extracellular matrix-associated genes that are commonly supposed to be expressed by fibroblasts, can be expressed also by ovarian cancer cells. This is of potential importance, as these gene were shown by us and also by others as related to poor prognosis.

References: 1. Lisowska KM et al (2016): JCancerResClinOncol 142: 1239-1252.

Acknowledgments: This study was supported from National Science Center grant 2012/04/M/NZ2/00133 to K.M.L.

. 105 55. INFLAMMASOME-RELATED GENES IN DIFFICULT WOUND HEALING CASES

Bartłomiej Skowronek1, Klaudia Simka1, Emilia Wojdas2, Bogdan Michalski3, Mateusz Michalski3, Barbara Kowolik3, Aleksandra Skubis2, Bartosz Sikora2, Teresa Kokot1, Urszula Mazurek2

1Department of Internal Medicine, School of Public Health in Bytom; 2Department of Molecular Biology, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec; 3Department of Gynaecology Oncological, School of Health Sciences in Katowice, Medical University of Silesia in Katowice

Wound healing is a process related with every surgical intervention. Typically, healing process is the most intensive during first 4 weeks after the wound occurrence. However, due to variety of factors such as: diet, infection, exposed bone or tendon, diabetes, ischemia, abnormal inflammation level etc., this process can be prolonged and result in dangerous complications. It is crucial to assess whether the wound is hard-to-heal, to introduce proper treatment as soon as possible to avoid deficient of excessive scar formation, proud flesh etc. Inflammasomes are a group of protein complexes connected with inflammation process and therefore can be related with wound healing process. The aim of this pilot study was to assess whether expression of genes coding inflammasome- forming proteins could be potentially used as a biomolecular marker allowing prediction whether wound would be hard-to-heal or not. Skin fragments were obtained during surgical intervention. Wound healing process was observed, and skin samples were divided into 2 groups: hard-to-heal wound (3 samples) or normal healing wound (1 sample). Total RNA was isolated from skin fragments and microarray experiment was performed using microarrays Affymetrix HG-U 133 A. List of 17 transcript IDs was created and were analysed in GeneSpring v. 12.0. During statistical analysis Hard-to-heal cases were compared to normal healing samples. Out of 17 IDs, statistically significant changes (p<0.05) between hard-to-heal wound cases and normally healing wound of 7 IDs (4 genes) were observed. Post-Hoc analysis revealed that only expression of NLRP1 gene was strongly and similarly underexpressed in all 3 cases. Underexpression of NLRP1, despite the fact that it can be a reason for problematic healing process, can serve as a potential marker of hard-to-heal wounds out of preselected set of genes.

Acknowledgements: This research was financed by the Medical University of Sillesia nr KNW - 1- 161/N/6/0.

106 . 56. STABILITY OF REFERENCE GENE EXPRESSION IN CELLS EXPOSED TO STRESSING FACTORS

Ewa Marek, Anna Lalik, Roman Jaksik

Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland

Introduction: Reference genes are used as an element of data standardization methods in many molecular biology experiments, including RT-qPCR, western blot, oligonucleotide microarrays or new generation sequencing. Selected from a group of housekeeping genes, with stable expression level, they are used to distinguish signal variations of biological origin from those which result from technical aspects of the biochemical procedures. However the assumption of stability of these genes is often not fulfilled, especially in experiments where cells are exposed to stress factors, like ionizing radiation. There are some statistical methods used for the evaluation of reference gene stability, including geNorm and NormFinder, however they are not applicable to case-control studies, used to test the influence of treating factors, focusing solely on the between cell line or between patient stability. Aim: The primary aim of this study is to develop a methodology for the analysis of gene stability in case-control experiments and to select the most suitable reference genes for studies in which cells are exposed to ionizing radiation. Secondary goal is to create software that can be used to analyze the stability of genes in experiments with treating factors. Materials and methods: The stability ranking was created using microarray data obtained from the ArrayExpress database and from experiments carried out at the Center of Oncology in Gliwice and Silesian University of Technology. We created a custom application for multi-experiment data preprocessing, and developed a stability index based on relative and absolute gene expression levels. Results: We created a stability ranking for nearly all human genes, using data obtained for various cell lines, radiation doses and times after treatment. Based on the results we determined that RPS11, ACTG1, RPL37A, RPL8, RPL13A are the most stable genes in cells exposed to ionizing radiation and that some of the most commonly used reference genes, including ACTB, TBP, RPL41 and previously suggested RNA18S5 (Banda et al. 2008) are not a good choice since they show variations in expression level at various time points after irradiation. Summary: The stability ranking which we created can be used to identify endogenous controls for molecular biology experiments, in which cells were exposed to ionizing radiation. Additionally the software which we created has a great potential for other applications such as analysis of gene stability in cells treated with chemicals, heat shock or other factors that induce stress response.

Acknowledgements: This work was supported by the BKM-514/RAu1/2015 . Calculations were carried out using the computer cluster Ziemowit (http://www.ziemowit.hpc.polsl.pl) funded by the Silesian BIO- FARMA project in the Computational Biology and Bioinformatics Laboratory of the Biotechnology Centre in the Silesian University of Technology.

. 107 57. CALCULATION OF NUCLEOTIDE SEQUENCE OCCURRENCE PROBABILITY IN HIGH-THROUGHPUT TCR SEQUENCING DATA TO EXAMINE EFFECT OF IRRADIATION

Justyna Mika1, Christophe Badie2, Serge Candéias3, Joanna Polanska1

1Data Mining Group, Faculty of Automatic Control, Electronics and Computer Science, Silesian University of Technology, Gliwice, Poland; 2Cancer Mechanisms and Biomarkers group, Radiation Effects, CRCE, Public Health England, Didcot, United Kingdom; 3Laboratory of Chemistry and Biology of Metals, French Alternative Energies and Atomic Energy Commission, Grenoble, France

T cell receptors are one of the molecules responsible for big variety of immune responses. Their Complementarity Determining Regions (CDR) are created during VDJ recombination, a process consisting of selection and assembly of genome-coded V, D and J gene segments. At first, one of two D genes and one of 14 J genes are selected and joined together in NHEJ (non-homologous end joining) process during which mostly random nucleotides are deleted and inserted between chosen segments, creating so called N1 nucleotide region. At second, one of 35 V genes is selected and joined to the previously created DJ-complex, similarly following NHEJ and forming so called N2 nucleotide region. It’s well known that ionizing radiation is a factor that causes a wide spectrum of DNA lesions, including single and double strand breaks and/or nucleotide insertions and deletions. The aim of the following study was to develop the procedure to estimate the probability of occurrence of certain nucleotide sequence in the nucleotide regions N1 or N2 and examine whether irradiation affects the creation of those regions. The study was performed on data coming from deep sequencing of T cell receptors of 10 mice irradiated with 0.1Gy, 10 with 1Gy and 10 control individuals. More than 0.5 billion sequences were obtained in total. Each sequence was categorized by the selected V, D and J gene and described with the starting positions of chosen gene segments, N1 and N2 nucleotide regions. To calculate the probability of occurrence of nucleotide sequence the empirical nucleotide frequencies were used. Sequences were categorized in accordance to irradiation dose. Frequencies of A, C, G and T nucleotides on next positions, starting from the first nucleotide flanking the end of gene segment, were estimated using observed sequences. Obtained frequencies were the basis for calculation of the reference probability. To estimate the probability of by chance occurrence of certain sequence, a binomial test was proposed with Bonferroni correction for multiple testing. Between samples comparisons were performed using Wilson (score) confidence intervals (for alpha=0.05). The described approach allowed to estimate if a certain nucleotide sequence was created by random nucleotide insertions and/or deletions during VDJ recombination process or was indicative of genome retention. It was observed that inserted N1 and N2 nucleotides are specific to the flanking gene segment. No effect of irradiation was observed.

Acknowledgements: The work was financially supported by NCN grant HARMONIA 4 register number 2013/08/M/ST6/00924.Calculations were carried out using infrastructure of GeCONiI (POIG.02.03.01- 24-099/13).

108 . 58. THE EXPRESSION PROFILE OF GENES ENCODING CASPASES IN WOUND HEALING PROCESS

Klaudia Simka1, Bartłomiej Skowronek1, Emilia Wojdas2, Aleksandra Skubis2, Bartosz Sikora2, Barbara Kowolik3, Mateusz Michalski3, Bogdan Michalski3, Małgorzata Muc-Wierzgoń1, Urszula Mazurek2

1Department of Internal Medicine, School of Public Health in Bytom; 2Department of Molecular Biology, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec; 3Department of Gynaecology Oncological, School of Health Sciences in Katowice, Medical University of Silesia in Katowice

Tissue regeneration is a very complicated process necessary to maintain tissue integration and homeostasis. Wound healing consists of three phases: inflammatory, proliferative and remodeling phase. It is regulated by balance of proliferation versus differentiation and cell survival versus cell death. There is evidence that apoptotic activity is involved in each phase. The key enzymes that carry out apoptosis are caspases, intracellular proteolytic enzymes with the pleiotropic action. Caspases also take part in the regulation of cell’s differentiation, proliferation, migration and induction of inflammatory response. Abnormalities in expression of genes encoding caspases lead to disfunctions in many molecular processes and occur in variety of diseases. The aim of the study was to evaluate whether the changes in transcriptional activity of genes encoding caspases might be associated with wound healing and to indicate which of these genes could be used as prognostic markers of wound healing process. The material for the study consisted of skin bioptats from patients with different types of chronic wounds that do not heal within six weeks and from patients who demonstrated normal healing during seven days (control). Total RNA was isolated from skin bioptats. The expression profile of genes encoding caspases was determined using oligonucleotide microarrays (HG- U133A, Affymetrix). Results were analyzed using GeneSpring 12.6.1. Analysis of 25 ID mRNAs encoding caspases indicates 10 statistically significant (p<0,05) differentiating ID mRNAs. Results revealed 3 ID mRNAs differentiating all tested groups from control. Those transcripts encoded 3 different genes, among which CASP2, CASP6 were overexpressed and CASP10 was downregulated. The expression profile of genes encoding caspases was different in skin bioptats from patients with hard-to-heal wounds compared to those with normal wound healing. Obtained results allow to formulate the hypothesis that overexpression of CASP2, CASP6 and downregulation of CASP10 could be used in prognosis of wound healing process.

Acknowledgements: This research was financed by the Medical University of Silesia nr KNW - 1- 161/N/6/0.

. 109 59. DOPAMINE-RELATED GENES AS POTENTIAL MOLECULAR MARKERS FOR ENDOMETRIAL CANCER

Nikola Zmarzły1, Agnieszka Jęda-Golonka2, Andrzej Witek2, Celina Kruszniewska-Rajs1, Joanna Gola1, Tomasz Janikowski1, Urszula Mazurek1

1Department of Molecular Biology, School of Pharmacy with Division of Laboratory Medicine in Sosnowiec, 2Department of Gynecology and Obstetrics, School of Medicine in Katowice, Medical University of Silesia in Katowice, Poland

Dopamine is an endogenous catecholamine that plays a very important role in neuronal and non-neuronal tissues. The studies suggest that dopamine can promote proliferation of non- transformed cells but shows antiproliferative effects on cancer cells. Endometrial cancer is the most common of the gynecologic malignancies, however, the exact causes of this cancer are still unknown. The purpose of the study was to evaluate transcriptional activity of dopamine-related genes in endometrial cancer with particular emphasis on tumor grading. The samples of normal endometrium (control group) and endometrial cancer, further divided according to the tumor grade G1, G2, G3 (study groups), were obtained at surgery in the Department of Gynecology and Obstetrics, Medical University of Silesia in Katowice. Samples had been collected and stored for the molecular analysis accordingly to the protocol for RNAlater. Total RNA was extracted from endometrial samples with the use of TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Expression profile of genes associated with dopamine was evaluated using HG-U133A oligonucleotide microarrays (Affymetrix, Santa Clara, CA). GeneSpring 12.0 platform (Agilent Technologies) was used for data analysis. Among 175 ID mRNA representing genes encoding proteins associated with dopamine, the level of 27 ID mRNA was changed (p<0,05) between the analyzed groups. The changed expression of dopamine-related genes was identified in each grade of endometrial cancer compared to control: G1 vs C (2 genes: PTPN11, GNAQ), G2 vs C (9 genes: GNB1, GNA11, HTT, CAV2, PALM, MAOB, SLC22A3, C5, SNCA), G3 vs C (2 genes: GNA11, SNCA). In this study, a significant up-regulation of dopamine-related genes involved in oncogenic transformation, proliferation and migration was observed, while genes associated with protein binding, immune surveillance and inflammation response were significantly down-regulated in endometrial cancer compared to control. The results from this study suggest that identified dopamine-related genes may be potential molecular markers for the presence and grading of endometrial cancer.

110 . 60. IDENTIFICATION OF SERUM PROTEINS AS POTENTIALLY USEFUL BIOMARKERS ASSOCIATED WITH RISK OF METASTASIS OF BREAST CANCER PATIENTS

Anna Walaszczyk1, Monika Pietrowska1, Anna Wojakowska1, Agata Abramowicz1, Paweł Rodziewicz2, Joanna Polańska3, Katarzyna Behrendt1, Elżbieta Nowicka1, Rafał Tarnawski1, Piotr Widłak1

1Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, 44-101 Gliwice, Poland; 2Institute of Bioorganic Chemistry, Polish Academy of Science, 61-704 Poznan, Poland; 3 Silesian University of Technology, 44-100 Gliwice, Poland

Background: Breast cancer diagnosed at early clinical stages is relatively well cured, yet even in this group some patients are at high risk of metastasis and failure of the treatment. Optimal selection of adjuvant treatment for these patients would be facilitated if reliable prognostic markers of risk of metastasis were available in clinical practice. Serum proteomics allows to characterize processes related to progression of cancer and its influence at patient’s organism. Study Aims: The major aim of this work was to analyze serum proteome of breast cancer patients in order to identify proteins that reflect high risk of metastasis and characterize systemic processes involved in spread of cancer cells. Methods and Materials: We selected a group of 15 patients who suffered from cancer relapse and metastasis during 5-year follow-up, and material from about 45 patients who benefited from successful treatment. Blood samples were collected before the start of the therapy, after the surgical resection of tumors and one year after the end of therapy. Patients with successful treatment were selected from larger group to provide relevant control/background for patients with failure (e.g. considering age, clinical features of the tumor). For the identification of multi-peptide signatures MALDI ToF/ToF mass spectrometry was exploited. To identify components of serum proteome we used LC-MS/MS. Results: We identified 24 serum proteins whose abundances changed between pre-treatment and post-treatment samples in the treatment failure group and 8 proteins in the control group; all differences detected by unsupervised clustering showed statistical significance. Conclusions: Serum proteome might be a source of knowledge about factors reflecting or enhancing spread of cancer cells.

Acknowledgements: This work was supported by Polish National Science Center, Grant UMO- 2012/05/N/NZ4/02307

. 111 61. SERUM LIPID PROFILE AS A BIOMARKER OF EARLY LUNG CANCER: DECREASED LEVELS OF LYSOPHOSPHATIDYLCHOLINES IN SERUM OF CANCER PATIENTS

Małgorzata Roś-Mazurczyk1, Monika Pietrowska1, Karol Jelonek1, Michał Marczyk2, Joanna Polańska2, Rafał Dziadziuszko3, Jacek Jassem3, Witold Rzyman3, Piotr Widłak1

1Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, ul. Wybrzeze Armii Krajowej 15, 44-100 Gliwice, Poland; 2Silesian University of Technology, ul. Akademicka 16, 44-100 Gliwice, Poland, 3Medical University of Gdansk, ul. Debinki 7, 80-211 Gdansk, Poland

The role of a low-dose computed tomography lung cancer screening remains a matter of controversy due to its low specificity and high cost. Screening complementation with blood-based biomarkers may allow more efficient pre-selection of candidates for imaging tests or discrimination between benign and malignant chest abnormalities detected by LD-CT. We aimed to identify molecular signature based on serum lipid profile distinguishing individuals with early lung cancer from healthy participants of the lung cancer screening program. Blood samples were collected from 100 patients with early stage lung cancer (including 31 screen-detected cases) and from a matched group of 300 healthy participants of the lung cancer screening program. MALDI-ToF mass spectrometry was used to analyze the molecular profile of lipid- containing organic extract of serum samples in the 320-1000 Da range. Serum levels of selected ions were additionally analyzed by nLC-ESI-IT mass spectrometry technique based on material from 20 patients and 20 controls. Several components of the serum lipidome were detected, with abundances discriminating patients with early lung cancer from high-risk smokers. This allowed the development of an effective cancer classifier with an area under the curve of 0.79 and 0.72 in the training and test groups, respectively, with corresponding negative predictive values of 100% and 92%, and positive predictive value of 28% each. Additionally, significantly decreased levels of lysophosphatidylcholines (18:2) and (18:1) in serum of early lung cancer patients were confirmed by chromatography-aided mass spectrometry. Lipid-based serum signature showed potential usefulness in discriminating early lung cancer patients from healthy individuals.

Acknowledgements: The project was supported by the National Science Centre, Grant 2013/11/N/NZ7/00770 and by the National Centre for Research and Development in Poland, Grant PBS3/A7/29/2015 (MoltestBis). The computation analyses were carried out using the GeCONiI infrastructure (NCBiR grant POIG.02.03.01-24-099).

112 . 62. MOLECULAR HETEROGENEITY OF ORAL SQUAMOUS CELL CARCINOMA AND DETECTION OF SIGNATURES DISCRIMINATING NORMAL AND CANCEROUS EPITHELIUM BY IMAGING MASS SPECTROMETRY

Magdalena Kalinowska-Herok1, Monika Pietrowska1, Marta Gawin1, Grzegorz Mrukwa2, Mykola Chekan1, Grzegorz Drazek2, Janusz Wierzgoń1, Joanna Polańska2, Piotr Widłak1

1Center of Oncology - Maria Sklodowska-Curie Memorial Institute, Gliwice, Poland; 2Silesian University of Technology, Gliwice, Poland

The particular advantage of Imaging Mass Spectrometry in cancer research stems out from allocation of molecular profiles to specific cell types, such as cancerous, preneoplastic or inflammatory. Moreover, IMS can be used in studies aimed at interfacing tumor and normal tissue (tumor niche) and intra-tumor heterogeneity. It is noteworthy that automated (unsupervised) methods of clustering of IMS data, particularly based on component analysis and spatial segmentation, appeared to be a particularly suitable approach in studies of intra-tumor heterogeneity and classification of tumor sub-regions. Here we aimed to characterize molecular profile of tumor and adjacent tissues in clinical material from patients with OSCC. Tissue samples (tumor and adjacent areas) resected from 5 patients with OSCC were fresh-frozen, cut in cryostat, placed on ITO-coated glass slides, and then dried in vacuum and covered by the methanol solution of 2,5-dihydroxybenzoic acid (50% methanol, 30 mg/mL DHB, 0.2% TFA) using ImagePrep device. Samples for peptide analysis were incubated overnight with trypsin prior to matrix deposition. Spectra were registered using UltrafleXtreme MALDI- ToF spectrometer in positive reflectron mode with 100 µm raster; peptide spectra were recorded in the 800-4000 Da. Tryptic peptides were identified in tissue lysates using LC-MS/MS approach and annotated to IMS data. Novel algorithm of IMS data analysis was developed and implemented, which included Gaussian mixture modeling for detection of spectral components and iterative k-means algorithm for unsupervised spectra clustering performed in domain reduced to a subset of the most dispersed components. About 4% of the detected peptides showed significantly different abundances between normal epithelium and tumor, and could be considered as a molecular signature of oral cancer. Moreover, unsupervised clustering revealed two major sub-regions within expert-defined tumor areas. One of them showed molecular similarity with histologically normal epithelium. The other one showed similarity with connective tissue, yet was markedly different from normal epithelium. Pathologist's re- inspection of tissue specimens confirmed distinct features in both tumor sub-regions: foci of actual cancer cells or cancer microenvironment-related cells prevailed in corresponding areas. Hence, molecular differences detected during automated segmentation of IMS data had an apparent reflection in real structures present in tumor.

