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Urokinazni plazminogenski aktivator (uPA) ili urokinaza zaslužan je za aktivaciju plazminogena i njegovu pretvorbu u plazmin, proteolitički enzim koji razgrađuje ekstracelularni matriks. Urokinaza je dio sustava koji obuhvaća i receptor uPA (uPAR) koji je veže na staničnu membranu te njene inhibitore, PAI-1 i PAI-2. Cilj ovog rada bio je klonirati sekvence koje kodiraju za urokinazu, njen receptor i inhibitor PAI-1 te ih eksprimirati u različitim staničnim linijama. Analiza ekstracelularne enzimske aktivnosti i ekspresije pojedinih elemenata sustava u različitim tipovima stanica ukazala bi na mehanizme regulacije urokinazne aktivnosti. Konstruirani su plazmidi sa sekvencama gena za uPA, PAI-1 i uPAR te transficirane stanične linije A1235, HeLa i HEK293. Stanice HEK293 i A1235 transficirane plazmidom sa sekvencom za uPA imale su bazalnu aktivnost urokinaze. Sojevi stanica A1235 i HEK293 transficirani plazmidom sa sekvencom za PAI-1, pokazali su smanjenu aktivnost urokinaze. Stanice HeLa nisu pokazale urokinaznu aktivnost. Unos plazmida sa sekvencom uPAR nije utjecao na urokinaznu aktivnost stanica. Analiza ekspresije proteina pokazala je da plazmidi sa sekvencom urokinaze nisu promijenili razinu ekspresije ovog proteina u staničnim lizatima. Kod stanica A1235 i HEK293 detektirano je povećanje ekspresije uPAR i PAI-1 nakon transfekcije plazmidima s njihovim sekvencama. Uzgojene stanične linije s različitom urokinaznom aktivnosti mogu poslužiti kao model za istraživanje utjecaja urokinaznog sustava na staničnu proliferaciju, migraciju i preživljenje. Urokinase plasminogen activator (uPA) or urokinase is responsible for the activation of plasminogen and its conversion into plasmin, a proteolytic enzyme that degrades the extracellular matrix. Urokinase is a part of a system that includes the uPA receptor (uPAR) that binds urokinase to the cell membrane and its inhibitors, PAI-1 and PAI-2. The aim of this study was to clone sequences encoding urokinase, its receptor and PAI-1, and to express them in different cell lines. Analysis of extracellular enzyme activity and expression of individual urokinase system elements in different cell types, could indicate the regulatory mechanisms of urokinase activity. Plasmids with gene sequences for uPA, PAI-1 and uPAR were constructed and A1235, HeLa and HEK293 cell lines were transfected with plasmids. HEK293 and A1235 cells transfected with the uPA sequence plasmid had basal urokinase activity. A1235 and HEK293 cell strains transfected with plasmid containing PAI-1 sequence showed reduced urokinase activity. HeLa cells did not have urokinase activity. Expression of plasmids with the uPAR sequence did not affect urokinase activity in the transfected cell lines. Analysis of protein expression showed that plasmids with the urokinase sequence did not change the expression levels of this protein in the cell lysates. In A1235 and HEK293 cells, an increase in uPAR and PAI-1 expression was detected after transfection with plasmids with corresponding sequences. Cell strains with different urokinase activity can serve as a model for investigating the impact of the urokinase system on cell proliferation, migration and survival.