Acknowledgement: The work was supported by National Science Center grant no 2011/03/D/NZ4/03507.

. 113 63. MOLECULAR COMPOSITION OF DEGENERATED RETINAL PIGMENT EPITHELIAL (RPE) CELLS AND THEIR SURROUNDINGS IN GEOGRAPHIC ATROPHY DETECTED BY MSI APPROACH

Jakub Hanus1, Monika Pietrowska2, Marta Gawin2, Łukasz Marczak3, Piotr Widłak2, Shusheng Wang1,4

1Department of Cell and Molecular Biology, Tulane University, New Orleans, USA; 2Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology Gliwice Branch, Gliwice, Poland; 3Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland; 4Department of Ophthalmology, Tulane University, New Orleans, USA

Age-related macular degeneration (AMD) is a degenerative disorder of the macula and the leading cause of irreversible central vision loss in elderly population in developed countries, affecting 30–50 million people worldwide. It is characterized by scattered or confluent areas of degeneration of retinal pigment epithelium (RPE) cells and the overlying photoreceptors which rely on RPE for trophic support. Currently, mechanism and pathogenesis of AMD are still unclear and therefore no causal treatment is available. The purpose of our study was to characterize the proteome of neighbouring healthy and diseased RPE cells surrounding atrophy sites. Comparison of the proteome of healthy and diseased RPE cells can lead to identification of signalling pathways that may help understand the disease process. Identification of molecular margins of RPE atrophy might be crucial for understanding AMD pathophysiology. Sodium iodate (NaIO3) injection has been extensively used as a preclinical model for RPE dystrophy and atrophy in AMD. It induces accumulation of ROS, however it is not clear if RPE cells in this model die from apoptosis or necrosis. To confirm oxidative stress-induced RPE cell death in vivo, we adopted a retro-orbital NaIO3 injection mouse model to induce RPE degeneration. Mouse eye tissues were collected 24 to 72 hours post injection and FFPE blocks were prepared. FFPE samples were cut into 10 μm sections, then placed onto ITO-coated conductive slides. Next, slides were coated with a solution of trypsin using ImagePrep device. After tryptic digestion methanolic solution of 2,5- dihydroxybenzoic acid was deposited onto tissue surface. Sections were subjected to peptide imaging with the use of a MALDI-ToF ultrafleXtreme mass spectrometer. Additionally protein extracts were prepared from consecutive FFPE tissue sections collected in 1.5 mL tubes and subjected to multi- enzyme digestion filter-aided sample preparation (MED-FASP) procedure. All fractions were separated using liquid chromatography and analysed by Orbitrap mass spectrometer for protein identification and label-free quantification. Abundance of identified proteins was estimated using MaxQuant software. After 24 hours from NaIO3 administration (20mg/kg), RPE cells showed signs of pigmentation lost with no effect on photoreceptor. At 24 to 72 hours post injection, RPE cells appeared swollen, vacuolized and started to break off from the RPE layer. Unsupervised analysis of MSI data performed in SCiLS Lab software classified samples in relation with time after NaIO3 injection. These results showed that changes induced by NaIO3 could be detected by MSI approach. We performed LC- MS/MS analysis and identified 3477 unique proteins in all samples. We made comprehensive analysis and functional classification of the identified proteins using Perseus software and found proteins whose change in level was statistically significant depending on the time after injection. We detected proteins exclusively present in samples after injection e.g.: Histone H3, Ceruloplasmin, Prominin-1, Proteasome activator complex subunit 2, Retinal-specific ATP-binding cassette transporter, Galectin- 1, Cell division control protein 42 homolog, Carboxylesterase 1D, Histidine-rich glycoprotein. The results of the performed functional classification of RPE-choroid proteome along with analysis of imaging data of mouse eye tissues subjected to oxidative stress are a promising and interesting starting point for deeper insight into macular degeneration.

114 .

Molecular markers and targets (64-69)

. 115 116 . 64. IMPACT OF POLYMORPHIC VARIANTS OF ABCB1, CBR1, CYP2C19, SLC22A16 GENES ON 5-FLUOROURACIL, DOXORUBICIN AND CYCLOPHOSPHAMIDE CHEMOTHERAPY IN BREAST CANCER PATIENTS

Karolina Tęcza, Jolanta Pamuła-Piłat, Joanna Łanuszewska, Dorota Butkiewicz, Lucyna Ponge, Iwona Domińczyk, Ewa Grzybowska

Center for Translational Research and Molecular Biology of Cancer, Maria Skłodowska-Curie Memorial Center and Institute of Oncology, Gliwice Branch, 44-101 Gliwice, ul.Wybrzeże Armii Krajowej 15, Poland

The effect of chemotherapy of breast cancer can be determined by genetic variations in drug metabolizing enzymes and transporters. Metabolism and distribution of drugs are mediated by drug- metabolizing enzymes such as cytochrome p450 (CYP), carbonyl reductase (CBR) and transporters such as ATP binding cassette (ABC) and solute carrier (SLC). The aim of study was to analyze a possible impact of genetic variants of ABCB1 c.1236 T>C p.Gly 412= (rs1128503), CBR1 c.627C>T p. Ala209= (rs20572), CYP2C19c.806C>T p. Lys262Arg (rs12248560), SLC22A16 p.312A>G p. Asn104= (rs6907567) and c.755C>T p.Val252Ala (rs723685) on 5-fluorouracil, doxorubicin and cyclophosphamide chemotherapy (FAC chemotherapy) in breast cancer patients. We evaluated the clinical response and treatment outcome of 324 women treated with FAC chemotherapy. Subjects were genotyped using polymerase chain reaction-restriction fragment length polymorphism. Patients carrying CT and TT genotypes of CYP2C19 c.806C>T were found to increase the risk of death, progression, relapse and second breast cancer [OR 2.46 (1,25-4,84); p=0,009]. Treatment failure-free survival risk (TFFS) for this group (CT+TT) was significantly increased [HR 3,29 (1.1-9.84); p=0.025]. This group of breast cancer patients was associated with higher risk of nephrotoxicity [OR=4.6 (1.24-17.5), p=0.021] than reference CC genotype patients group.For theSLC22A16 (rs6907567) GG genotype was associated with shorter overall survival (OS) [OR 2.51 (0.99-6.3), p=0.05] and progression-free survival (PFS) [OR 2.7 (1.1-6.87), p=0.029]. Patients carrying AG or GG genotypes of SLC22A16 (rs6907567) were found to be associated with hepatotoxicity compared with dominant AA genotype [OR 4.4 (1.0-18.36), p=0,039]. Polymorphisms of CYP2C19 (rs12248560) and CBR1 (rs20572) were correlated with gastrointestinal symptoms. Patients with two unfavorable genotypes of CYP2C19 (CT + TT) and CBR1(CT + TT) were related with shorter TFFS [HR 9,7 (3.5- 26.79), p=000001]. Our results confirm that polymorphisms in genes belonging to ADME genes are associated with the response of chemotherapy. This study indicates that genotyping the ADME genes may be useful in choosing the most optimal therapy in breast cancer patients.

Acknowledgements: This work was financially supported by National Science Centre grant no. 2012/07/N/NZ5/00026

. 117 65. PRELIMINARY EVALUATION OF POLYMORPHISM T-129C OF THE GENE ABCB1 IN GROUP OF PATIENTS WITH MULTIPLE MYELOMA

Katarzyna Niebudek, Maria Fijałkowska, Ewa Balcerczak, Marta Żebrowska

Laboratory of Molecular Diagnostics and Pharmacogenomics, Department of Pharmaceutical Biochemistry and Molecular Diagnostics, Interfaculty Cathedral of Laboratory and Molecular Diagnostics, Medical University of Lodz; Muszynskiego 1, 90-151 Lodz, Poland

Multiple myeloma (lat. myeloma multiplex; MM) is a malignant tumour of blood cells coming from the bone marrow, that is characterized by a clonal proliferation of atypical plasma cells usually producing the monoclonal immunoglobulin (protein M). Multiple myeloma accounts for about 1-2% of all diagnosed cancers. MM occupies the second place, after leukemia, on the list of haematological malignancies. The recent studies showing the increased incidence of cases of diagnosing the multiple myeloma much more often and earlier (in patients under 40 years of age). Despite the steady progress of medicine MM consistently remains incurable disease with an average of 3-4 years of patient survival. The aim of our study was a preliminary evaluation of T-129 C polymorphism in the promoter of ABCB1 gene in patients with multiple myeloma. Material for the study included DNA isolated from peripheral blood patients diagnosed with multiple myeloma (investigated group) and from healthy people (control group) [Consent of Bioethics Committee of Medical University of Lodz No: RNN/88/16/KE; RNN/285/13/KE]. The polymorphism study was conducted applying the PCR-RFLP technique. In the group of patients with multiple myeloma TT genotype demonstrated the dominance with 97%. No statistically significant differences between patients with multiple myeloma and control group were found (p=0.7560). Moreover, research also showed no significant differences for the analysis of the relationship between the investigated SNP T-129C and gender in the MM cohort. The examined polymorphism seems not to correlate with the development of the multiple myeloma. However, the obtained results require confirmation in further research on the larger group of patients.

Acknowledgments: Research supported by statutory funds of Department of Pharmaceutical Biochemistry and Molecular Diagnostics, Medical University of Lodz 503/3-015-02/503-31-001; and Grant UMED 564/3-000-00/564-20-013

118 . 66. ASSESSMENT OF ABCB1 GENE POLYMORPHISM AT POSITION -129C

Katarzyna Mazur1 , Rafał Świechowski1 , Agnieszka Jeleń1 , Monika Talarowska2 , Piotr Gałecki2, Ewa Balcerczak1 Marta Żebrowska1

1Laboratory of Molecular Diagnostics and Pharmacogenomics, Department of Pharmaceutical Biochemistry and Molecular Diagnostics, Interfaculty Cathedral of Laboratory and Molecular Diagnostics, Medical University of Lodz; Muszynskiego 1, 90-151 Lodz, Poland; 2Department of Adult Psychiatry, Medical University of Lodz, Aleksandrowska 159, 91-229 Lodz Poland

Depression is one of the most common mental diseases. The causes of depression development are not fully known. It is believed that beyond such factors as: chronic stress, high concentration of proinflammatory cytokines or lack of monoamine responsible for the regulation of the mood also genetic factors may increase susceptibility to the development of depression. One of the genetic risk factors may be the gene encoding a protein co-creating the blood-brain barrier (BBB), which is an important element in protecting the brain and affecting the transport of substrates from and into the brain (such as ABCB1 gene encoding P-glycoprotein). ABCB1 gene is highly polymorphic. Over 50 single nucleotide polymorphisms (SNP) of this gene have been found. One of them is SNP at position -129C

Acknowledgments: Research supported by statutory funds of Department of Pharmaceutical Biochemistry and Molecular Diagnostics, Medical University of Lodz 503/3-015-02/503-31-001; new research tasks on Faculty of Pharmacy no. 502-03/3-015-02/502-34-085; no: 502-03/3-015-02/502- 34-087; NCN grant no:2011/01/D/HS6/05484 and no.2012/05/B/NZ5/01452 and Grant UMED 564/3- 000-00/564-20-012

. 119 67. AQUAPORINS AS A POTENTIAL DRUG TARGET IN OVARIAN CANCER AND PCOS

Łukasz Blukacz1, Agata Wawrzkiewicz-Jałowiecka2, Karolina Kowalczyk1, Dagmara Pluta1, Paweł Madej1

1Department of Gynecological Endocrinology, Medical Faculty in Katowice, Medical University of Silesia, 40-752 Katowice; 2Department of Physical Chemistry and Technology of Polymers, Faculty of Chemistry, Silesian University of Technology, 44-100 Gliwice, Strzody 9, Poland

A Aquaporins (AQPs) are pore-forming transmembrane proteins that regulate water flow across cell membranes. Due to their ubiquitous expression, high permeability and selectivity, AQPs are involved in a large number of processes connected to water and/or glycerol homeostasis. In our investigation we wanted to emphasize the significant therapeutic potential of different types of aquaporins in a broad range of diseases of female reproductive system. Here, we choose two examples of them - polycystic ovary syndrome PCOS and ovarian cancer. We aimed to turn attention on the possible additional, non-hormonal molecular basis of these diseases’ pathogenesis. There is strong evidence that ovarian cancer may be associated with dysregulated aquaporins AQPs 1 and 5 expression. Analogously, pathogenesis of PCOS seem to be directly linked with the altered AQPs 7-9 level. We postulate possible mechanisms standing behind the influence of aquaporins’ dysfunctions (or changes in their amount) on the considered diseases. We also answer here a question why controlling the functioning of aquaporins is a promising method of PCOS treatment and why it can can effectively support ovary cancer prophylaxis.

120 . 68. THE ROLE OF AQUAPORINS IN PCOS, ENDOMETRIAL HYPERPLASIA AND ENDOMETRIAL CANCER

Karolina Kowalczyk1, Agata Wawrzkiewicz-Jałowiecka2, Łukasz Blukacz1, Dagmara Pluta1, Agnieszka Jęda3, Paweł Madej1

1Department of Endocrinological Gynaecology, Medical University of Silesia in Katowice Medical University of Silesia, 40-752 Katowice; 2Department of Physical Chemistry and Technology of Polymers, Faculty of Chemistry, Silesian University of Technology, 44-100 Gliwice, Strzody 9, Poland; 3Department of Gynecology and Obstetrics, Medical University of Silesia in Katowice, 40-752 Poland

Aquaporins (AQPs) are pore-forming integral membrane proteins which are able to conduct water selectively across cell membranes. Due to their wide distribution in living organisms and involvement in a large number of processes, their potential role as a drug target arises. Many diseases of the reproductive organs are correlated with changes of AQPs expression and their malfunction. That is the case in our previous investigations, in which we have postulated a new direction in research on the pathogenesis of the polycystic ovary syndrome (PCOS), and a novel way of PCOS treatment, which is focused on the AQPs 7-9 modulation. Here, we would like to broaden our consideration and analyze the impact of aquaporins dysfunction and changes of their distribution in endometrial hyperplasia and endometrial cancer. The incidence of these two diseases increases in the course of PCOS. That is the reason why AQPs 7-9 are the natural candidates to be involved in the development of the endometrial hyperplasia. To gain the first premise of connection between aquaglyceroporins AQP-7 and AQP-9, and considered endometrium abnormality, we checked the correlation between obesity, endometrial hyperplasia and endometrial cancer. We comment the results not only in the global context of obesity as a pathogenic factor, but explain the recognized linkage on the molecular level - in the context of improper water and glycerol homeostasis, induced by changes in AQPs 7 and 9 levels. We also postulate mechanisms in which changes in the functioning of aquaporins may lead to improper thickness of the endometrium, and eventually to tumor formation. We suggest that preventing the results of changes in aquaporins expression (at least AQPs 1.7-9) may constitute an alternative (or at least be supportive for), to therapy in PCOS and endometrial hyperplasia, and may foster the prophylaxis of endometrial cancer.

. 121 69. OMENTAL-DERIVED METASTATIC OVARIAN CANCER SAMPLES SHOW HIGHER EXPRESSION OF FIBRONECTIN (FN1) THAN PRIMARY OVARIAN CANCER SAMPLES

Katarzyna A. Kujawa1, Ewa Zembala-Nożyńska2, Alexander J. Cortez1, Patrycja A. Tudrej1, Jolanta Kupryjańczyk3, Katarzyna M. Lisowska1

1Center for Translational Research and Molecular Biology of Cancer; 2Tumor Pathology Department, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch; 3Tumor Pathology Department, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland

In our previous microarray study we identified a set of genes related with different survival of the patients with high-grade serous ovarian cancer [1]. Fibronectin (FN1) was among the identified gene signature; higher expression of FN1 in the tumor was correlated with shorter overall survival. FN1 is important for the structure and functioning of extracellular matrix, it also interacts with specific integrin receptors and in this way can activate signaling pathways. We decided to further evaluate the prognostic significance of FN1 by immunohistochemistry (IHC). We analyzed the formalin-fixed paraffin-embedded tissue sections from 108 ovarian cancer patients, using rabbit polyclonal antibody against FN1 (A0245, Dako). The results of IHC were examined together with an experienced pathologist. The association between FN1 expression and clinico-pathological parameters was evaluated using Chi-square test. Survival of patients was plotted using the Kaplan-Meier method and analyzed by the log-rank test. We found that patients with higher FN1 expression in tumor stroma had significantly shorter overall survival (p=0.003). There was no difference in FN1 expression according to disease free survival. All tumor samples were from patients with late stage (FIGO IIIC) disease and with high grade tumors (grade 3 and 4) so we could not analyze correlation between FN1 expression and these features. Interestingly, we found that FN1 expression level was related to source of tumor samples (ovary vs omentum). We observed more tumors with higher FN1 expression among the samples derived from omental metastatic disease than in samples from primary site (p=0.024). In addition, we found that FN1 expression was positively correlated with degree of desmoplasia (p=10-4). In summary, we found that increased stromal expression of FN1in high-grade serous ovarian cancer is indicative of shorter survival. In addition, metastatic tumors more frequently have high expression of FN1 than primary ovarian cancers.

References: 1. Lisowska KM et al (2016): JCancerResClinOncol 142: 1239-1252.

Acknowledgments: This study was supported from grant 2012/04/M/NZ2/00133 from National Science Center to K.M. Lisowska.

122 .

New methods, new molecules and new approaches (70-81)

. 123 124 . 70. COMPARISON OF RIGID AND NON-RIGID IMAGE REGISTRATION ALGORITHMS FOR MRI IMAGE DISTORTION ASSESSMENT AND CORRECTION

Paweł Bzowski1,2, Rafał Panek3, Maria Schmidt3, Damian Borys1,2

1Institute of Automatic Control, Silesian University of Technology, Akademicka 16, 44-100 Gliwice, Poland 2Biotechnology Centre, Silesian University of Technology, Krzywoustego 8, 44-100 Gliwice, Poland 3Institute of Cancer Research, 15 Cotswold Road, SM2 5NG, London, United Kingdom

Medical imaging techniques, such as magnetic resonance imaging (MRI) and computed tomography (CT), are commonly used to non-invasively detect and diagnose anomalies of anatomical structures. MRI images are particularly useful in assessment of soft tissues and can provide information on functional properties of tissue (i.e. diffusion and perfusion), whereas CT provides a high resolution imaging of bone structures at high geometrical precision. This complementary information can be used together and MRI-CT image co-registration is increasingly used. However, there are many causes of potential image dissimilarities. MRI images may not be geometrically accurate in the case of large field of view examinations or at the presence of metallic implants. Another concern is difference in patient positioning in different imaging sessions. As a result, the obtained image can be deformed or displaced and might not match the anatomical reference. Image co-registration methods, such as rigid and non-rigid transformations can be used to correct for these differences. The main aim of this work was to implement an algorithm for non-rigid registration based on Navier-Stokes equation [1]. This algorithm would be able to find an optimal transformation for match of two different studies in different modalities. Algorithm was implemented in MATLAB and the sum of squared difference and mutual information was used as quality measure [2,3]. In this work we used LEGO phantom and Linear Test Object – LTO [4,5], with dimensions 440x270x360 mm. Images were acquired with Philips 3T MRI scanner and GE CT. These images were transformed using rigid transformation with mutual information as a quality measure. Then the new data set was transformed using non-rigid elastic method based on Navier-Stokes equation. As a result, we obtained a rigid and non-rigid transformation of phantom images. The rigid method is a good choice for preliminary processing but some inaccuracies occurs, especially in the off center areas. Results were visually evaluated as well as MI measure was used to assess the quality of image matching in both rigid and non-rigid algorithms. The non-rigid transformation allowed for a successful co-registration of both, distorted MRI and non-distorted CT data in the whole large field of view region.

This work was supported by the Polish National Center of Research and Development grant no. STRATEGMED2/267398/4/NCBR/2015 (MILESTONE - Molecular diagnostics and imaging in individualized therapy for breast, thyroid and prostate cancer). Calculations were performed on the Ziemowit computer cluster in the Laboratory of Bioinformatics and Computational Biology, created in the EU Innovative Economy Programme POIG.02.01.00-00-166/08 and expanded in the POIG.02.03.01-00-040/13 project.

References: 1. M.Bro-Nielsen, C.Gramkow. Fast Fluid Registration of Medical Images. Proc. Visualization in Biomedical Computing (VBC’96), Lecture Notes in Computer Science, vol. 1131, pp. 267-276, Springer (1996) 2. J. Modersitzki, Numerical Methods for Image Registration, Oxford University Press, New York, NY (2004) 3. D’Agostino, E., Maes, F., Vandermeulen, D., Suetens, P. A viscous fluid model for multimodal non-rigid image registration using mutual information. Medical Image Analysis. 7:565–575 (2003) 4. S.J. Doran, S. Reinsberg, L. Charles-Edwards, M.O. Leach, A complete distortion correction for MR images: I. Gradient warp correction, Phys. Med. Biol. 50(7), 1343–1361 (2005) 5. S.A. Reinsberg, S.J. Doran, E.M. Charles-Edwards, M.O. Leach, A complete distortion correction for MR images: II. Rectification of static-field inhomogeneities by similarity-based profile mapping, Phys. Med. Biol. 50(11), 2651–2661 (2005)

125 . 71. A NEW ALGORITHM FOR ILLUMINA SEQUENCING READS CORRECTION

Maciej Długosz, Sebastian Deorowicz

Institute of Informatics, Faculty of Automatic Control, Electronics and Computer Science, Silesian University of Technology, 44-100 Gliwice, ul. Akademicka 16, Poland

DNA sequencing is dominated by next-generation sequencing (NGS) technologies. Their ability to produce huge amount of data at moderated cost enabled various genomic data applications in biomedical sciences. Unfortunately, reads produced by NGS techniques are affected by significant amount of errors. This drawback causes many problems in reads utilization. Algorithms processing defected reads produce results with lower quality and often require more computational resources, especially huge amount of memory. Accuracy of the reads can be improved by performing read error correction. We present a new algorithm for correction of whole genome seqencing (WGS) reads produced by Illumina's sequencers. The algorithm – called RECKONER – is a backtracking algorithm and belongs to k-mer spectrum-based algorithms. RECKONER is based on correction algorithm BLESS [1] and exploits tools KMC 2 [2] for k-mer counting and storing a k-mers database and KMC TOOLS for k-mers database manipulating. The idea of RECKONER is to utilize both quality indicators (delivered with reads from input FASTQ files) and numbers of k-mers occurrences in the input reads. RECKONER also introduces a new method of rating potential single read corrections allowing to choose the best one. We performed comparison of a few state-of-the-art correction algorithms (including RECKONER) in terms of correction quality and resource consumption on both simulated and real sequencing data. The results show, that RECKONER is able to detect and correct similar or higher number of erroneous reads than the competiting algorithms. Tests also proved positive impact of error correction on typical genomic data processes, like de novo assembly and reassembly. Moreover, RECKONER was able to correct set of reads from eucaryotic-sized organism with genome of length 500 Mbp in about half an hour on 16-core CPU with consumption 4 GB of memory, what makes possible to run RECKONER on a desktop computer.

References: 1. Heo Y, Wu X-L, Chen D, Ma J, Hwu W-M (2014): Bioinformatics 30: 1354. 2. Deorowicz S, Kokot M, Grabowski S, Debudaj-Grabysz A (2015): Bioinformatics 31: 1569.

Acknowledgements: This work was supported by Silesian University of Technology grant no. BKM507/RAU2/2016.

126 . 72. MEASUREMENT OF PARTIAL-VOLUME EFFECT USING CONTINUOUS TABLE MOVEMENT TECHNIQUE IN PET/CT

Nikol Niejadlik1, Damian Borys1,2, Izabela Gorczewska1, Kamil Gorczewski1

1Department of PET Diagnostics, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Wybrzeże Armii Krajowej 15, 44-101 Gliwice, Poland; 2Institute of Automatic Control, Silesian University of Technology, ul. Akademicka 16,44-100 Gliwice, Poland

Partial-volume effect (PVE) is a phenomenon occurring in PET/CT tumor imaging. PVE introduces signal loss and blurring effect in PET/CT images which are causes of large biases in the context of radiotherapy treatment planning[1]. The intensity of the effect is related to the volume of a lesion. Therefore, the therapy monitoring should correct for this effect when tracer uptake is compared over time. Conventional PET acquisition consists of several overlapping measurements that are afterwards combined into a volumetric result. Recently introduced FLOW acquisition technique[2] allows for continuous patient movement under the PET detector with no need of measurement overlapping. The aim of the study was to quantify the intensity of the PVE effect with continuous bed movement acquisition and compare those results with conventional acquisition mode that were published. The phantom consisting of 6 spheres of different diameters filled F-18 with 27 kBq/ml, and surrounding background with C-11 with 13 kBq/ml was acquired in FLOW mode. Single CT scan was performed at the beginning of measurements for attenuation correction and sphere delineation. The PET measurements were repeated 11 times every 6 minutes. The use of F-18 and C-11 allow to observe changes in contrast between spheres and background, since the half-life of F-18 is 109,8 minutes and of C-11 is 20,3 minutes. The Recovery Coefficients (RC) [3] were evaluated for each of 11 time points. The signal loss due to PVE effect was calculated for each sphere of each time point. Resulting RCs were in the range from 2 - 12,5. The maximal signal loss was found in the smallest sphere at RC of 0.46, which is consistent with results presented in [3]. The smallest RCs are the most important from the clinical perspective. As a tumor is treated, it loses both the volume and the metabolic uptake. The signal loss in PET can have those two sources. It is important to distinguish those sources since only lower metabolic uptake verifies the positive outcome of therapy. The signal decrease due to volume shrinkage is often a false-positive response and has to be distinguished from the true metabolic effect.

References: 1. Soret M et al (2007): Partial-Volume Effect in PET Tumor Imaging. JNuclMed 48: 932-945. 2. Osborne DR et al (2015): Quantitative and qualitative comparison continuous bed motion and traditional step and shoot PET/CT. AmJNuclMedMolImaging 5: 55-64. 3. Gallivanone F et al (2011): PVE Correction in PET-CT Whole-Body Oncological Studies From PVE-Affected Images. IEEETransactNuclSci 58: 736-747.

. 127 73. STRUCTURE AND PROPERTIES OF TEETH ORIGIN HYDROXYAPATITE – POTENTIAL AUGUMENTATION MATERIAL IN SMALL BONE DEFECTS

Adrianna Słotwińska3, Ewa Żytecka3, Katarzyna Latusek3, Dorota Łyko-Morawska1 Work’s tutor: prof. dr hab. n. med. Iwona Niedzielska1, dr inż. Andrzej Hudecki2

1Department of Cranio- Maxillo- Facial and Oral Surgery, School of Medicine in Zabrze, Medical University of Silesia, Katowice; 2Institute of Non Ferrous Metals Gliwice; 3Student’s Association, Department of Cranio- Maxillo- Facial and Oral Surgery, School of Medicine in Zabrze

Introduction: Augmentation of alveolar bone process loss is one of more commonly used surgical procedures, hence the great interest of researchers seeking new technological solutions in that area. The aim: New innovative method to obtain composites from extracted teeth was evaluate. Materials structure and properties were explored and compared to commercial available material. Material and Methods: 16 teeth were included into examination in accordance with eligibility criteria. Tooth were preparated using authors’ standards (cleaning, rinsing, sintering) and divided into two separate study groups. First group of tooth material was grinded, the second was grinded and sintered into powdered form. The control group was a commercially available HA-based augumentation material. The material structure and features were analyzed by scanning electron microscopy (SEM), spectrophotometry and differential calorymetry. Results: The difference in electrical conductivity was shown. For the specimens obtained from synthetic hydroxyapatite conductivity was lower than from natural hydroxyapatite. Teeth orgin composites samples had 26.1 % weight loss in calorimetry. The observations using scanning electron microscopy confirmed that increasing milling time reduces grain size of the powder but the structures of composites (teeth vs. synthetic) are totally different. Conclusion: As the main aim is to find materials which are cheap, easy to manufacture and autogenic the teeth-originated hydroxyapatite shows a good perspective in clinical use.

Keywords: augumential material, alveolar bone loss, hydroxyapatite, teeth

128 . 74. THERMOREVERSIBLE SOFT MATRICES FOR 3D CELL CULTURE BASED ON GLYCOSYLATED PNIPAM MICROGELS

Małgorzata Burek1, Sylwia Waśkiewicz2, Anna Lalik3, Sebastian Student3, Tadeusz Bieg1, Ilona Wandzik1

1Silesian University of Technology, Faculty of Chemistry, Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, 4 B. Krzywoustego Street, 44 100 Gliwice, Poland; 2Silesian University of Technology, Faculty of Chemistry, Department of Physical Chemistry and Technology of Polymers, 9 M. Strzody Street, 44 100 Gliwice, Poland; 3Silesian University of Technology, Institute of Automatic Control, Systems Engineering Group, 8 B. Krzywoustego, 44 100 Gliwice, Poland

Cancer research commonly relies on standard two-dimensional (2D) cultures, where cells are grown as monolayers on stiff glass or plastic surfaces. However, under such conditions cells show abnormal polarization and flattened shape as they are exposed to non-physiologically homogenous environment with enough nutrients and oxygen. It results in loss of differentiated phenotype, which along with lack of cell-cell and cell-matrix interactions do not reflect the essential features of native tumor and affect the biological characteristics such as proliferation, migration, and therapeutic resistance. Therefore, to overcome the limitations of monolayer cultures and better simulate the in vivo microenvironment of tumor, where cells are embedded in three-dimensional (3D) manner surrounded by extracellular matrix (ECM) components, various strategies have been developed. The most well- known techniques commonly used in 3D cultures involve cultivation in hanging drop, microfluidic devices, or hydrogel matrices. Especially, use of hydrogels, water-swollen polymeric networks, that have a soft tissue-like elastic properties and aim to mimic ECM have emerged as an attractive option. Herein, a series of thermoresponsive glycomicrogels with trehalose in the cross-links or with trehalose in the cross-links and as pending moieties were synthesized. These materials were obtained by surfactant-free precipitation copolymerization of N-isopropylacrylamide and various amounts of trehalose monomers. The resultant particles showed spherical shape and submicrometer hydrodynamic size with narrow size distribution. At 25 ºC, glycomicrogels in solutions with physiological ionic strength formed stable colloids, which further gelled upon heating to physiological temperature forming macroscopic hydrogel with interconnected porous structure. These extremely soft matrices with dynamic storage modulus in the range 9-70 Pa were examined in 3D culture systems for HeLa cell culture in comparison to traditional 2D mode. They showed relatively low syneresis over time, especially when glycomicrogels with high content of hydrophilic trehalose were used as building blocks. Incorporated pending trehalose composed of two α,α’-1,1’-linked D-glucose moieties was used with the intention of providing multivalent interactions with glucose transporters (GLUTs) expressed on the cell surface. Better cell viability was observed when soft hydrogel with the highest content of trehalose and the lowest syneresis was used as a matrix compared to 2D control assay.

Acknowledgements: This work was financially supported by the National Science Centre, Poland under the project 2014/15/N/ST8/02707. Experiments were performed in the Biotechnology Center of Silesian University of Technology in Gliwice using equipment financed by Silesian BIO-FARMA Project (POIG.02.01.00-00-166/08)

. 129 75. USAGE OF APPLICATION OSIRIX IN THE ASSESSMENT OF THE AORTIC CONTRACTILITY IN PATIENTS WITH AORTIC BICUSPID EXAMINED BY COMPUTER HEART TOMOGRAPHY

Róża Dzierżak1, Ryszard Maciejewski2

1Lublin University of Technology, Faculty of Electrical Engineering and Computer Science, Lublin; 2Medical University of Lublin, Department of Normal Anatomy, Lublin

The paper presents a method for the aortic contractility evaluation of that is wholly different than the commonly used ones. The evaluation is based on the cardiac CT scan images and uses the OsiriX medical imaging application for the DICOM file processing. The contractility testing has been performed on a group of patients with a congenital bicuspid aortic valve (BAV) disease that occurs in ca 1% of the population. The testing has been also applied to a control group of patients with the normal tricuspid valve configuration.

Key words: bicuspid aortic valve (BAV), computed tomography (CT), aortic contractility, OSIRIX application

130 . 76. STUDY OF ANOMALOUS AND NORMAL TWO-DIMENSIONAL DIFFUSIVE MOTION OF A TRACER PARTICLE IN MEMBRANE

Monika Krasowska1, Anna Strzelewicz1, Gabriela Dudek1, Michał Cieśla2

1Department of Physical Chemistry and Technology of Polymers, Faculty of Chemistry, Silesian University of Technology, Strzody 9, 44-100 Gliwice, Poland; 2M. Smoluchowski Institute of Physics, Jagiellonian University, Łojasiewicza 11, 30-348 Kraków, Poland

A subject of intense current investigation in the area of anomalous diffusion focus on the question of how the environment affects the dynamic properties of diffusing particles. Motivated by our recent research we report results of simulations of the motion of a finite sized tracer particle in heterogeneous environment made up of a polymeric membrane with inorganic filler. The examined membranes are composed of polymer (like chitosan, ethylcellulose etc.) and magnetic powder. The structure of pattern formed by the magnetic powder used in the membrane matrix depends on many parameters such as polymeric matrix used, type of powder, its amount, granulation and molecular clusters (if any). The comparision of diffusion driven by Gaussian random walk and Lévy flights was studied. Subdiffusion has been observed for Gaussian random walk for short times, however, superdiffusion has been noted for Lévy flights. In the case of long times, subdiffusion has been observed for both Gaussian random walk and Lévy flights. The median of mean squared displacement (MSD) and effective diffusion exponent were investigated. It was shown that the effective exponent of the median of square displacement at the long time limit does not depend significantly on the character of motion, but mainly on the morphology of the environment. It is expected that obtained results will contribute to further progress in the design and preparation of membrane structures with specific diffusion properties.

References: 1. Krasowska M, Rybak A, Dudek G, Strzelewicz A, Pawelek K, Grzywna ZJ (2012): Structure morphology problems in the air separation by magnetic membranes. JMembrSci 415-6: 864-870. 2. Strzelewicz A, Krasowska M, Dudek G, Rybak A, Turczyn R, Cieśla M (2013): Anomalous diffusion on fractal structure of magnetic membranes. ActaPhysPolonB 44: 955-965. 3. Cieśla M, Dybiec B, Sokolov I, Gudowska-Nowak E (2015): Taming Lévy flights in confined crowded geometries. JChemPhys 142: 16904. 4. Krasowska M, Strzelewicz A, Rybak A, Dudek G, Cieśla M (2016): Structure and transport properties of ethylcellulose membranes with different types and granulation of magnetic powder. PhysicaA, 452: 241-250. 5. Metzler R, Klafter J (2000): The random walk’s guide to anomalous diffusion: a fractional dynamics approach. PhysicsRep 339: 1-77.

. 131 77. ENZYMATIC DEGRADATION OF COPOLYMERS PREPARED FROM Ε-CAPROLACTONE AND TERT-BUTYL METHACRYLATE

Anna Mielańczyk, Monika Hohn, Dorota Neugebauer

Department of Physical Chemistry and Technology of Polymers, Faculty of Chemistry, Silesian University of Technology, ul. M. Strzody 9, 44-100 Gliwice, Poland

Biocompatible polymers including poly(ε-caprolactone) (PCL) have received considerable attention in the field of temporary therapeutic applications such as sustained drug delivery systems, surgical sutures, osteosynthetic devices, scaffolds for tissue repair and regeneration [1]. This work is a continuation of our previous research, which concerned the synthesis of amphiphilic miktoarm star copolymers with two PCL and six polymethacrylate arms, their self-assembly and entrapment of anticancer drugs such as doxorubicin or camptothecin, followed by drug release studies [2]. The aim of the present studies was to investigate the degradation mechanism of PCL and its copolymers with tert-butyl methacrylate, in order to determine the influence of the topology (linear block copolymers, miktoarm stars) and composition of copolymers on the degradation process as well as to evaluate the selectivity of enzyme Novozyme 435 toward different types of ester groups which are present in the main chain of PCL or/and pending tert-butyl ester groups.