Svaki organizam ima regulacijske mehanizme koji kontroliraju proliferaciju, diferencijaciju i preživljavanje stanice. Razni faktori poput kemijskih karcinogena, UV zračenja, hormona, virusa i bakterija negativno utječu na regulacijske mehanizme što uglavnom dovodi do razvoja stanica malignog karaktera. U kulturi tumorskih stanica vidljiv je neprekidan rast stanica koji je posljedica njihove sposobnosti samostalne produkcije faktora rasta. Karcinogeneza je složen proces u kojem važnu ulogu igraju onkogeni te tumor-supresorski geni. Onkogeni nastaju iz protoonkogena mutacijskog aktivacijom, dok tumor-supresori svojom inaktivacijom ili gubitkom funkcije vode razvoju zloćudnih tumora. Kod tumora se uočava neregulirana kontrola staničnog ciklusa pri čemu izbjegavaju kontrolna mjesta tzv. „checkpoints“ koja zaustavljaju proliferaciju u prisutnosti oštećene DNA. Cilj ovog završnog rada je opisati vrste tumora, objasniti njihov razvoj i svojstva, različite uzroke te gene povezane s njihovim nastankom. Poseban je naglasak stavljen na različite pristupe pri liječenju i prevenciji tumora., Every organism has regulatory mechanisms that control cell proliferation, differentiation, and survival. Various factors such as chemical carcinogens, UV radiation, hormones, viruses and bacteria have a negative effect on the regulatory mechanisms, leading mainly to the development of malignant cells. In tumor cell cultures, continuous growth of cells can be observed as they are able to produce growth factors independently. Carcinogenesis is a complex process in which oncogenes and tumor suppressor genes play an important role. Oncogenes arise from protooncogenes through mutational activation, whereas tumor suppressors lead to the development of malignant tumors through their inactivation or loss of function. In tumors, unregulated control of the cell cycle is observed, with checkpoints that stop proliferation in the presence of damaged DNA. The aim of this thesis is to describe the types of tumors, explain their development and characteristics, various causes and genes associated with their development. Special emphasis is given to the different approaches for the treatment and prevention of tumors.

Serozni karcinomi jajnika najčešći su oblik epitelnih tumora jajnika. Uglavnom se dijagnosticiraju u uznapredovalom stadiju bolesti i imaju visoku stopu smrtnosti. Kod raka jajnika primijećena je poremećena aktivacija više signalnih puteva među kojima je i signalni put Hedgehog (Hh). Uloga signalnog puta Hh u raku jajnika, kao i u pojedinim histološkim podtipovima raka jajnika, nije dovoljno istražena. U ovoj disertaciji istražili smo ulogu proteina PTCH1, SHh i IHh, kao i promjene u metilacijskom statusu promotora istoimenih gena, u seroznim karcinomima jajnika. Istraživanje je provedeno na uzorcima seroznih karcinoma jajnika niskog (LGSC) i visokog (HGSC) stupnja malignosti, dok su uzorci zdravih jajnika i jajovoda služili kao kontrole. U istraživanju su korištene i stanične linije HGSC-a, OVCAR5, OVCAR8 i OVSHAO, i kontrolna stanična linija normalnih epitelnih stanica jajovoda, FNE1. Ekspresija proteina PTCH1, SHh i IHh u tkivnim uzorcima analizirana je metodom imunohistokemije, dok je u staničnim linijama ispitana uz pomoć imunofluorescencije i Western blota. Metilacijski obrazac DNA u promotorima gena PTCH1, SHh i IHh analizirali smo koristeći PCR ovisan o metilaciji (MSP). Ekspresija proteina PTCH1, SHh i IHh bila je značajno veća u karcinomima HGSC i LGSC u odnosu na kontrolna tkiva, kao i u karcinomskim u odnosu na kontrolnu staničnu liniju. U tumorskom tkivu i tumorskim stanicama u kulturi bila je izražena nuklearna ekspresija proteina PTCH1 što navodi na zaključak da bi navedeni protein mogao igrati aktivnu, tumor-promotorsku ulogu u jezgri malignih stanica seroznih karcinoma jajnika. U staničnim linijama detektirani su fragmenti proteina PTCH1 različite molekularne mase što upućuje na mogućnost proteolitičkog cijepanja ovog proteina. Autokrina signalizacija ovisna o SHh-ligandu utvrđena je u karcinomima HGSC i LGSC, dok su autokrina i parakrina signalizacija ovisna o IHh-ligandu uočene u karcinomima HGSC. Metilacija DNA u promotorima gena PTCH1, SHh i IHh nije bila povezana s razinom ekspresije ovih proteina što navodi na zaključak da ovaj mehanizam nije uključen u patogenezu seroznih karcinoma jajnika. Dobiveni rezultati upućuju na aktivnu ulogu proteina PTCH1, SHh i IHh u patogenezi seroznih karcinoma jajnika te doprinose boljem razumijevanju molekularne patogeneze ovih karcinoma., Serous ovarian carcinomas are the most common type of epithelial ovarian cancers. They are mostly diagnosed in the advanced stage of the disease and have a high mortality rate. In ovarian cancer, aberrant activation of several signaling pathways, including the Hh signaling pathway, has been observed. The role of Hh signaling pathway in ovarian cancer, as well as in certain histological subtypes of ovarian cancer, has not been sufficiently investigated. In this dissertation, we investigated the role of PTCH1, SHh and IHh proteins, as