References: 1. Woodruff MA, Hutmacher DW (2010): ProgPolymSci 35: 1217–1256. 2. Mielańczyk A Odrobińska J Grządka S Mielańczyk Ł Neugebauer D IntJPharm http://dx.doi.org/10.1016/j.ijpharm.2016.10.034

132 . 78. MODIFIED BIOPOLYMERS AS SORBENTS FOR THE REMOVAL OF BORON FROM AQUEOUS SOLUTION

Joanna Kluczka1, Małgorzata Gnus2, Gabriela Dudek2, Roman Turczyn2

Silesian University of Technology Faculty of Chemistry:, 1Department of Inorganic, Analytical Chemistry and Electrochemistry, Krzywoustego 6 Street; 2Department of Physical Chemistry and Technology of Polymers, Marcina Strzody 9 Street, 44-100 Gliwice, Poland

Boron is a naturally occurring element that is widely distributed at low concentrations in the environment. Boric acid and borate salt have extensive industrial uses in the manufacture of glass and porcelain, in wire drawing, production of leather, carpet and photographic chemicals. Compounds of boron are used in organic synthesis, in the manufacture of a particular type of glasses, and as wood preservatives. Boron filaments are used for advanced aerospace structure. Although boron is essential for plant growth, in excess of 2.0 mg/L in irrigation water it is deleterious to certain plant species. Boron deficiencies cause growth problems and difficulties in sugar mobilization. Other plants may be affected adversely by concentration as low as 1.0 mg/l. The human body contains approximately 0.7 ppm of boron, an element that is not considered a dietary requirement. Still, humans absorb this element from foodstuffs, because it is a dietary requirement for plants. Daily intake is approximately 2 mg. At a daily intake of over 5 g of boric acid the human body is clearly negatively influenced, causing nausea, vomiting, diarrhoea and blood clotting. Amounts over 20 g are life-threatening. Boron dissolved in surface and ground waters is present at concentration levels of 0.3–100 mg/L and higher (1), while recommended level of boron in drinking and irrigating water and wastewaters discarded to the environment is 1.0 mg/L in Poland and EU. The adsorption process is the most studied and applied method for removing boron from aqueous solution. Chitosan, being cheap, environment-friendly and possessing good ability to adsorb pollutants, has attracted great interest in water and wastewater treatment. However, adsorption of boron species onto the chitosan beads occurred with physical forces and characterised with not high enough capacity (3). An Al, Fe, Ce and Zr oxide/hydroxide adsorbent demonstrates high efficiency for boron removal (4-6). However, post-treatment requires the elimination of particles as colloidal precipitates using additional processes such as coagulation, sedimentation and filtration. In order to avoid this step, metal-oxide nanoparticles may be immobilized on inert surfaces, such as activated carbon, foams or nanofibers. The objective of this research was study the efficiency of adsorption technology to reduce or remove boron from water using several kinds of oxides/hydroxide (titanium, chromium and iron oxide/hydroxide) doped into chitosan gel and determine the effect of influence of operating parameters like pH, temperature, sorption time and sorption dose on boron adsorption from aqueous solution. Boron removal was examined in the batch system. The experimental data were evaluated by the equilibrium isotherm and kinetic models. By combining the advantageous properties of chitosan and metal nanooxides, an effective adsorbents for boron were developed. The results show that composite chitosan beds doped with titanium dioxide and iron(III) hydroxide nanoparticles can be interesting new sorbents in surface water treatment to remove boron. Besides of many advantages of matrix, e.g. non-toxicity, bacteriostatic and fungistatic properties, new sorbents were characterized by good affinity to boron species.

References: 1. Hilal et al. (2011): Desalination 273: 23–35. 2. Bobrowska-Grzesik et al. (2013): Chemical elements. Compendium, Cesky Tesin, 2 Theta 3. Bursali et al EA (2011): JApplPolymerSci 122: 657-665. 4. Demetriou A, Pashalidis J (2012): DesalWatTreat 37: 315-320. 5. Öztürk N, Kavak D (2008): Desalination 223: 106-112. 6. Kluczka J (2015): IntJEnvironRes 9: 711-720.

Acknowledgements: The autors would like to thank the Silesia University of Technology for providing financial support under the project BKM-526/RCH4/2016.

. 133 79. THE PREPARATION AND CHARACTERIZATION OF THE MAGNETIC HYBRID MEMBRANES BASED ON MODIFIED PPO AND LPI POLYMER MATRICES FOR AIR SEPARATION

Aleksandra Rybak1, Zbigniew J. Grzywna1, Petr Sysel2, Waldemar Kaszuwara3

1Department of Physical Chemistry and Technology of Polymers, Faculty of Chemistry, Silesian University of Technology, Strzody 9, 44-100 Gliwice, Poland; 2Department of Polymers, Faculty of Chemical Technology, University of Chemistry and Technology, Technická 5, CZ-166 28, Prague, Czech Republic; 3Faculty of Materials Science and Engineering, Warsaw University of Technology, Woloska 141, 02-507 Warszawa, Poland

The major issue of current membrane research is developing highly permeable, selective and resistant membrane materials with many medical and industrial applications (air separation, hydrogen recovery and CO2 removal). For this purpose, the polymers can be modified by various substitutions, insertion of the metal cations, production of copolymers or carbon membranes [1, 2]. Another strategy for improving the mass transport through polymer films is the incorporation of inorganic materials (zeolites, carbon molecular sieves, silica nanoparticles, carbon nanotubes, metal organic framework and clay layered silicates) into a polymer matrix. Such functional inorganic-organic hybrid composites are materials of great interest because of their adjusted properties obtained by control of the composition, content and morphology of the particle addition, usage of different processing techniques or by modification of the polymer matrix [3, 4]. This work is the continuation of our earlier research [5-8], which confirmed that the incorporation of magnetic particles into the polymer matrix enhanced the hybrid membrane’s gas transport properties. We have examined the gas separation properties of homogeneous FeSPPO and APTES-endcapped LPI membranes and the magnetic hybrid inorganic-organic membranes (filled with modified magnetic powders) based on these modified polymer matrices. The magnetic powders were covered with silica shell via hydrolysis of TMOS using modified Stöber’s method to improve the interaction between the organic and inorganic phase. APTES acted as a coupling agent, chemically bonding the organic polymer with the inorganic filler. In turn, the magnetic matrix of FeSPPO membranes influenced the introduced magnetic particles, resulting in membranes with improved gas transport and magnetic properties. The results showed that the modification, both of the polymer matrix and the magnetic particles caused the increase in gas permeability and diffusivity, while their permselectivity and solubility were rather maintained or slightly increased. It was found that the magnetic membrane’s gas transport properties were improved with the increase of magnetic particle filling and decrease in powder particle size.

References: 1. Li NN, Fane AG, Winston Ho WS, Matsuura T (2008): Advanced membrane technology and applications. Hoboken, John Wiley & Sons Ltd. 2. Kruczek B, Matsuura T (2000): JMembrSci 167: 203. 3. Sysel P Minko E, Čechová R (2009): e-Polymers 9: 976-985. 4. Minko E, Sysel P, Hauf M, Brus J, Kobera L (2010): MacromolSymp 295: 88-93. 5. Rybak A, Dudek G, Krasowska M, Strzelewicz A, Grzywna ZJ (2014): SepSciTechnol 49: 1729. 6. Rybak A, Grzywna ZJ (2009): Kaszuwara W JMembrSci 336: 79. 7. Rybak A, Dudek G, Krasowska M, Strzelewicz A, Grzywna ZJ (2012): SepSciTechnol 47: 1395. 8. Rybak A, Grzywna ZJ, Sysel P (2013): SepPurifTechn 118: 424-431.

134 . 80. INJECTABLE NANOCOMPOSITES CONTAINING PREFERENTIALLY BIODEGRADABLE POLY(ESTER-ANHYDRIDE) MICROSPHERES

Monika Śmiga-Matuszowicz1, Katarzyna Jaszcz1, Marcin Kaczmarek2, Ryszard Pilawka3, Marta Lesiak4, Aleksander L. Sieroń4, Damian Kusz5

1Silesian University of Technology, Department of Physical Chemistry and Technology of Polymers, PL 44-100 Gliwice, ul. M. Strzody 9, Poland; 2Silesian University of Technology, Department of Biomaterials and Medical Devices Engineering, PL 41-800 Zabrze, ul. de Gaulle`a 66, Poland; 3West Pomeranian University of Technology, Polymer Institute, PL 70-322 Szczecin, ul. Pułaskiego 10, Poland; 4Medical University of Silesia, Department of General and Molecular Biology and Genetics, PL 40-752 Katowice, ul. Medyków 18, Poland; 5Medical University of Silesia, Department of Orthopedics and Traumatology, PL 40-635 Katowice, ul. Ziołowa 45, Poland

The bone defects caused by tumors or trauma often require implantation of synthetic bone substitutes favouring bone tissue regeneration. Polymeric injectable bone substitutes (IBS) offer a major advantage, compared to prefabricated 3D scaffolds for orthopedic applications, by providing the ability to use minimally invasive treatment methods. Biodegradable liquid IBS are cured in site of injection and are capable of filling irregular skeletal defects providing immediate structural support and then allow bone tissue regeneration. For proper bone formation, the material should be porous to allow metabolism and migration of cells [1]. The novel interesting IBS are composite materials composed of biodegradable polymeric matrix and biodegradable microspheres and can serve as a scaffold combined with drug delivery system [2]. This study was aimed to prepare novel in situ forming biodegradable nanocomposites based on poly(3-allyloxy-1,2-propylene) succinate (PSAGE) and nanosized hydroxyapatite (HA). These nanocomposite materials contain poly(ester-anhydride) (PEA) microspheres embedded in polyester matrix prepared by crosslinking PSAGE with oligo(1,2-propylene maleate) and methacrylic monomers. Methyl methacrylate and one of hydrophilic oligo(ethylene glycol) methacrylates with different functionality and various length of oligooxyethylene chains were used as polymerizable diluents. In situ formation of pores has been achieved by preferential degradation of dispersed phase in a biphasic polymer system. The obtained materials are liquid before curing and harden in several minutes with moderate exothermic effect. The effect of the composition of nanocomposite materials on selected properties, such as water sorption, mechanical strength, porosity and hydrolytic degradation behaviour was investigated. Analysis by energy dispersive spectroscopy confirmed the presence of characteristic features of HA in the nanocomposite materials. The morphology of the cured nanocomposites subjected to hydrolytic degradation was evaluated by SEM. The MTS cytotoxicity assay was carried out for extracts from crosslinked materials using hFOB1.19 cells. It was found that the extracts exhibit a dose-depended cytotoxic response.

References: 1. Karageorgiou V, Kaplan D (2005) Biomaterials 26: 5474. 2. Li B, Yoshii T, Hafeman AE, Nyman JS, Wenke JC, Guelcher SA (2009) Biomaterials 30: 6768.

. 135 81. INJECTABLE BIOCOMPOSITES FOR BONE AUGMENTATION PREPARED FROM ISOSORBIDE-BASED POLYMERIZABLE RESINS

Monika Śmiga-Matuszowicz1, Bartosz Janicki1, Bożena Nowak2, Ryszard Pilawka3, Marcin Basiaga4

1Silesian University of Technology, Department of Physical Chemistry and Technology of Polymers, PL 44-100 Gliwice, ul. M. Strzody 9, Poland; 2University of Silesia, Department of Biochemistry, 40-032 Katowice, Jagiellońska Street 28, Poland; 3West Pomeranian University of Technology, Polymer Institute, PL 70-322 Szczecin, ul. Pułaskiego 10, Poland; 4Silesian University of Technology, Department of Biomaterials and Medical Devices Engineering, PL 41-800 Zabrze, ul. de Gaulle`a 66, Poland

Currently, the most commonly used in situ curable biomaterials in bone and vertebral fractures caused by osteoporosis, tumors or trauma are injectable bone cements based on poly(methyl methacrylate) (PMMA). The efficient procedure for the treatment of painful vertebral fractures is percutaneous vertebroplasty and kyphoplasty. Liquid materials harden through free radical polymerization in several minutes after application and assume the mechanical function of damaged bone, but their high curing temperatures can cause necrosis of the adjacent bone tissue. Additionally, the mechanical properties of the cured material are far in excess of the trabecular bone, leading to possible fractures provoked by overload of adjacent vertebrae [1,2]. Thus, PMMA bone cements seem to be successful, but not optimal materials. The aim of presented work was to prepare and characterize new composite materials obtained by crosslinking of two novel resins based on bioderived alicyclic diol - isosorbide. The mixture of biodegradable oligo(isosorbide maleate) (OMIS), dimethacrylate resin - 2,5-bis(2-hydroxy-3- methacryloyloxypropoxy)-1,4:3,6-dianhydro-sorbitol (ISDGMA) and methyl methacrylate was studied as a polymer matrix of partially biodegradable, injectable biomaterials for bone augmentation. Cement formulations contained also radiopaque agent – zirconium dioxide (ZOX) and one of bioactive fillers such as nanosized hydroxyapatite (nHA) or microsized calcium carbonate (mCC). In this study selected properties of tested materials were compared with the results obtained for commercial BIOMET Bone Cement® V. Observed maximum curing temperature of the cement formulations was close to 40 oC thus being considerably lower than 90 oC specified as acceptable for commercial PMMA bone cements. The compressive modulus (Ec) of tested cured formulations was very high in comparison to BIOMET ® Bone Cement V, but after water uptake a significant drop of Ec values was observed for all materials as they were more close to modulus of trabecular bone. The morphology of solidified materials, before and after hydrolytic degradation, the release profile of bactericidal agent - ofloxacin (OFX) and its activity against Staphylococcus aureus and Escherichia coli were examined.

References: 1. Verlaan J-J, Oner FC, Dhert WJA (2006): Biomaterials 27: 290. 2. Heini PF, Berlemann U (2001) EurSpineJ 10: S205.

136 .

New molecules and new therapies (82-101)

. 137 138 . 82. RESEARCH ON THE SYNTHESIS OF BISPHOSPHONATE MODELS USED AS CONTRAST AGENTS IN MAGNETIC RESONANCE IMAGING

Anna Kuźnik1,2, Sylwia Korusiewicz1

1Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Silesian University of Technology, B. Krzywoustego 4, 44-100 Gliwice, Poland; 2Biotechnology Center of Silesian University of Technology, B. Krzywoustego 8, 44-100 Gliwice, Poland

Bisphosphonates (BPs), used as inhibitors of bone resorption, contain two phosphonate groups attached to a single carbon atom, forming a P-C-P moiety. Consequently, BPs as structural analogues of pyrophosphate, exhibit characteristic features such as: high affinity for calcium ions that are components of bone hydroxyapatite and resistance to enzymatic degradation. Furthermore, the structure of the side chain makes BPs a large group of compounds of various physical, chemical and biological properties [1]. Several bisphosphonates are applied as effective therapeutic agents in clinical disorders such as: osteoporosis, Paget’s disease, myeloma, bone metastasis, brittle bone disease, fibrous dysplasia and others [2]. However, α-aminomethylidenebisphosphonates with a suitably protected amino group could be also potentially used as substrates in the synthesis of compounds that are used in the magnetic resonance imaging [3]. Thus, the aim of our research was the synthesis of bisphosphonate models 2 and 3 with amino group protected with chloroacetyl or 4-chlorobutyryl group (Scheme). These types of BPs may be attached to the macrocyclic complexes of paramagnetic ions for their further use as contrast agents in MRI.

Scheme

We have elaborated a new effective procedure for the synthesis of expected bisphosphonate models 2 and 3 comprising in preparation of tetraethylα-(N-phenylacetylamino)methylidenebisphos- phonate 1, followed by its enzymatic hydrolysis catalyzed by penicillin acylase G (PGA, EC 3.5.1.11) and subsequent protection of the free amino group with selected chloroacyl chlorides. The simplicity of this synthetic route as well as mild reaction conditions make it an alternative pathway for the synthesis of these types of BPs. Those compounds will be next used in the synthesis of ligands for MRI to improve the clinical diagnostic imaging of bone metabolism, pathological calcifications or bone metastases.

References: 1. Szpak M, Kurzak B (2014): Chemik, 68: 321-328. 2. Romanenko VD, Kukhar VP (2012): Arkivoc 4: 127–166. 3. Kubicek V, Rudovsky J, Kotek J, Hermann P, Elst L, Muller R, Kolar Z, Wolterbeek H, Peters J, Lukes I (2005): JAmChemSoc 127: 16477-16485.

. 139 83. SPILANTHOL OF ACMELLA OLERACEA AS NATURAL PHARMACEUTICAL AND DERMATOLOGICAL AGENT

Mirosława Grymel1,2, Karolina Kałuża1, Roman Mazurkiewicz1,2, Przemysław Kołak3

1Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Silesian University of Technology B. Krzywoustego 4, 44-100 Gliwice, Poland; 2Biotechnology Center of Silesian University of Technology, B. Krzywoustego 8, 44-100 Gliwice, Poland; 3NATUU Sp. z o.o. Koryciny 73, 17-315 Grodzisk, Poland

Acmella oleracea (syn. Spilanthes acmella, Spilanthes oleracea, Jambu) is a plant which is a representative of species of Asteraceae family. The herb grows in America, Africa and India where is used in traditional folk medicine. It is characterized by a yellow-red flower head and height from 30-60 cm. In pharmaceutical study of Acmella oleracea it turned out that this herb has very important properties which are used in various areas of human activity. Flowers of this plant show antinociception effect by inhibiting prostaglandin synthesis and exerting antihistamine activity. The plant has found applications in pharmaceuticals as an antitoothache formulation for pain relief, swelling and gum infections and periodontosis. In addition, the plant extract was also used for stimulating, reorganizing and strengthening the collagen network in anti-age applications [1]. Bioactive compounds such as: N-alkylamides 1-5, β-sitostenone, vanilic acid, scopoletin, trans-ferulic acid, 3-acetylaleuritolic acid and trans-isoferulic acid that are responsible for properties of the plant are isolated in the extract of Acmella oleracea. The most important bioactive compound of these N-alkylamides group is spilanthol 4 (Fig.1) [2].

O O 6 5 3 1' R R 1 NH NH 11 4 2 2'

3' 1 5,6-dehydro (Z), R = Me 4 R = Me 2 R = Me 5 R = Et 3 R= Et

Fig. 1. The structures of N-alkylamides

Spilanthol can exert a variety of biological and pharmacological effects such as local anesthetic, analgesic, bacteriostatic, antipyretic, anti-inflammatory, antioxidant, diuretic, antimutagenic and anti-cancer agent. It is also possible that spilanthol has important effects on the central nervous system (CNS) and immune system [2]. Due to the fact that spilanthol is an amphiphilic compound it can be extracted from the plant using alcohols, hexane or carbondioxide. We have demonstrated that the efficient method of isolating spilanthol of Acmella oleracea is extraction using ethanol. Moreover, we have developed rapid method of analysis of spilanthol content in ethanolic extract from Acmella oleracea using nuclear magnetic resonance technique (1H NMR).

References: 1. Cheng Y-B, Chang F-R et al (2015): Alkylamides of Acmella oleracea. Molecules 20: 6970-6977. 2. Barbosa AF, Carvalho MG, et al (2016): Spilanthol: occurrence, extraction, chemistry and biological activities. RevistaBrasilFarmacog 26: 128-133.

140 . 84. COMPLEXATION OF SELECTED METABOLITES OF CATECHOLAMINES WITH Β-CYCLODEXTRIN – A REEXAMINATION

Anna Korytkowska-Wałach1, Beata Dubrawska1, Monika Śmiga-Matuszowicz2, Tadeusz Bieg1

1Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Silesian University of Technology, 44-100 Gliwice, Krzywoustego 4, Poland; 2Department of Physical Chemistry and Technology of Polymers, Silesian University of Technology, 44-100 Gliwice, Strzody 9, , Poland

Metabolites of catecholamines have great importance for the diagnosis of tumours of the sympathoadrenal system. Polymers containing β-CD moieties for enhancing entrapment of catecholamine metabolites from aqueous solutions are of interest to us. In this study inclusion complexes formed between metabolites of catecholamines, i.e. vanillylmandelic acid (VMA), homovanillic acid (HVA) as well as vanillin (VA) with β-cyclodextrin (β-CD) were tested using NMR spectroscopy. Stoichiometry and association constants of the complexes of β-CD with VMA, HVA and VA respectively were determined by using continuous variation and 1H NMR titration methods. The association constants of the inclusion complexes were calculated by using curve fitting method, which requires no approximations and allows unrestricted distribution of reagents. The data obtained from the titration experiments were fitted to a 1:1 binding model, using WinEQNMR computer program. Significant discrepancies were pointed out depending on used referencing method. In this study water solution of 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt as an external reference was used to avoid errors in the determination of association constants. Our results show that the most stable cyclodextrin inclusion complexes are formed with VA and HVA molecules whilst smallest value of association constant was determined for the VMA/β-CD complex. Established geometry of inclusion complexes based on two-dimensional rotating-frame Overhauser effect spectroscopy (2D ROESY) results shows two possibilities of arranging complexed molecules in the β-cyclodextrin cavity. It is concluded that β-CD moiety introduced into polymeric materials could be effective for vanillin and homovanillic acid entrapping.

. 141 85. DEVELOPMENT OF BIOCONJUGATES VIA SITE-SPECIFIC CONDENSATION OF HYDRAZIDES WITH N-TERMINAL ALDEHYDES

Radosław Kwapiszewski, Ilona Marszałek, Beata Chęcińska, Urszula Konopacka, Anna Molga, Albert Jaworski, Sebastian Pawlak, Michał Szymanik

Drug Discovery Department, Adamed Group, 05-152 Czosnów, Pieńków 149, Poland

Bioconjugation is a versatile technique used for biological interactions discovery, biochemical assays, diagnostic applications, in vivo imaging, or drug development. However, efficient control of bioconjugation process remains a challenge. Poor control over the modification site often results in heterogeneous derivatives generation or loss of the target biomolecule activity. The aim of our project is the development of bioconjugates as novel anti-cancer drugs. The challenges are avoidance of non-specific conjugation, and separation of the conjugated protein from non-conjugated fraction. Here we present a highly efficient and selective approach of recombinant proteins modification. A two-step bioconjugation method is based on oxidation of N-terminal serine or threonine to aldehyde, and subsequent condensation with hydrazine or hydroxylamine derivatives. The resulting bioconjugate can be further reduced with sodium borohydride to increase the linkage stability. The influence of such factors as oxidant concentration, incubation time, temperature, pH, ionic strength and type of catalyst was investigated. 80% bioconjugation yield was achieved for recombinant EGF ligand and CFTM568 hydrazide, as determined by UV-VIS spectrum and RP-HPLC analysis. The high specificity (no other bioconjugation products) was confirmed by ESI-TOF mass spectrometry. The conjugated and non-conjugated proteins were mostly separated using hydrophobic interactions liquid chromatography. The presented approach is a great alternative to traditional bioconjugation reactions involving cysteine or lysine residues modification if high specificity and efficiency are of value. Our experience shows that it can be successfully applied to drug development. Moreover, we present the utility and potential of various analytical techniques in bioconjugates analysis.

142 . 86. A NOVEL BISANTHRACYCLINE WP760 SUPPRESSES MELANOMA CELL GROWTH DUE TO INHIBITION OF TRANSCRIPTION AND P53 ACTIVATION

Magdalena Olbryt1, Aleksandra Rusin1,2, Izabela Fokt2, Anna Habryka1, Patrycja Tudrej1, Sebastian Student3, Aleksander Sochanik1, Waldemar Priebe2

1Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch Center for Translational Research and Molecular Biology of Cancer; 2Laboratory of Chemistry and Biology, MD Anderson Cancer Center, Houston, USA; 3Institute of Automatic Control, Silesian University of Technology, Akademicka 16, 44-100 Gliwice, Poland

Anthracycline antibiotics, e.g.doxorubicin or daunorubicin have well established position in chemothetharapy of many malignancies due to a broad spectrum of activity. Although the mechanism of action of anthracyclines is multifactorial, the prevailing modes of their cytotoxic effects are attributed to intercalating into DNA, interfering with topoisomerases activity and introducing double-strand breaks into DNA. However, modifications of a molecule profoundly alter their mechanism of action and spectrum of activity. The main aim of this work was to elucidate mechanism of action and indicate molecular targets of a novel bisanthracycline WP760 which demonstrates significant anti-melanoma effects in low nanomolar concentrations. In vitro model comprised a panel of cell lines derived from melanoma at different pathologic stages and with various mutation characteristics. WP760 inhibited proliferation of melanoma cells in vitro with IC50 in the 1-99 nM range, impaired clonogenic survival of cells when used at 100 nM concentration, and potently inhibited spheroid growth at 300 nM. WP760 did not induce double-strand breaks in DNA but it profoundly inhibited transcription, caused nucleolar stress and led to re-activation of p53 pathway. Additionally, molecular analysis with the use of PCR Arrays revealed that WP760 suppressed transcription of 10 genes (ABCC1, MTOR, IGF1R, EGFR, GRB2, PRKCA, PRKCE, HDAC4, TXNRD1, AKT1) and increased PLK2 expression consistently in at least 3 melanoma cell lines. To sum up, WP760 is a potent melanoma cell growth inhibitor activating p53 pathway which seems to be of great importance for its potential therapeutic application.

. 143 87. IN VITRO CYTOTOXICITY EVALUATION OF GLYCOSYLATED THIOSEMICARBAZIDES AND THEIR ANALOGS AS NEW COMPOUNDS AGAINST LEUKEMIA CELL LINE

Anna Czubatka-Bieńkowska1, Zbigniew Witczak2, Tomasz Popławski1

1Department of Molecular Genetics, University of Lodz, Lodz, 90-236, Poland; 2Department of Pharmaceutical Sciences, Nesbitt School of Pharmacy, Wilkes University, Wilkes-Barre, PA 18766, USA

Design and synthesis of new structures useful as anticancer agents is a challenge in the field of cancer treatment and organic medicinal chemistry. Several studies about potential medical applications of thiosemicarbazides and their analogs have been reported. Anticancer properties of thiosemicarbazide analogs encored us to design and synthetize new compounds from this group and evaluate their biological properties. A set of glycosylated thiosemicarbazides and their analogs (named from FCP14 to FCP22 presented in figure 1) were designed and synthesized to evaluate their biological activities as anticancer agents. Cytotoxic activity of the synthesized compounds FCP14-FCP22 was assessed against HL-60 (human acute promyelocytic leukemia) cancer cell line and normal blood lymphocytes. The cytotoxic effect of studied FCPs was determined by using the cell viability assay CCK-8.

Fig. 1. Tested thiosemicarbazides and their analogs.

All tested compounds decreased HL-60 cell viability with IC50 value ranging from 30 μM (FCP16A) to 441 μM (FCP22) following 72 h of incubation with compounds. Unfortunately they had also significant cytotoxic effects against normal blood lymphocytes with IC50 values ranging from 57 μM to 207 μM after 72 h of FCPs incubation. These data clearly suggest that FCP16A has anticancer potential. However, its mechanism of anticancer action must be still (and will be) evaluated. This would help us to modify its structure to increase its selectivity toward cancer cells.

Acknowledgements: This work was supported by the The National Science Centre, decision UMO- 2014/15/N/NZ7/02949

144 . 88. NEW DERIVATIVES OF 3,4-DICHLORO-5-HYDROXY-2(5H)-FURANONE AS COMPOUNDS WITH PROAPOPTOTIC PROPERTIES

Anna Byczek-Wyrostek1,2, Klaudia Rumak1, Anna Kasprzycka1,2, Krzysztof Walczak1,2

1Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Faculty of Chemistry, Silesian University of Technology, 44-100 Gliwice, Krzywoustego 4, Poland; 2Centre of Biotechnology, Silesian University of Technology, Krzywoustego 8, 44-100 Gliwice, Poland

Background: The 2(5H)-furanone moiety occurs in many natural products exhibiting various biological activities, namely antibiotic, cytotoxic and antitumor. Vitamin C is one of the most known antioxidants containing a 2(5H)-furanone ring in its structure. Modifications of parent compound leads to several bioactive derivatives. Thus, 5-butoxy-3,4-dichloro-2(5H)-furanone is cytotoxic at 3 mM concentration against MAC 13 and MAC 16 murine colon cancer cell lines. Further improvements of the leading structure brought to 3,4-dichloro-5-(oxiran-2-ylmethoxy)-2(5H)-furanone derivative showing cytotoxicity in nanomolar range on the same cancer cell lines. A broad antibiotic activity against Staphylococcus aureus ATCC 25923 and other bacteria strains has been observed in the case of 4- amino-5- hydroxy-2(5H)- furanones. Another one, 3-aryl-4-alkylamino-2(5H)-furanone has antifungal properties. It has also been proven that derivatives of 3-alkanoilo-5-hydroxymethyl-tetronic acid cause inhibition of the enzyme which catalyzes the last stage of the replication cycle of the HIV-1 virus. It has been reported that 2(5H)-furanone itself has anticancer properties. Materials and methods: An array of 5-alkoxy derivatives of 3,4-dichloromuco acid has been obtained and subjected to modification in the C-4 position of 2(5H)-furanone ring. The series of 4- aminoaryl derivatives has been obtained in the reaction of nucleophilic substitution of the chlorine atom by molecule of an aromatic amine. The cytotoxicity of obtained compounds was checked by MTT assay, and the impact of the derivatives on the cell cycle was determined by flow cytometry and analysis of microscopic slides. Genotoxic properties were tested through micronucleus test, comet assay test and clonogenicity. Results: The modification of compounds in the C-4 position of 2(5H)-furanone ring did not improve their antiproliferative properties. The derivatives which contained a branched alkoxy substitute in the C-5 position have shown the best anticancer properties. These compounds have caused a strong block of cell cycle in the G2 phase and growth in number of apoptotic cells.

. 145 89. STABILITY STUDIES OF TRAIL-DERIVED ANTICANCER BIOMOLECULE AD-O51.4 AS AN IMPORTANT STAGE OF PRECLINICAL EVALUATION OF NEW DRUG CANDIDATE

Anna Molga, Bartłomiej Żerek, Sebastian Pawlak, Piotr Rózga, Katarzyna Poleszak, Marlena Gałązka, Katarzyna Bukato, Małgorzata Teska-Kamińska, Albert Jaworski, Michał Szymanik

Drug Discovery Department, Adamed Group, Poland

The Apo2L/TRAIL (tumour necrosis factor-related apoptosis-inducing ligand) exhibits significant ability to induce apoptosis in cancer cells but has no toxic effect on normal cells. The efficacy of TRAIL was however insufficient to become a remarkable single therapy agent. For last few years, during the ONCO-3CLA project, over four hundred of TRAIL-based recombinant proteins and their variants were developed. We worked to improve TRAIL effectiveness by its fusion with various effectors. One of them – AD-O51.4, was chosen as the most promising anticancer agent. The AD-O51.4 molecule is a variant of TRAIL, to which doubled effector peptide derived from a vascular endothelial growth factor (VEGF) ligand was added. We proved that this fusion molecule binds to death and VEGF receptors and has pro-apoptotic and anti-angiogenic activity. Results of our experiments confirmed that AD-O51.4 has high anticancer potential, much higher than the potential of TRAIL molecule. This was shown on large panel of established cancer cell lines, and patient-derived cancer cells in vitro as well as in vivo on mouse xenograft models including patient-derived xenografts. After development research, AD-O51.4 enters preclinical experiments necessary before start of clinical trials. The GMP production process of AD-051.4 is currently improved by CMO partner and production of toxicology batch of this molecule will start shortly. Preclinical and clinical development of AD-051.4 together with establishment of the first in Poland center for early phase oncology clinical trials is the aim of the project that Adamed realizes together with a group of polish medical institutes within the ONCOTRAIL consortium which leader is Maria Skłodowska Curie Memorial Cancer Centre and Institute of Oncology. Within the preclinical research of AD-051.4 the most important will be toxicology studies. Before that we need however to obtain toxicology batch of the protein and define its storage conditions. We are still working then on the stable formulation for this protein. In this poster we present stability analysis of AD-051.4 at 20°C and 37°C in two formulations including evaluation of oligomeric state of the molecule in different incubation times using size exclusion chromatography and protein aggregation assay.

146 . 90. SYNTHESIS AND IN VITRO ANTIPROLIFERATIVE ACTIVITY OF NOVEL PHENYL RING- SUBSTITUTED TETRACYCLIC PHENOTHIAZINE DERIVATIVES

Anna Nycz1, Małgorzata Latocha2, Aleksander Sochanik3, Andrzej Zięba1

1Department of Organic Chemistry; 2Department of Cell Biology, School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia in Katowice, Jagiellońska 4, 41-200 Sosnowiec, Poland; 3Center for Translational Research and Molecular Biology of Cancer, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Wybrzeże AK 15, 44-101 Gliwice, Poland

Phenothiazine is a prototypical pharmaceutical lead structure in modern medicinal chemistry. Structural modifications of phenothiazine and azaphenothiazine have been achieved by introducing substituents and functional moieties mainly at the thiazine nitrogen or, less often, into benzene ring or nitrogen-containing heterocycles. The compounds reported so far exhibit neuroleptic, antimalarial, immunopotentiating, antibacterial, antiviral, antifungal, anti-proliferative or antitumor activities and can mediate reversal of multidrug resistance. A new method of 1,4-thiazine system synthesis was developed by us, consisting of hydrogen atom substitution in the phenyl ring by thiolate sulfur atom. The mechanism of cyclization reaction was studied. Using this new method several novel tetracyclic phenothiazine derivatives were synthesized that contain a quinoline moiety [1-2]. The structure of particular compounds was modified by introducing several different substituents or functional groups into the quinobenzothiazine system [3]. 1 R : Y N H N (CH ) N 2 n R1 _ Y Y (CH ) N Cl 2 n X S + R Y = CH, N, O 1 X = CH, N Anti-proliferative effect of the synthesized compounds was studied using cultured neoplastic cell lines (MDA-MB-231, SNB-19, and C-32). Four most promising compounds were selected and investigated in more detail in terms of cytotoxicity and effect upon transcriptional activity of genes regulating cell cycle (TP53), apoptosis (BAX, BCL-2) and proliferation (H3). Finally, ability of the selected compounds to bind DNA was checked in the presence of ethidium bromide [3]. More advanced biological studies are underway.

References: 1. Zięba A, Sochanik A, Szurko A, Rams M, Mrozek A, Cmoch P (2010): EurJMedChem 45: 4733. 2. Zięba A, Latocha M, Sochanik A (2013): MedChemRes 22: 4158. 3. Zięba A, Latocha M, Sochanik A, Nycz A, Kuśmierz D (2016): Molecules (in press).

Acknowledgements: This study has been supported by the Medical University of Silesia in Katowice, Poland (Grant No. KNW-006-/K/6/O).

. 147 91. SYNTHESIS OF 8-HYDROXYQUINOLINE GLYCOCONJUGATES AND PRELIMINARY EVALUATION OF THEIR BIOLOGICAL ACTIVITY

Monika Krawczyk1, Karolina Górecka1, Gabriela Pastuch-Gawołek1,2, Ilona Wandzik1,2, Urszula Nawrot3

1Department of Organic Chem. Bioorganic Chem. and Biotechnol., Silesian University of Technology, 44-100 Gliwice, ul. Krzywoustego 4, Poland; 2Biotechnology Centre, The Silesian University of Technology, Krzywoustego 8, 44-100 Gliwice, Poland; 3Department of Pharmaceutical Microbiology and Parasitology, Wroclaw Medical University, Borowska 211A, 50-368 Wrocław, Poland

8-Hydroxyquinoline (OHQ) scaffold can be found in many biologically active compounds used as anticancer, antimicrobial, antifungals, as well as antituberculotic agents [1-3]. Furthermore, derivatives of OHQ have recently met with interest as therapeutic agents for Alzheimer’s disease [4]. However, structures designed on the core of OHQ suffers from poor bioavailability/membrane transport. Several reports indicate that glycoconjugation of 8-hydroxyquinoline derivatives could be a powerful strategy for improving of their solubility as well as biological activities [5-7]. This prompted us to elaborate a simple and efficient way to synthesize of OHQ glycosides. In our experiments 8-hydroxyquinoline and 2-methyl-8-hydroxyquinoline was used as glycosyl acceptor in glycosylation with a per-O-acetylated glucopyranosyl or galactopyranosyl bromides. Previously described glycosylation method, performed in the classical two-phase system using K2CO3 as a base and TBABr as phase transfer catalyst, was compared to the other phenols glycosylation methods [8- 9]. The obtained quinoline glycosides were tested for their antifungal activity, as well as for their ability to inhibit β-1,4-Galactosyltransferase. The results of the initial assessment of the biological activity of these compounds will be presented.

References: 1. Darby CM, Nathan CF (2010): J Antimicrob Chemother 65: 1424-1427. 2. Jiang H, Taggart J, Zhang X, Benbrook DM, Lind SE, Ding W-Q (2011): CancerLett 312: 11-17. 3. Hongmanee P, Rukseree K, Buabut B, Somsri B, Palittapongarnpim P (2007): AntimicrobAgentsChemother 51: 1105–1106. 4. Hung LWH, Barnham JK (2012): FutureMedChem 4: 955-969. 5. Oliveri V, Giuffrida ML, Vecchio G, Aiello C, Viale M (2012): DaltonTranslation 41: 4530-4535. 6. Oliveri V, Viale M, Caron G, Aiello C, Gangemi R, Vecchio G (2013): DaltonTranslation 42: 2023-2034. 7. Oliveri V, Viale M, Aiello C, Vecchio G (2015): JInorgBiochem 142: 101-108. 8. Talisman IJ, Kumar V, Razzaghy J, Malhotra SV (2011): CarbohydrRes 346: 883-890. 9. Kur’yanov VO, Priskoka US, Chupakhina TA, Chirva VYa (2005): RusJBioorgChem 31: 300-301.

148 . 92. THIODISACCHARIDES AS ANTICANCER AGENTS IN ASTROCYTOMA TREATMENT

Joanna Sarnik1, Monika Toma1, Tomasz Śliwiński1, Zbigniew J. Witczak2, Tomasz Popławski1

1Department of Molecular Genetics, University of Lodz, Lodz 90-236, Poland; 2Department of Pharmaceutical Sciences, Nesbitt School of Pharmacy, Wilkes University, Wilkes Barre, Pennsylvania, United States

Astrocytomas are graded from I to IV, with grade I and II being slow growing astrocytomas, grade III - anaplastic astrocytomas and grade IV being glioblastoma multiforme (GBM) – the most common (65%) and most malignant form of brain tumors. The prognosis of GBM patients is very poor, largely owing to early invasion into the central nervous system, making a surgical cure nearly impossible. Novel treatment modalities, such as concomitant radiotherapy and chemotherapy, are now considered standard for GBM treatment. Despite recent advances in cancer biology, treatment outcomes have not changed significantly over the past decade and high-grade gliomas remain fatal only 10% of those patients, including all post-treatment living conditions, survive five years after diagnosis. GBMs are highly drug-resistant, limiting the effectiveness of chemotherapy. In this study we tested carbohydrate-drugs with a modification in the form of sulfur atom presence (thiosugars). Thiosugars are biologically active with many important effects pharmacologically distinctive from their oxygen analogs. They possess important biological properties including resistance to carbohydrate degrading enzymes and enzyme inhibitory activity. Recent evidence suggests that these carbohydrate analogs may have the therapeutic potential in the treatment of cancer and infectious diseases. Tested carbohydrate derivatives is a group of thiodisaccharides denoted as FCP6 (1,6- anhydro-3-deoxy-4-S-(2,3,4,6-tetra-Oacetyl-β-D-glucopyranosyl)-4-thio-β-D-erythro-hexopyranos-2- ulose); FCP7 (1,6-anhydro-3-deoxy-4-S-(β-Dglucopyranosyl) -4-thio-β-D-erythro-hexopyranos-2- ulose); FCP8 (1,6-anhydro-3-deoxy-4-S-(salicylyl)-4-thio-β-D-erythrohexopyranos-2-ulose); FCP9 (1,6-anhydro-3-deoxy-4-S-(β-D-galactopyranosyl)-4-thio-β-D-erythro-hexopyranos-2-ulose). The chosen cancer cell lines represents astrocytoma cells at GIV stage of cancer development. We used well described HTB-14 cell line and cell line which was newly established from patient with glioblastoma. The aim of this research was to determine the cell viability after treatment with thiodisaccharides. To achieve the results we used colorimetric method based on dehydrogenase activity (CCK-8) and trypan blue exclusion test of cell viability. We observed decrease in cell viability in micromolar concentration of FCP. The most active compounds were FCP6 and FCP9 (respectively IC50= 135.2 µM and IC50= 97 µM for cell line newly established from patient). Experimental result conducted on HTB-14 cell line confirmed our observations on anticancer activity of FCPs. The rResults suggest that these new functionalized carbohydrates are promising therapeutic agents in the treatment of astrocytomas.