well as changes in the promoter methylation status of the corresponding genes, in serous ovarian carcinomas. Low- (LGSC) and high-grade (HGSC) serous ovarian carcinoma tissue samples were used in this study, while normal ovarian and fallopian tube tissue samples served as controls. HGSC cell lines, OVCAR5, OVCAR8 and OVSHAO, and control cell line, normal fallopian tube non-ciliated epithelium cell line FNE1, were also used in the study. PTCH1, SHh and IHh protein expression levels were analyzed using immunohistochemistry in tissue samples, and by immunofluorescence and Western blot in cell lines. DNA methylation pattern of PTCH1, SHh and IHh genes were analyzed by methylation-specific PCR (MSP). PTCH1, SHh and IHh protein expression levels were significantly higher in HGSCs and LGSCs compared to control tissues, as well as in cancer cell lines compared to control cell line. Nuclear expression of the PTCH1 protein in tumor tissue and cultured tumor cells suggests that this protein could play an active, tumor-promoter role in the nuclei of serous ovarian cancer cells. PTCH1 protein fragments of different molecular weight were detected in the cell lines, indicating possible proteolytic cleavage of this protein. Autocrine SHh signaling was found in HGSCs and LGSCs, while autocrine and paracrine IHh signaling were observed in HGSCs. DNA methylation of PTCH1, SHh, and IHh gene promoters were not in line with the expression levels of these proteins, leading to the conclusion that this mechanism is not involved in the pathogenesis of serous ovarian carcinoma. Our results indicate that PTCH1, SHh and IHh proteins play an active role in pathogenesis of serous ovarian carcinoma, and contribute to a better understanding of the molecular pathogenesis of these cancers.

Karakteristika svih somatskih stanica je njihov ograničeni životni vijek te regulirana proliferacija, diferencijacija i preživljavanje što je potrebno da bi čitav organizam mogao normalno funkcionirati. Nakupljanje stanica koje čine tkivo bez fiziološke funkcije naziva se tumor ili neoplazma. Jedno od temeljnih svojstava tumora je klonalnost, nastanak iz jedne stanice koja počinje proliferirati. Nekontroliran rast karakterističan za tumorske stanice nastaje kao posljedica poremećaja regulacije staničnih sustava. Nastanak raka je proces niza promjena u kojem određene stanice postupno postaju zloćudne. Tumori koji imaju potencijal širenja po organizmu u kojem su nastali smatraju se malignima. Uzroci malignih transformacija povezuju se s vanjskim faktorima kao što su mutageni, odnosno karcinogeni, pogreškama u mehanizmima same stanice poput greške u replikaciji, a jedni od najvažnijih uzročnika su virusi. Onkolitički virusi su virusi koji se umnažaju u tumorskim stanicama, ubijaju ih i šire se među njima a da ne oštećuju ili inficiraju netumorske stanice. Tumorski virusi nisu važni samo zato što uzrokuju bolesti u ljudi, već i zato što imaju ključnu ulogu u istraživanju raka služeći kao modeli u molekularnim i staničnim istraživanjima transformacije stanice te zahvaljujući napretku u molekularnoj biologiji raka došlo je do poboljšanja u razvoju novih pristupa u liječenju raka. Cilj ovog rada je opisati svojstva tumorskih stanica, njihovo nastajanje i rast, objasniti utjecaj virusa na nastajanje tumora, pristupe u liječenju tumora te važnost cijepljenja u svrhu prevencije od bolesti., The characteristics of all somatic cells is their limited life duration and regulated proliferation, differentiation and survival necessary for the organism to function properly. The accumulation of cells that make up tissue without physiological function is called a tumor or neoplasm. One of the fundamental properties of a tumor is clonality, the emergence from a single cell that begins to proliferate. The uncontrolled growth characteristic of tumor cells arises as a result of impaired regulation of cellular systems. Cancer is the process of a series of changes in which certain cells gradually become malignant. Tumors that have the potential to spread to the organism in which they are produced are considered malignant. The causes of malignant transformations are related to external factors such as mutagens, that is, carcinogens, to errors in the mechanisms of the cell itself, such as a replication error, and viruses are one of the most important causes. Oncolytic viruses are viruses that multiply in tumor cells, kill them and spread among them without damaging or infecting non-tumor cells. Tumor viruses are not only important because they cause pain in humans, but also because they play a key role in cancer research by serving as models in molecular and cellular cell transformation studies, and thanks to advances in molecular cancer biology, improvements have been made in developing new approaches to cancer treatment . The objective of this thesis is to explain the properties of tumor cells, their formation and growth, to explain the impact of the virus on tumor formation, approaches to tumor treatment, and the importance of vaccination for the prevention of disease.

Karakteristika svih somatskih stanica je njihov ograničeni životni vijek te regulirana proliferacija, diferencijacija i preživljavanje što je potrebno da bi organizam mogao funkcionirati. Kao posljedica djelovanja unutrašnjih i vanjskih faktora na organizam i zbog nepravilne regulacije kontrolnih mehanizama, stanice dobivaju nove biološke osobine odnosno maligni fenotip. Tumorske stanice imaju svojstvo klonalnosti što znači da tumor nastaje nekontroliranom proliferacijom jedne stanice. Proces karcinogeneze nastaje kao posljedica djelovanja karcinogena koji oštećuju DNA i induciraju mutacije te promotora koji stimuliraju proliferaciju stanica. U kontroli staničnih procesa, ali i u nastanku raka sudjeluju proteini kodirani genima. Onkogeni su geni koji potiču diobu stanica, dok tumor supresor geni inhibiraju proliferaciju i kontroliraju stanični ciklus. Cilj ovog rada je opisati svojstva tumorskih stanica, njihovo nastajanje i rast, objasniti gene uključene u nastanak raka, pristupe u liječenju tumora te važnost pravilne i raznolike prehrane, odnosno važnost hrane za očuvanje zdravlja i prevenciju nastanka tumora., The characteristics of all somatic cells is their limited life duration and regulated proliferation, differentiation and survival necessary for the organism to function properly. As a result of internal and external factors on the organism, along with inappropriate regulation of control mechanism, the cells receive new biological characteristics, also known as malignant. Tumor cells can duplicate meaning that the tumor acquires an ability for uncontrollable proliferation of cells. The process of carcinogenesis begins as a result of carcinogens that damage DNA and induce mutations and promoters that stimulate the proliferation of cells. In the control of cellular processes, the proteins encoded by genes participate in cancer formation. Oncogenes are genes which stimulate cell division, while tumor suppressor genes inhibit proliferation and control the cell’s life cycle. The objective of this thesis is to explain the properties of tumor cells, their formation, growth, and also to explain genes that are involved in the formation of cancer, approaches to treatment of tumors and the importance of a proper diet, as well as the importance of food as a preventative mean to the formation of tumors.

U uvodnim poglavljima izložena je motivacija problema. Kratko su opisani biološki koncepti čije je poznavanje važno u kasnijim fazama. Glavna tema rada je razvoj generičkog klasifikatora. Naglasak se stavlja na mogućnost određivanja tipova i podtipova tumorskih stanica. U inkrementalnom postupku razvoja koristi se algoritam stroja potpornih vektora (SVM). Prikazan je način optimizacije parametara SVM-a uz pomoć genetskog algoritma. Opisana je implementirana paralelizacija posla pri učenju. Izgrađeni klasifikator ispitan je na različitim vrstama skupova podataka. Ukratko su opisane strategije višerazredne klasifikacije. Predstavljen je način procjene vjerojatnosti pripadnosti uzoraka pojedinim razredima klasifikacije u vidu Plattovog skaliranja. Izloženi su rezultati i uspješnost konačnog modela. Motivation for this assignment’s problem is presented in introductory chapters. Biological concepts, key to understanding later work, are briefly described. Main topic in this assignment is development of generic classifier. Emphasis is put on possibility of determining types and subtypes of tumor cells. Algorithm Support Vector Machine is used in incremental building procedure. Optimization of SVM’s parameters with genetic algorithm is described thoroughly. Implementation of parallel computing for work needed for training is also presented. Built classifier is tested on various types of datasets. Strategies of multiclass classification are shortly described. A way of creating a probability of class association approximation is shown by method of Platt scaling. Results and achievements of final model are stated in the end.