Acknowledgements: This work was supported by the National Science Center decision no. DEC- 2014/15/N/NZ7/02948

. 149 93. NEW PORPHYRIN-TYPE PHOTOSENSITIZER FOR PHOTODYNAMIC THERAPY; PHOTOPHYSICAL AND ANTICANCER PROPERTIES

Sandra Michalak1, Marlena Golec1, Agnieszka Szurko1, Alicja Ratuszna1, Dorota Zygadło1, Piotr Kuś2

1Department of Solid State Physics, August Chełkowski Institute of Physics, University of Silesia, Silesian Center for Education and Interdisciplinary Research, 75 Pułku Piechoty 1A, 41-500 Chorzów, Poland; 2Department of Organic Synthesis, Institute of Chemistry, University of Silesia, 40-003 Katowice ul. Szkolna 9, Poland

The photodynamic therapy (PDT) is an approved treatment method for certain types of cancer, precancerous conditions and certain non-malignant diseases. This method makes use of the light- sensitive chemical compounds called photosensitizers, which are able to penetrate to pathological tissue and be excited by radiation of a specific wavelength causing destruction of this tissue. In this context, physical-chemical properties and biological activity of a new porphyrin derivative of were characterized. Measurements of the absorption and emission spectra of the tested compound in distilled water and in chloroform were performed and fluorescence quantum yield and the quantum yield of singlet oxygen generation were investigated. In biological studies the kinetics of porphyrin penetration into the HCT116 cells, as well as the toxicity and phototoxicity were determined with MTS assay. Based on the studies of porphyrin penetration kinetics, it has been determined that this compound accumulates with the maximum concentration insideof HCT116 cells after 12 hours of incubation. The phototoxicity and cytotoxicity studies of the new tested porphyrin derivate of have shown a high therapeutic effect in the absence of toxicity. The measured quantum yield of singlet oxygen generation for the tested porphyrin was 27.5%. As part of further research the second mechanism for generating oxygen, the so-called free radical mechanism will be checked. The results are very promising and show that the compound could potentially be applied, especially in the diagnosis (PDD).

References: 1. Debele TA, Peng S, Tsai H (2015): Drug Carrier for Photodynamic Cancer Therapy. IntJMolSci 16: 22094-22136. 2. Bacellar IO, Tsubone T, Pavani C, Baptista M (2015): Photodynamic Efficiency: From Molecular Photochemistry to Cell Death. IntJMolSci 16: 20523-20559.

150 . 94. NOVEL GALLIUM PHTHALOCYANINE AS POTENTIAL PRO-APOPTOTIC DRUG AGAINST MELANOMA ME45 CANCER CELLS

Magdalena Skonieczna1,2, Marta Kliber-Jasik3, Joanna Nackiewicz3

1Biosystems Group, Institute of Automatic Control, Silesian University of Technology, Akademicka 16, 44-100 Gliwice, Poland; 2Biotechnology Centre, Silesian University of Technology, 44-100 Gliwice, ul. Krzywoustego 8, Poland; 3 Faculty of Chemistry, University of Opole, Oleska 48, Opole 45-052, Poland

Metallophthalocyanines (MPcs) are well known synthetic porphyrin analogs, which have been used in a wide range of different applications due to their specific physicochemical properties [1, 2]. In recent years MPcs have been intensively studied, as highly promising second-generation photosensitizers for photodynamic therapy (PDT) [3]. This is due to their strong absorption in the phototherapeutic window 600–900nm, low dark toxicity, easy modification, high phototoxicity and high efficiency of generating reactive oxygen species (ROS) [4]. The subject of our research is gallium phthalocyanine, which has eight carboxylic groups (fig. 1). The compound is well-soluble in water which is advantageous from the viewpoint of PDT. Gallium phthalocyanine is particularly interesting, because this molecule has a high triplet quantum yield what ensures high cytotoxicity against tumours.

Fig. 1. Structure of octacarboxyphthalocyanine, M–Ga.

In our studies we have tested pro-apoptotic activity of gallium phthalocyanine (M-Ga) and we have found, that at 30 µM concentration of and after irradiation the compound induced early apoptosis, followed by Anexin-V apoptosis assay. The pro-apoptotic activity was tested against three cell lines in vitro, normal human keratinocytes HaCaT, fibroblasts NHDF and cancer melanoma Me45. The obtained results show visible selectivity and the highest apoptotic cell population was observed in Me45 cells. These findings correlate also with the most effective ROS production in Me45 after M-Ga and IRF exposure. We believe, the novel M-Ga is a good photosensitizer for photodynamic therapy against melanoma, with low toxicity against normal cells.

References: 1. Gottfried JM (2015): SurfSciRep 70: 259–379. 2. Gregory P (2000): JPorphyrinsPhthalocyanines 4: 432–437. 3. Ҫakɪr V et al (2015): JOrganometChem 783: 120–129. 4. Kɪrbaҫ E et al (2014): JOrganometChem 752: 115–122. 5. Karaoğlan GK et al (2011): DyesPigm 88: 247–256.

Acknowledgements: This project was funded by the grant SUT BK-277/Rau1/2015 t.3 from the Silesian University of Technology in Gliwice, Poland (M.S.).

. 151 95. SILSESQUIOXANES- A NEW APPROACH IN DRUG DELIVERY

Ewelina Sobierajska, Małgorzata Konopka

Department of General Biophysics, Faculty of Biology and Environmental Protection University of Lodz, 141/143 Pomorska str., 90-236 Lodz, Poland

Nowadays, drug delivery system is increasingly gaining in importance, particularly in terms of cancer treatment. Numerous nanoparticles are being studied as carriers of drugs, including polymers, dendrimers or liposomes. However, there are still major problems concerning side effects, poor effectiveness and high costs. Therefore, searching for extraordinary solutions is indispensable. Recently, polyhedral oligomeric silsesquioxane (POSS) has attracted significant interest in the biological field owing to the unique structure, nanoscale size (diameters in the range of 1-3 nm) and easy chemical modification. POSS are nanocages consisting of an inorganic silicon and oxygen inner core, while the outer shell is made up of organic functional groups The aim of our study was to verify the effectiveness of POSS as carriers of doxorubicin. In order to do that we undertook a toxicity evaluation of POSS-DOX conjugates in four different proportions (1:1; 1:2, 1:4, 1:8) towards three cell lines: microvascular endothelial cells (HMEC-1), breast cancer cells (MCF-7) and human cervical cancer endothelial cells (HeLa). Moreover, we evaluated the rate of penetration of the compounds into the cells. The results show that all three cell lines were more sensitive to POSS-DOX conjugates at concentrations from 1 to 100 µM than free doxorubicin. Moreover, the intracellular uptake of conjugates was much faster when compared to free drug. A cytotoxicity and uptake profile of silsesquioxanes makes them promising platforms for cell- specific targeting.

152 . 96. ANTICANCER POTENTIAL OF NEW XANTHONE DERIVATIVES IN COLON AND OVARIAN CANCER CELL LINES

Daniel Sypniewski1, Paulina Borzdziłowska1, Magdalena Połowczuk1, Natalia Szkaradek2, Agnieszka Gunia-Krzyżak2, Henryk Marona2, Ilona Bednarek1

1School of Pharmacy with the Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Dept. of Biotechnology and Genetic Engineering, ul. Jedności 8, 41-200 Sosnowiec, Poland; 2Dept. of Bioorganic Chemistry, Collegium Medicum of the Jagiellonian University, 30-688 Kraków, ul. Medyczna 9, Poland

Colon cancer and ovarian cancer are some of the most common causes of cancer death in the developed countries posing a serious threat to public health. Plants of the family Clusiaceae Lindl. are the source of pharmacologically active compounds termed xanthones. Both natural xanthones and their synthetic derivatives display valuable anticancer features The most studied natural xanthones are α-mangostin and gambogic acid. Gambogic acid has been subjected to clinical trials in cancer patients. The aim of the study was to estimate the anticancer potential of newly synthesized xanthone derivatives toward the selected colon and ovarian cancer cell lines. The following cell lines were used: Caco2, HT-29 (colorectal adenocarcinoma), A2780, and OVP10 (ovarian carcinoma). The study included four synthetic derivatives (compounds marked as 1, 2, 3, and 4) synthesized at the Dept. of Bioorganic Chemistry of the Jagiellonian University (Kraków), and two natural derivatives (α- mangostin, gambogic acid). Their influence on cancer cells was established and compared to reference drugs commonly used in the chemotherapy of colon cancer (cisplatin, 5-fluorouracil) and ovarian cancer (cisplatin and etoposide). XTT test was used to determine IC50 values. The cytotoxic effect of xanthones was evaluated by LDH release test, and cell proliferation was analyzed microscopically using the test Click-iT® EdU Imaging Kits (Invitrogen). The results demonstrate that IC50 values for all the analyzed xanthone derivatives were within a micromolar range indicating potential anticancer efficiency of these compounds toward the studied cancer types. In ovarian cancer IC50 values of the tested compounds were significantly lower in A2780 than OVP10 cell line, but in both cell lines the highest value of IC50 was observed for the compound 3 (IC50 for OVP10: 67.61 μM; for A2780: 36.31 μM). In HT-29 cell line IC50 values of all synthetic xanthones were significantly higher than the IC50 values for natural compounds: IC50 for α- mangostin and gambogic acid were respectively 7.08 μM and 10.47 μM; IC50 for synthetic derivatives were between 51.29 μM and 63.10 μM. There was a correlation between the results obtained using the XTT and LDH assays for all the cell lines tested (r=0.633, p<0.0001). Analysis of cell proliferation further supported those results indicating that the most active compounds (gambogic acid, α- mangostin, and compound 3) efficiently decreased proliferation in the studied cell cultures.

Acknowledgements: The study was supported by Medical University of Silesia funding (KNW-1- 028/N/5/0, KNW-1-043/N/6/B).

. 153 97. EVALUATION OF 3,3',4,4'-BENZOPHENONETETRACARBOXYLIC DIPHTHALIMIDE DERIVATIVES AS HEXOKINASE II INHIBITORS

Mateusz D. Tomczyk1, Krzysztof Kochel2, Rui F. Simoes3, Tomasz Fraczek4, Adrian Sobon5, Paulo J. Oliveira4, Krzysztof Z. Walczak1, Aneta Koceva-Chyla2

1Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Faculty of Chemistry, Silesian University of Technology, Krzywoustego 4, 44-100 Gliwice, Poland; 2Department of Medical Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland; 3CNC – Center for Neuroscience and Cell Biology, University of Coimbra, UC- Biotech, Biocant Park, 3060-197 Cantanhede, Portugal; 4Institute of Applied Radiation Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland; 5Department of Industrial Microbiology and Biotechnology, Institute of Microbiology, Biotechnology and Immunology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland Contact: [email protected]

Hexokinase II (HKII) an isozyme of phosphorylase is responsible for phosphorylation of terminal 6-hydroxyl group in glucose, the process which begins the aerobic glycolysis in many cells. HKII plays an important role in the fast proliferating malignant tumor cells and can be involve in an inhibition of an apoptosis process. This points out that interference with mitochondrial-bound HKII might represent a potential target for cancer therapy. In the present study we used the P19 embryonal carcinoma cell line as a biological target for novel inhibitors of HKII elaborated by us. In the first stage of our investigation the docking study with Glide and Virtual High Throughput Screening (VHTS) was performed to open conformation of HKII. Inspection of the results led us to design and synthesize some new derivatives containing a 3,3',4,4'- benzophenonetetracarboxylic dianhydride (BTDA) core. All the obtained derivatives were then tested as HKII inhibitors and studied for their biological properties: changes in cellular protein content (SRB), mitochondrial membrane potential (MMP) and level of mitochondrial superoxide anion. The results show that one of the obtained compounds inhibits activity of HKII by 50% at a 500 times lower concentration (100 µM) than 2-deoxyglucose (50 mM) best known as an inhibitor of glucose metabolism. This derivative could be considered as a starting point in the rational design for novel, potent HKII inhibitors.

154 . 98. DIFFERENTIAL NOTCH SIGNALING AS PREDICTIVE VALUES OF RENAL CLEAR CARCINOMA SUBTYPES

Dorota Jędroszka, Magdalena Orzechowska, Izabela Baryła, Katarzyna Kośla, Magdalena Nowakowska, Elżbieta Płuciennik, Karolina Pospiech, Ewa Styczeń-Binkowska, Andrzej K. Bednarek1

1Department of Molecular Carcinogenesis, Medical University of Łódź, 90-643 Łódź, ul. Żeligowskiego 7/9, Poland

Kidney cancer is one of the most lethal malignancies due to late-stage diagnosis and poor treatment options. The most common type of kidney cancer is renal cell carcinoma (RCC) with the two subtypes called clear cell carcinoma and papillary carcinoma. Notch signalling pathway is a critical regulator of tissues differentiation and organs development, including kidney by establishing proximal tubular epithelial cell fate and cell type specification in the renal collecting system. Although aberrant Notch is associated with several human malignancies, the prognostic value of individual Notch pathway members in RCC subtypes remains indefinable. In the present study, we aimed to assess whether differential expression of Notch members has contrary effect on disease-free survival (DFS) in clear renal cell carcinoma (KIRC) and papillary cell renal cell carcinoma (KIRP). We analysed data of 533 KIRC and 289 KIRP cohorts from TCGA (RNAseqV2, RSEM normalized) and determined the relevance of 19 Notch members differential expression on tumour recurrence. DFS analysis was performed using freely available Cutoff Finder (log-rank test, p<0.001). To integrate the relationship between the groups of variables (recurrence, stage, genes expression) describing the individuals (patients) we applied Multiple Factor Analysis (MFA). The analysis was perform with R and the packages FactoMineR and Factoextra. The result show that most of Notch members, including NOTCH1, 3 and 4 receptors, exhibit the same effect and their lowered expression is correlated with favourable prognosis both in KIRP and KIRC. An exception are APH1B whose high expression is favourable for KIRC and KIRP and also PSEN1 whose high expression means good prognosis. Moreover, two other Notch members, NUMB and PSEN2 showed contrary Notch profiles across renal carcinoma subtypes. MFA analysis confirmed that in most cases of partition of variables, the higher stage (III and IV) are assigned to patients with recurrence, whereas lower stage to patients with no recurrence. Our results suggest that expression profiles of Notch pathway members have significant impact on DFS in renal cell carcinoma subtypes and that expression of particular genes may serve as prediction factor of relapse of the disease in patients with KIRC and KIRP.

. 155 99. NOTCH SIGNALLING HAS PREDICTIVE POTENTIAL THAT CORRELATES WITH RECURRENCE AND STAGE OF LUNG CANCERS

Magdalena Orzechowska, Dorota Jędroszka, Elżbieta Płuciennik, Katarzyna Kośla, Magdalena Nowakowska, Izabela Baryła, Karolina Pospiech, Ewa Styczeń-Binkowska, Andrzej K. Bednarek

Department of Molecular Carcinogenesis, Medical University of Lodz, 90-752 Łódź, ul. Żeligowskiego 7/9, Poland

Lung malignancies are lethal and aggressive tumours remaining a leading cancer-related death cause worldwide with lung squamous cell carcinoma (LUSC) and adenocarcinoma (LUAD) accounting for majority of cases. Notch signalling is evolutionarily-conserved pathway regulating essential cellular processes, however the association between disease recurrence and Notch signalling in lung cancer remains unclear. Therefore, the aim of our study was to investigate associations of Notch differential expression with disease – free survival (DFS) in LUSC and LUAD. We assessed the role of 19 Notch members in LUAD and LUSC cohorts from TCGA. Results in the form of Kaplan – Meier plots (log – rank test, p<0.05) enabled us to split patients into favourable/unfavourable prognosis groups regarding expression of Notch genes. For additional grouping of patients according to a considered set of variables (recurrence, stage, Notch genes) we employed Multiple Factor Analysis (MFA) provided in the form of FactoMineR R package. Our results show that expression of particular Notch member is significantly correlated with specific subtype of lung carcinoma. In particular, lowered expression of NOTCH4, DLL4, JAG1 and HES5 is correlated with good prognosis in LUAD and LUSC. MFA resulted in distinct grouping of LUAD and LUSC patients dependently on disease recurrence that correlated with stage (Ia - IIb with relapse free and III - VI with relapse) of lung cancer. Our analysis could be valuable for better understanding of the molecular mechanism of lung carcinoma as well as Notch signalling in lung cancer with emphasis on pathway gene expression as a useful biomarker for predicting favourable/unfavourable recurrence prognosis in patients with LUAD and LUSC.

156 . 100. USE OF MESENCHYMAL STEM CELLS AS CARRIERS OF ONCOLYTIC MYXOMA VIRUS FOR MELANOMA THERAPY

Joanna Jazowiecka-Rakus1, Aleksandra Rusin1, Aleksander Sochanik1, Iwona Mitrus2, Wojciech Fidyk2, Grant McFadden3

1Center for Translational Research and Molecular Biology of Cancer, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Branch Gliwice, Poland; 2Department of Bone Marrow Transplantation and Oncohematology, Cancer Center and Institute of Oncology, Branch Gliwice, Poland; 3Deptartment of Molecular Genetics and Microbiology, College of Medicine, University of Florida Gainesville, FL (USA)

Early efforts to employ viruses for eliminating neoplastic tumors were unsuccessful due to low specificity of such viruses and high risk associated with treatment. In recent years oncolytic virotherapy has enjoyed renewed interest as biotechnology developments have made feasible vironcolytic treatment of poorly accessible/aggressive neoplasms. Systemic (intravenous) administration of oncolytic viruses alone elicits production of antibodies specific towards them and mobilizes the phagocytic system (MPS) leading to virus sequestration. Thus, effective delivery of oncolytic viruses into neoplastic foci would require shielding of viruses during systemic transit, e.g. by providing a protective carrier. Among the promising candidates are mesenchymal stem cells (MSC). They can be preloaded with virus ex vivo, delivered iv. to the recipient, migrate to the tumor site and release the oncolytic cargo. MSC cells show low immunogenicity (low expression of MHC I and II). Due to release of prostaglandins and interleukins, they can regulate immune response by forming a tolerogenic milieu, inhibiting activity of dendritic cells (DC), natural killer (NK), CD8+ and CD4+ cells. We have tried to implement oncolytic therapy with myxoma virus (MYXV), a representative of poxviruses. MYXV is not pathogenic to humans and other vertebrates (except rabbits). This virus has great therapeutic potential as it shows tropism to various types of cancer cells, selectively infecting and replicating within them, which leads to these cells’ rupture. This should lead in vivo to elimination (oncolysis) of tumor mass, without adverse effects for adjacent tissues. We evaluated applicability of MSC carrier cells for shielding the transiting MYXV cargo in anticipation of experimental metastatic melanoma therapy. Methods: MSC cells were obtained from healthy donor bone marrow. Following MSCs isolation, their phenotype was confirmed using Human MSC Analysis Kit and flow cytometry. vMyx- GFP was produced in RK13 rabbit cells and used to infect murine (B16-F10) and human (451Lu, WM35, WM793B) melanoma, as well as MSC cells. B16-F10 and WM793B cells stably expressing mRFP were used (retrovirus-based pLNCX2 system derived from MLV). MYXV infection was monitored by fluorescence microscopy, virus titering and flow cytometry; cell viability was measured by Alamar Blue assay. Results: The adopted method of isolating MSC cells allowed derivation of cell types capable of differentiating into adipocytes or osteocytes; phenotype of isolated has shown them as suitable for further experiments. Our data confirmed that the murine melanoma and the three human melanoma cell lines used as well as MSC cells are permissive to MYXV infection. We thus examined the effect of MYXV infection on the viability of MSCs, B16-F10 and RK13 cells (the latter used for virus propagation). We observed ~98% MSC cell survival at MOI=10 as compared to uninfected MSC cells (MOI=0). Murine and human melanoma cell lines, as well as rabbit RK13 cells were susceptible to MYXV-mediated killing. We also observed that MYXV-infected MSCs successfully cross-infected murine and human melanoma cells after in vitro co-culturing. Morphology of nuclei in infected melanoma cells hints to apoptotic death with consistent condensation seen. Conclusion: These results suggest that carrier MSC cells (“Trojan horse”) might be indeed suitable for attempts to improve MYXV-mediated oncolysis of metastatic melanoma tumors.

. 157 101. 3D CULTURE IMPACT TYROSINE KINASE INHIBITORS ACTIVITY AGAINST RENAL CELL CANCER CANCER STEM CELLS

Anna M. Czarnecka1, Zofia F. Bielecka1,2, Klaudia K. Brodaczewska1, Kamila Maliszewska1, Agata Malinowska3, Paweł Krasowski1,4, Wojciech Solarek1,2, Anna Kornakiewicz1,2, Elżbieta Grzesiuk4, Jan Piwowarski4, Cezary Szczylik1

1Military Institute of Medicine, Department of Oncology with Laboratory of Molecular Oncology, Szaserów 128, 04-141 Warsaw, Poland; 2Warsaw Medical University, School of Molecular Medicine, Księcia Trojdena 2a, 02-091 Warsaw, Poland; 3Polish Academy of Sciences, Institute of Biochemistry and Biophysics, Environmental Laboratory of Mass Spectrometry, Pawińskiego 5a, 02-106 Warsaw, Poland; 4Polish Academy of Sciences, Institute of Biochemistry and Biophysics, Department of Molecular Biology, Pawińskiego 5a, 02-106 Warsaw, Poland

Renal cancers account for 2 to 3% malignancies in adult population and 80% of primary renal cancers are renal cell carcinomas (RCCs). RCC is currently treated with tyrosine kinase inhibitor (TKIs). The clinical and biological anti-cancer effects produced by tyrosine kinase inhibitors in RCC are the result of their inhibitory activities on a variety of cell receptors on cancer cells, endothelial cells, pericytes, and stromal cells in 3D tumor environment. The targeted effects of TKIs are dependent on the inhibition of downstream mediators up-regulated in response to molecular abnormalities (i.e. VHL, c-MET) in RCC. This goal of our study was to verify the toxicity of TKI against clear cell and papillary renal cell cancer cells along and RCC cancer stem cells in 3D cell culture model. Multiple cell lines used in the study included: primary cancer cell lines 769-P and 786-0, and metastasis derived cell lines including: Caki-1 – derived form metastasis to the skin, ACHN – pleural metastasis or SK-RT-42 – from bone metastasis; and Human Kidney Cancer Stem Cells (HKCSC) isolated from nephrectomy specimen of papillary and clear cell renal cell carcinoma. Control conditions included RMPI/FBS 2D monolayer culture and in order to mimic intratumoral conditions cells were cultured in 3D spheres. Moreover 3D structures were obtained on laminin, collagen I and/or poly-D-lysine matrix and in hypoxia as in vivo. RCC cultures were treated with TKIs - sunitinib, sorafenib or axitinib and proliferation was evaluated. It was shown that RCC cells and HKCSC are direct targets of TKI. Low oxygen availability further decreases proliferation of RCC cells, but may promote HKCSCs. Growth in 3D structures (spheres) promotes changes in expression of stem-like phenotype related genes including Sox-2, Oct-4, E-cadh, N-cadh, CD105 or CD133. Moreover, in hypoxia downregulation of expression of eukaryotic translation initiation factor B (encoded EIF4B gene) and dual specificity mitogen-activated protein kinase 1 (encoded by MAP2K1 gene) found. In summary, anti-angiogenic agents generate intratumoral hypoxia, modulating disease progression and metastatic process and stimulating cancer stem cells (CSC) phenotype. Currently, determining the most effective application of TKIs clinically in RCC treatment is imperative to investigate the mechanisms underlying their efficacy.

Acknowledgments: This research was supported by LIDER/031/625/L-4/NCBR/2013 grant from the National Centre for Research and Development in Poland (NCBiR).

158 . 102. PHYSICAL PROPERTIES OF LYMPHOID CELL LYMPHOMA WITH HIGH B CELLS LOCATED IN LYMPH NODE OR PRIMARY AND SECONDARY EXTRANODAL LYMPHOMA

Dorota Zygadło1, Agnieszka Szurko1, Karolina Bukowska-Strakova2,3, Alicja Józkowicz2, Katarzyna Balin1, Jacek Szade1, Wojtek Spychałowicz†4, Alicja Ratuszna*1

1August Chełkowski Institute of Physics, University of Silesia, Uniwersytecka 4, 40-007 Katowice, Silesia Center for Education and Interdisciplinary Research, 75 Pułku Piechoty 1A, 41-500 Chorzów, Poland; 2Department of Medical Biotechnology, Faculty Biochemistry Biophysics & Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland; 3Department of Clinical Immunology, Institute Pediatric, College Medical, Jagiellonian University, 30-688 Krakow, Poland; 4Department Internal Medicine and Oncology, Silesian Medical University, Medyków 18, 40-752 Katowice, Poland *mail: [email protected] †mail:[email protected]

Non-Hodgkin lymphoma of the large B-cell lymphoma (DLBCL) can be divided into nodal and extranodal lymphomas. Lymphoma most frequently develops within the structure of the lymph nodes that are part of the lymphatic system. By means of the lymphatic system lymphoma cells may spread to distant nodes. In addition, via blood system these cells can also reach other places and give secondary extranodal organ involvement. Lymphomas, which originally formed outside the lymph nodes, are called primary extranodal lymphoma (e.g. the stomach, small intestine, skin, thyroid and testes). Register analysis of DLBCL lymphoma led to the concept of dividing the above-mentioned groups. Despite a number of different clinical observations, there is no known molecular marker differentiating these subgroups. Our study aimed to search for differences in B-cell extranodal DLBCL in different locations compared to the nodal DLBCL. In addition, we wanted to answer the question whether investigated properties have an impact on the local specificity of the formation of extranodal DLBCL. Another studied phenomenon was the impact of the test model with the microenvironment and intercellular communication. The object of our research are lymphoma cells (large B cells) isolated from clinical specimens from patients with a defined subtype of lymphoma (DLBCL). Nodal lymphomas are a reference because they seem to have no specificity to the microenvironment. Another studied group was B cells isolated from patients with extranodal lymphoma, with different locations, i.e. skin, thyroid and testis. B lymphocytes isolated from the peripheral blood of individual patients were used as control cells. We present the results of setting up optimal conditions for assessing clinical material by spectroscopic methods. Cells obtained from a patient in the form of aspirate were separated with a panel of fluorescent-labeled antibodies. Sorting process takes place in a flow cytometer equipped with sorter (FACS, fluorescence activated cell sorting). Next, the isolated population of B lymphoma cells were prepared for the measurement techniques such as TOF-SIMS, Confocal Raman Spectroscopy and AFM. These studies are aimed at the search for differences in physical properties of our research model. Raman Spectroscopy allows for the measurement of vibrational spectra of molecules giving the information about composition of the substance, interactions of molecules and their organization. The secondary ion mass spectrometry (TOF-SIMS) gives information about chemical composition and map of the distribution of particular ions. Atomic Force Microscopy AFM allows us to measure the topography of cells with nanometric resolution and estimate the elasticity of the cell by measuring the Young’s modulus. We determined the procedure for preparing the material for measurement in a liquid, and ultra high vacuum by chemical fixation, dehydration, freezing in liquid nitrogen and attachment of live cells in a medium for measurement of appropriately functionalized substrate. The preliminary results are very promising and show that it is possible to investigate both the living non-adherent, as well as fixed cells.

. 159 103. CHARACTERYSTICS OF BIOLOGICAL ANTITUMOR ACTIVITY OF TWO NEW GOLD(III) COMPLEXES INCORPORATING 2,2’:6’,2”-TERPYRIDINE LIGAND FRAMEWORK

Marlena Golec1, Dorota Zygadło1, Magdalena Skonieczna2,3, Katarzyna Czerwińska4, Joanna Palion-Gazda4, Agata Szlapa-Kula5, Stanisław Krompiec5, Sandra Michalak1, Alicja Ratuszna1, Barbara Machura†4, Agnieszka Szurko*1

1August Chełkowski Institute of Physics, University of Silesia, Uniwersytecka 4, 40-007 Katowice, Silesia Center for Education and Interdisciplinary Research, 75 Pułku Piechoty 1A, 41-500 Chorzów, Poland; 2Silesian University of Technology, Center Biotechnology Bioengineering and Bioinformatics, Gliwice, Poland; 3Silesian University of Technology, Institute of Automatic Control, Gliwice, Poland; 4Department of Crystallography, Institute of Chemistry, University of Silesia, 9th Szkolna St, 40-006 Katowice, Poland; 5Department of Inorganic, Organometallic Chemistry and Catalysis, Institute of Chemistry, University of Silesia, 9th Szkolna St., 40-006 Katowice, Poland *mail: [email protected] †mail: [email protected]

Antitumor activity related to cisplatin chemotherapy is well known. The use of compounds containing gold atoms increased substantially in recent years. Published reports of biological activity show that curative results for complexes are significantly better as compared to cisplatin. [1-4]. We present results of antitumor activity of two new gold(III) complexes incorporating 2,2′:6′,2′′- terpyridine ligand framework. In particular, we aimed to elucidate the possibility of fine-tuning cytotoxic activity of gold(III)-terpyridine complexes by structural modifications of terpy ligand. Cytotoxicity of gold complexes was estimated using tumor and normal cell lines and standard MTS assay. Tumor lines tested were HCT116 (Human Colon Adenocarcinoma), HCT116p53-/- (Human Colon Adenocarcinoma with p53 gene silenced), MCF-7 (Human Breast Adenocarcinoma) and A549 (Adenocarcinomic Human Alveolar Basal Epithelial Cells). As control normal cell lines were selected: NHDF (Normal Human Dermal Fibroblasts) and BEAS 2B (Human Bronchial Epithelial Cell Line). Cytotoxicity of cisplatin was used for all the selected cell lines as reference. IC50 values obtained for gold complexes for all the cell lines indicate a higher killing potential of these complexes compared to free ligands and the standard anticancer drug, cisplatin. Furthermore, cytotoxicity results obtained for human normal fibroblasts indicate selectivity of the tested compound. In addition, cell cycle and apoptosis/necrosis analyses as well as intracellular level of reactive oxygen species (ROS) after treatment with gold complexes were carried out. In order to better understand the mechanism of the cell cycle, cell death and ROS-production, we also studied mitochondrial membrane potential (Δψm) and mass. The results are very promising and show that gold(III) complexes could be applied as potential drugs in anticancer chemotherapy.

References: 1. Amani V, Abedi A, Ghabeshi S, Khavasi HR, Hosseini SM, Safari N (2014): Polyhedron 79: 104. 2. Arsenijević M, Milovanovic M, Volarevic V, Djeković A, Kanjevac T, et al. (2012): JMedChem 8: 2. 3. Iwashita S, Saito Y, Ohtsu H, Tsuge K (2014): DaltonTrans 43: 15719. 4. Martín-Santos C, Michelucci E, Marzo T, Messori L, Szumlas P, et al. JInorgBiochem (2015): 153: 339.

160 . 104. THE SYNTHESIS OF NEW POTENTIAL PHOTOSENSITIZERS

Violetta Kozik1*, Andrzej Bąk2†, Danuta Pentak3‡, Marcin Rojkiewicz1*, Aleksander Sochanik4± Piotr Kuś1*

1Department of Synthesis Chemistry, Institute of Chemistry, University of Silesia, 40-006 Katowice, Poland; 2Department of Organic Chemistry, Institute of Chemistry, University of Silesia, 40-006 Katowice, Poland; 3Department of Materials Chemistry and Chemical Technology, Institute of Chemistry University of Silesia, 40-006 Katowice, Poland; 4Center for Translational Research and Molecular Biology of Cancer, Maria Skłodowska-Curie Memorial Cancer Centre and Institute of Oncology,44-100 Gliwice, Poland * mail: [email protected], [email protected], [email protected] †mail: [email protected] mail: [email protected] ±mail:[email protected]

Six tetraphenyl derivatives of arylpropionic acids (profens) were synthesized in search for improved novel photosensitizers that might find application in photodynamic therapy (PDT) of various cancers. Our goal was to alter hydrophobic-hydrophylic characteristics of the tetraphenylporphyrin ring by substitution with propionyl derivatives. As starting materials we used profens known primarily from their widespread use as non-steroidal anti-inflammatory drugs (ibuprofen, naproxen, ketoprofen, fenoprofen, fluorbiprofen) and 2-phenylpropionic acid (model compound). The new compounds were characterized using spectroscopic and photophysical methods with focus given to measurement of molecules' lifetime in excited triplet state, time resolved singlet oxygen phosphorescence and measurement of photostability. Fluorescence quantum yields were also determined; data revealed no planarity changes in the porphyrin molecule following substitution with profens.

. 161

Authors’ index

Abramowicz A 98, 111 Dołbniak M 52 Adamczyk W 55, 59 Domińczyk I 117 Aldaz M 23 Drazek G 113 Aqeilan RI 24 Dubrawska B 141 Badie C 104, 108 Dudek G 131, 133 Bagnall J 74 Durbas M 84, 88 Balcerczak E 118, 119 Dworakowska B 97 Balin K 159 Dziadziuszko R 112 Bandoła D 57 Dzierżak R 130 Barco A 29 Farshadi E 15 Baryła I 25, 90, 91, 92, 93, 96, 155, 156 Feillet C 16 Basiaga M 136 Fidyk W 157 Bednarczyk K 61 Fijałkowska M 118 Bednarczyk M 87 Flyamer IM 26 Bednarek AK 25, 90, 91, 92, 93, 96, 155, 156 Fokt I 143 Bednarek I 80, 94, 95, 153 Fraczek T 154 Behrendt K 111 Fujarewicz K 18, 33, 47, 48, 49 Bernaczek K 65 Funk S 103 Biecek P 8 Gajda K 72 Bieg D 94, 95 Gałązka M 146 Bieg T 129, 141 Gałdyszyńska M 92 Bielecka ZF 158 Gałecki P 119 Bil P 72 Gąsior M 59 Binczyk F 61 Gavrilov AA 26 Blukacz Ł 120, 121 Gawin M 103, 113, 114 Boots AMH 83 Gdowicz-Kłosok A 85 Boratyn E 84, 88 Gelfand MS 26 Borowski T 64 Ghavami S 32, 39 Borys D 18, 60, 125, 127 Ghobria RM 40 Borzdziłowska P 153 Głowala-Kosińska M 75 Bouffler S 104 Gnus M 133 Brasier AR 52 Gogler-Pigłowska A 10 Brodaczewska KK 158 Gola J 110 Brouwer E 83 Golda A 55 Bukato K 146 Goldbeter A 15 Bukowska-Strakova K 159 Golec M 150, 160 Burek M 129 Góra A 64 Butkiewicz D 117 Gorczewska I 127 Byczek-Wyrostek A 145 Gorczewski K 127 Bzowski P 125 Górecka K 148 Candéias S 108 Gracka M 55, 56 Cecotka A 104 Grymel M 140 Chadalski M 77 Grzesiuk E 158 Chaves I 15 Grzybowska E 117 Chęcińska B 70, 142 Grzywna Z 65, 97, 134 Chekan M 113 Gunia-Krzyżak A 153 Chertovich A 26 Habryka A 143 Chylinski K 31 Hanus J 114 Cieśla M 131 Hartman ML 86 Cieślar-Pobuda A 32, 39 Hashim A 38 Collas P 38 Hejmo T 71 Cortez AJ 76, 79, 105, 122 Herok M 8 Czarnecka AM 158 Hohn M 132 Czerwińska K 160 Horwacik I 84, 88 Czerwińska P 8 Hudy D 71 Czubatka-Bieńkowska A 144 Jain MV 32 Czyż M 86 Jaksik R 18, 107 Danch-Wierzchowska M 60 Janicki B 136 Daniłowicz A 72 Janikowski T 89, 110 de Jong D 83 Janus P 9 Deorowicz S 126 Jasiński A 70 Długosz M 126 Jassem J 112 Jaszcz K 135 Kuś P 150 Jaworski A 70, 142, 146 Kusz D 135 Jazowiecka-Rakus J 157 Kuźnik A 139 Jęda-Golonka A 89, 110, 121 Kwapiszewski R 70, 142 Jędroszka D 25, 90, 91, 92, 93, 96, 155, 156 Łabaj W 101 Jeleń A 119 Łakomiec K 47 Jellema P 83 Lalik A 107, 129 Jelonek K 112 Łanuszewska J 117 Józkowicz A 159 Latoch M 147 Kaczmarek M 135 Latusek K 128 Kała S 72 Leclerc P 15 Kalinowska-Herok M 113 Lesiak M 135 Kałuża K 140 Levi F 14 Kantidze OL 62 Likus W 32 Kardyńska M 50 Lisowska KM 76, 79, 105, 122 Kasprzycka A 145 Loch T 80 Kaszuwara W 134 Łos MJ 32 Khrameeva EE 26 Luzhin AV 62 Kimmel M 52, 74 Łyko-Morawska D 128 Kimsa M 82 Łysek-Gładysińska M 75 Klamka J 53 Machura B 160 Klarzyńska K 10 Macieja A 69 Kliber-Jasik M 151 Maciejewski R 130 Klimczak M 8 Mac-Marcjanek K 93 Kloc M 40 Madej P 120, 121 Kluczka J 133 Malinowska A 158 Kluiver J 83 Maliszewska K 158 Klyszcz K 75, 76 Malmberg KJ 38 Koceva-Chyla A 154 Manning G 104 Kochel K 154 Marczak Ł 103, 114 Kokot T 106 Marczyk M 63, 102, 112 Kołak P 140 Marek E 107 Kondera-Anasz Z 94, 95 Marona H 80, 153 Konopacka U 142 Marszałek I 142 Konopka M 152 Matuszczyk I 85 Konopka W 34 Matyśniak D 75 Korfanty J 9, 73, 77 Mazur K 119 Kornakiewicz A 158 Mazurek U 81, 82, 87, 89, 106, 109, 110 Korusiewicz S 139 Mazurkiewicz R 140 Korytkowska-Wałach A 141 McFadden G 157 Kos P 26 Melka B 54, 55 Kosiński M 8 Michalak S 150, 160 Kośla K 25, 90, 91, 92, 93, 96, 155, 156 Michalski B 106, 109 Kowalczyk K 120, 121 Michalski M 106, 109 Kowolik B 106, 109 Mielańczyk A 41, 65, 132 Kozieł P 87 Mielańczyk Ł 41 Krajewski P 55 Mielczarek A 86 Krasowska M 131 Mielczarek-Palacz A 94 Krasowski P 158 Mika J 108 Kratz G 39 Miłaczewska A 64 Krawczyk M 148 Mitrus I 157 Krawczyk Z 10 Molga A 70, 142, 146 Kristiansen HA 38 Mrozek D 18 Kroesen B-J 83 Mrukwa G 113 Krompiec S 160 Muc-Wierzgoń M 87, 109 Kruszniewska-Rajs C 110 Multhoff G 5 Krześniak M 85 Nackiewicz J 151 Krzywoń A 48, 49 Naumowicz A 74, 77 Krzyzosiak WJ 30 Nawrot U 148 Kubiak JZ 40 Nejc D 86 Kujawa KA 76, 79, 105, 122 Neugebauer D 41, 65, 132 Kupryjańczyk J 122 Niebudek K 118 Kurasz K 48 Niejadlik N 127 Kurpas M 52 Nikiel B 105 Nowak AJ 55 Sharee A 32 Nowak B 136 Sherman MY 7 Nowak E 94, 95 Shevelyov YY 26 Nowak I 84, 88 Shevtsov M. 5 Nowakowska M 25, 90, 91, 92, 93, 96, 155, 156 Sieroń AL 135 Nowicka E 111 Sikora B 81, 82, 106, 109 Nycz A 147 Sikora J 95 Ochab M 53 Simka K 106, 109 Oei V 38 Simoes RF 154 Olbryt M 79, 105, 143 Sistonen L. 6 Oliveira PJ 154 Skonieczna M 41, 71, 151, 160 Olszewski MB 8 Skowronek B 106, 109 Orzechowska M 25, 90, 91, 92, 93, 96, 155, 156 Skubis A 81, 82, 106, 109 Osadnik T 59 Slezak-Prochazka I 83 Ostrowski Z 55, 59 Śliwiński T 149 Palion-Gazda J 160 Słotwińska A 128 Pamuła-Piłat J 117 Śmieja J 52, 50 Panek R 125 Śmiga-Matuszowicz M 135, 136, 141 Papiez A 101 Smigielska-Czepiel K 83 Pastuch-Gawołek G 148 Sobierajska E 152 Paszek P 74 Sobon A 154 Pawlak S 70, 142, 146 Sochanik A 143, 147, 157 Pietrowska M 98, 103, 111, 112, 113, 114 Sojka D 10, 75 Pilawka R 135, 136 Solarek W 158 Piwowarski J 158 Spychałowicz W 159 Płuciennik E 25, 90, 91, 92, 93, 96, 155, 156 Staerk J 38 Pluta D 120, 121 Stańczak A 64 Poczęta M 94 Stangl S 5 Poczęta M 95 Stokowy T 73, 78 Polanska J 61, 101, 102, 104, 108, 111, 112, 113 Strzelewicz A 131 Poleszak K 146 Student S 18, 129, 143 Połowczuk M 153 Styczeń-Binkowska E 25, 90, 91, 92, 93, 96, 155, Ponge L 117 156 Popęda M 92 Svaerd O 38 Popławski T 69, 144, 149 Świechowski R 119 Pospiech K 25, 90, 91, 92, 93, 96, 155, 156 Świerniak A 18, 60 Poterała-Hejmo A 71, 72 Sypniewski D 80, 94, 95, 153 Priebe W 143 Sysel P 134 Psiuk-Maksymowicz K 18, 58 Szade J 159 Puchała M 69 Szapski M 80 Puszyński K 53 Szczygieł I 59 Rafat M 37 Szczylik C 158 Ratuszna A 150, 159, 160 Szkaradek N 80, 153 Razin SV 26, 62 Szlapa-Kula A 160 Reinke H 13 Sztiller-Sikorska M 86 Rodacka A 69 Szurko A 150, 159,160 Rodziewicz P 111 Szymanik M 70, 142, 146 Rogne M 38 Talar B 86 Rojczyk D 59 Talarowska M 119 Rojczyk M 55, 59 Tarnawski R 111 Rokita H 84, 88 Tęcza K 117 Roś-Mazurczyk M 112 Teska-Kamińska M 70, 146 Różanowski B 81, 82 Teteloshvili N 83 Rózga P 70, 146 Tian B 52 Rumak K 145 Tinhofer-Keilholz I 98 Rusin A 79, 143, 157 Tobiasz J 78 Rusin M 85 Toma M 149 Rutgers B 83 Toma-Jonik A 9, 77, 78 Rybak A 134 Tomczyk MD 154 Rzeszowska-Wolny J 17, 39, 71, 72 Tracz-Gaszewska Z 8 Rzyman W 112 Tudrej P 76, 79, 105, 122, 143 Sarnik J 149 Turczyn R 133 Ścieglińska D 10, 75 Ulianow SV 26 Seitz A 83 van den Berg A 83 van der Horst GTJ 15 van der Lei RJ 83 Velichko AK 62 Vilas Jain M 39 Vydra N 9, 77, 78 Walaszczyk A 111 Walczak K 145, 154 Walkiewicz K 87 Wandzik I 129, 148 Wang S 114 Wasilewski J 59 Waśkiewicz S 129 Waszkielewicz AM 80 Wawrzkiewicz-Jałowiecka A 97, 120, 121 Wawrzynow B 8 White M 74 Whiteside T 103 Wideł M 49 Widlak P 98, 103, 111, 112, 113, 114 Widłak W 9, 73, 74, 77, 78 Wiech M 8 Wiechec E 39 Wierzgoń J 113 Witczak Z 144, 149 Witek A 89, 110 Witkowska A 82 Wiznerowicz M 8 Wojakowska A 111 Wojdas E 89, 106, 109 Yan J 15 Yuan Y 83 Zajkowicz A 85 Żebrowska M 118, 119 Zembala-Nożyńska E 105, 122 Żerek B 70, 146 Zięba A 147 Zieleniak A 93 Zmarzły N 89, 110 Żurawska-Kliś M 93 Żurawski M 19 Zygadło D 150, 159, 160 Żyła J 102 Żylicz A 8 Żylicz M 8 Żytecka E 128 Participant affiliation and e-mail addresses

Partici- Last name First name City E-mail pation1 Abramowicz Agata P Gliwice [email protected]

Adamczyk Wojciech P Gliwice [email protected]

Aldaz Marcelo L Houston [email protected]

Aqeilan Rami L Jeruzalem [email protected]

Babilas Dorota P Gliwice [email protected]

Bąk Andrzej P Katowice [email protected]

Bandoła Dominika P Gliwice [email protected]

Barco Angel L Alicante [email protected]

Baryła Izabela P Łódź [email protected]

Bednarczyk Martyna P Sosnowiec [email protected]

Bednarczyk Katarzyna P Gliwice [email protected]

Bednarek Andrzej L Łódź [email protected]

Bernaczek Katarzyna P Gliwice [email protected]

Bil Patryk Gliwice [email protected]

Binczyk Franciszek Gliwice [email protected]

Blukacz Łukasz P Katowice [email protected]

Boratyn Elżbieta P Kraków [email protected]

Burek Małgorzata P Gliwice [email protected]

Byczek-Wyrostek Anna P Gliwice [email protected]

Bzówka Maria P Gliwice [email protected]

Bzowski Paweł P Gliwice [email protected]

Cecotka Agnieszka P Gliwice [email protected]

Chadalski Marek P Gliwice [email protected]

Chęcińska Beata P Czosnów [email protected]

Chyliński Krzysztof L Vienna [email protected]

Cortez Alexander P Gliwice [email protected]

Czarnecka Anna P Warszawa [email protected]

Czubatka-Bieńkowska Anna P Łódź [email protected]

Danch-Wierzchowska Marta P Gliwice [email protected]

Delaunay Franck C Nice [email protected]

Dołbniak Marzena P Gliwice [email protected]

Dzierżak Róża P Lublin [email protected]

Długosz Maciej P Gliwice [email protected]

Farshaid Elham L Rotterdam [email protected]

Feillet Celine L Nice [email protected] Fujarewicz Krzysztof L Gliwice [email protected]

Gajda Karolina Gliwice [email protected]

Gawin Marta P Gliwice [email protected]

Gnus Małgorzata Gliwice [email protected]

Golda Adam Pilchowice [email protected]

Grabarek Beniamin P Sosnowiec [email protected]

Gracka Maria P Gliwice [email protected]

Grzybowska Ewa Gliwice [email protected]

Hamouz Raneem Łódź [email protected]

Hartman Mariusz P Łódź [email protected]

Hejmo Tomasz P Zabrze [email protected]

Herok Marcin Warszawa [email protected]

Hohn Monika P Gliwice [email protected]

Hopko Katarzyna P Gliwice [email protected]

Hudy Dorota Gliwice [email protected]

Jaksik Roman Gliwice [email protected]

Janikowski Tomasz Sosnowiec [email protected]

Janus Patryk P Gliwice [email protected]

Jazowiecka-Rakus Joanna P Gliwice [email protected]

Jędroszka Dorota P Lódź [email protected]

Jelonek Karol P Gliwice [email protected]

Kalinowska-Herok Magdalena P Gliwice [email protected]

Kardyńska Małgorzata P Gliwice [email protected]

Kała Sylwia P Gliwice [email protected]

Kałuża Karolina P Gliwice [email protected]

Klim Jakub P Gliwice [email protected]

Klimczak Marta Warszawa [email protected]

Kokot Marek P Gliwice [email protected]

Konopacka Urszula P Czosnów [email protected]

Konopka Małgorzata P Łódź [email protected]

Konopka Witold L Warszawa [email protected]

Korfanty Joanna P Gliwice [email protected]

Korusiewicz Sylwia P Gliwice [email protected]

Korytkowska-Wałach Anna P Gliwice [email protected]

Kośla Katarzyna P Łódź [email protected]

Kowalczyk Karolina P Katowice [email protected]

Kozik Violetta P Katowice [email protected]

Krajewski Piotr Gliwice [email protected] Krasowska Monika P Gliwice [email protected]

Krawczyk Zdzisław C Gliwice [email protected]

Krześniak Małgorzata P Gliwice [email protected]

Krzywon Aleksandra P Gliwice [email protected]

Krzyżosiak Włodzimierz L Poznań [email protected]

Kubiak Jacek L Paris [email protected]

Kujawa Katarzyna P Gliwice [email protected]

Kupczak Maria Gliwice [email protected]

Kurasz Karolina P Gliwice [email protected]

Kurczyk Agata P Gliwice [email protected]

Kurpas Monika P Gliwice [email protected]

Kwapiszewski Radosław Czosnów k. W-wy [email protected]

Latusek Katarzyna Katowice [email protected]

Leszczorz Kinga P Siemianowice Śląskie [email protected]

Levi Francis L Coventry [email protected]

Lisowska Katarzyna P Gliwice [email protected]

Los Marek L Linkoping [email protected]

Luzhin Artem P Moscow [email protected]

Łakomiec Krzysztof P Gliwice [email protected]

Łanuszewska Joanna Gliwice [email protected]

Macieja Anna P Łódź [email protected]

Mączyńska Justyna P Wrocław [email protected]

Malarz Katarzyna Katowice [email protected]

Marczyk Michal P Gliwice [email protected]

Marek Ewa P Gliwice [email protected]

Mazur Katarzyna P Łódź [email protected]

Melka Bartlomiej P Gliwice [email protected]

Michalak Sandra P Katowice [email protected]

Mielańczyk Anna L Gliwice [email protected]

Mielczarek Aleksandra Łódź [email protected]

Mika Bożena P Mikołów [email protected]

Mika Justyna P Gliwice [email protected]

Mitusińska Karolina P Gliwice [email protected]

Molga Anna P Czosnów k. W-wy [email protected]

Multhoff Gabrielle L Munich [email protected]

Naumowicz Anna P Gliwice [email protected]

Niebudek Katarzyna P Lodz [email protected]

Niejadlik Nikol P Gliwice [email protected] Nowak Andrzej J. P Gliwice [email protected]

Nowak Iwona P Cracow [email protected]

Nowak Marcin Gliwice [email protected]

Nowakowska Magdalena Łódź [email protected]

Ochab Magdalena P Gliwice [email protected]

Olbryt Magdalena P Gliwice [email protected]

Orzechowska Magdalena P Łódź [email protected]

Ostrowski Ziemowit P Gliwice [email protected]

Pacholczyk Marcin Gliwice [email protected]

Pala Paweł P Bielsko-Biała [email protected]

Pamuła-Piłat Jolanta P Gliwice [email protected]

Papiez Anna P Gliwice [email protected]

Pastuch-Gawołek Gabriela P Gliwice [email protected]

Pietrowska Monika P Gliwice [email protected]

Popławski Tomasz P Łódź [email protected]

Pospiech Karolina P Łódź [email protected]

Poterała-Hejmo Aleksandra Gliwice [email protected]

Psiuk-Maksymowicz Krzysztof L Gliwice [email protected]

Płuciennik Elżbieta P Łódź [email protected]

Rafat Mehrdad L Linkoping [email protected]

Razin Sergey L Moscow [email protected]

Reinke Hans L Düsseldorf [email protected]

Rejmund Marta P Katowice [email protected]

Rogolinski Jacek Gliwice [email protected]

Rojczyk Marek P Gliwice [email protected]

Rolnik Bożena P Gliwice [email protected]

Roś-Mazurczyk Małgorzata P Gliwice [email protected]

Rotarska-Mizera Anna P Gliwice [email protected]

Różanowski Bartosz Kraków [email protected]

Rybak Aleksandra P Gliwice [email protected]

Rzeszowska-Wolny Joanna L Gliwice [email protected]

Sarnik Joanna P Łódź [email protected]

Sherman Michael Y. L Boston [email protected]

Sieradzka Katarzyna P Gliwice [email protected]

Sikora Bartosz P Sosnowiec [email protected]

Simka Klaudia P Katowice [email protected]

Sistonen Lea L Turku [email protected]

Skonieczna Magdalena P Gliwice [email protected] Skowronek Bartłomiej P Katowice [email protected]

Skubis Aleksandra P Sosnowiec [email protected]

Slezak-Prochazka Izabella P Gliwice [email protected]

Śmiga-Matuszowicz Monika P Gliwice [email protected]

Śmigielska-Czepiel Katarzyna P Groningen [email protected]

Sobierajska Ewelina P Bratoszewice [email protected]

Staerk Judith L Oslo [email protected]

Stalica Paweł Izabelin [email protected]

Stańczak Agnieszka P Gliwice [email protected]

Strzelewicz Anna P Gliwice [email protected]

Styczeń-Binkowska Ewa P Łódź [email protected]

Sypniewski Daniel P Katowice [email protected]

Słotwińska Adrianna Katowice [email protected]

Ścieglińska Dorota L Gliwice [email protected]

Toma-Jonik Agnieszka P Gliwice [email protected]

Tomczyk Mateusz P Gliwice mateusz.d.tomczyk

Tudrej Patrycja P Gliwice [email protected]

Walaszczyk Anna P Gliwice [email protected]

Wawrzkiewicz-Jałowiecka Agata P Gliwice [email protected]

Wawrzynów Bartosz Warszawa [email protected]

Widłak Piotr Gliwice Piotr.Widł[email protected]

Widłak Wiesława L Gliwice Wiesława.Widł[email protected]

Wiechec Emilia L Linkoping [email protected]

Wojdas Emilia P Katowice [email protected]

Zmarzły Nikola P Sosnowiec [email protected]

Zygadło Dorota P Katowice [email protected]

Zyla Joanna P Gliwice [email protected]

Żurawski Marek L Kraków [email protected]

Żylicz Alicja Warszawa [email protected]

Żylicz Maciej L Warszawa [email protected]

Żytecka Ewa P Katowice [email protected]

1C-chairman; L-lecture; P-poster

Sonia Jamroży, mezzosoprano graduate of Karol Szymanowski Academy of Music Katowice, Poland performing in Poland and the United States of America

Alexandr Vovk Katarina Vovková Dušan Růžička baritone – Soloist, soprano – Soloist, tenor – Soloist Opava Opera Theatre, Opava Opera Theatre, Opava Opera Theatre, Czech Republic Czech Republic Czech Republic

Program

1. Sonia Jamroży: Azucena's aria "Stride la vampa" from the opera “Il Trovatore” (Trubadur) by Giuseppe Verdi 2. Katarina Vovková: Lauretta's aria "O mio babbino caro" from the opera “Gianni Schicchi” by Giacomo Puccini 3. Dušan Růžička: Nemorino's aria "Una furtiva lagrima" from the opera “L'elisir d'amore” (Napój Miłosny) by Gaetano Donizetti 4. Alexandr Vovk: Escamillo’s aria "Votre toast" (The Toreador Song) from the opera “Carmen” by Georges Bizet 5. Katarína Vovková: Marica's aria (Slovak text) from the operetta “Countess Maritza” (Hrabina Marica) by Emmerich Kálmán 6. Dušan Růžička: Jenik’s aria from the opera ”The Bartered Bride” (Sprzedana Narzeczona) by Bedřích Smetana 7. Alexandr Vovk: Kecal’s aria from the opera “The Bartered Bride” (Sprzedana Narzeczona) by Bedřích Smetana. 8. Sonia Jamroży: Eliza's song “I could have danced all night" from the musical “My Fair Lady" by Frederick Loewe 9. Alexandr Vovk and Dušan Růžička: Kecal and Jenik duet from the opera “The Bartered Bride” (Sprzedana Narzeczona) by Bedřích Smetana. 10. Katarína Vovková and Dušan Růžička: Violetta and Alfredo duet in "Brindisi" (Drinking Song) from the opera “La Traviata” (Traviata) by Giuseppe Verdi

PROJECT FINANCED BY THE NATIONAL CENTRE FOR RESEARCH AND DEVELOPMENT UNDER THE APPLIED RESEARCH PROGRAMME

Narodowe Centrum Badań i Rozwoju

BioTest - platform for remote testing and analyzing of biomedical data

Platform features: • Collection, integration and preprocessing of biomedical data (including proteomic, transcriptomic and genomic data) • Implementation of a multiscale model providing variety of data analysis, with posible return to the previous analysis • Virtualization and creation of remote work environment • Presentation, visualization, and reporting the results of analysis

Institutions involved in the project: • Silesian University of Technology, Department of Automatic Control, Electronics and Computer Science • Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology - Branch Gliwice • WASKO Sp. z o.o

www.biotest.polsl.pl