Cirkulirajuće tumorske stanice (CTC) najvjerojatnije imaju ključnu ulogu u posredovanju nastanka metastaza. CTC-i putuju kroz krvotok i limfni sustav. Hipoteze današnjih istraživanja jesu da CTC-i idu na udaljena mjesta u različite organe i tkiva. Moraju preživjeti u stranom mikrookolišu a zatim se razmnožavaju, čime se stvaraju mikroskopske, potom makroskopske, a potom i kliničke značajne neoplastične izrasline., Circulating tumor cells (CTC) most likely play a key role in mediating the formation metastasis. CTCs travel through the blood stream and lymphatic system. Today Hypotheses of many researches are that CTCs will go to distant places in different organs and tissues. They have to survive in a foreign microenvironment and then proliferate, thus creating a microscopic than macroscopic, and then clinically significant neoplastic growths.

Sustav urokinaznoga plazminogenskoga aktivatora (uPA) regulira razgradnju izvanstaničnog matriksa aktivacijom sveprisutne proteaze plazmina. Sudjeluje u nizu fizioloških i patoloških procesa te stanično je specifično reguliran u organizmu na razini ekspresije proenzima, inhibitora i receptora. U ovom je radu opisana regulacija sustava uPA kod stanica glioblastoma A1235, manjkavih u popravku alkilirajućeg oštećenja, nakon obrade inhibitorom poli(ADP-ribozil) polimeraze-1 (PARP-1) i alkilirajućim agensom N-metil-N'-nitro-N-nitrozoguanidinom (MNNG). MNNG je povećao uPA aktivnost u uskom rasponu koncentracija, u uvjetima visoke vijabilnosti, ali i zaustavljenog staničnog ciklusa. U tim je uvjetima inhibicija PARP-1 modulirala indukciju uPA aktivnosti inhibicijom popravka oštećenja DNA. Aktivacija uPA bila je posljedica promjene u odnosu ekspresije uPA i njegovog inhibitora PAI-1 u čemu je sudjelovao niz signalnih puteva. U drugom dijelu istraživanja našli smo da je derivat aspirina, natrijev salicilat (NaS) kod stanica tumora dojke MDA MB-231 promijenio ekspresiju uPA i PAI-1 i na taj način smanjio uPA aktivnost u ovisnosti o koncentraciji i vremenu tretmana. NaS je također smanjio ekspresiju integrinskih podjedinica αV i β5, zaustavio stanični rast i inducirao morfološke promjene. Ovi rezultati svjedoče o složenoj regulaciji sustava uPA koja je stanično specifična i uključuje niz signalnih puteva. Urokinase plasminogen activator (uPA) system regulates extracellular matrix remodeling by activating ubiquitous protease plasmin. The system is involved in various physiological and pathological processes and its regulation is cell specific involving the regulation of proenzyme, its inhibitors and receptor. This thesis describes the regulation of uPA system in A1235 glioblastoma cells, deficient in alkylation damage repair, after the treatment with poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor and alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MNNG has induced an increase in uPA activity in the narrow range of concentrations, under the conditions of high viability and the cell cycle arrest. In these experimental conditions, inhibition of PARP-1 has modulated induced uPA activity influencing the process of DNA repair. Increase in uPA activity was a consequence of change in the balance between uPA and its inhibitor PAI-1 expression and involved various signaling pathways. In the second part of this research, we found that sodium salicylate (NaS), a derivative of aspirin, has changed the expression of uPA and PAI-1 in MDA MB-231 breast cancer cells and inhibited uPA activity in a dose- and time-dependent manner. NaS has also inhibited the expression of αV i β5 integrin subunits, arrested cell growth and induced morphological changes. These results altogether confirmed the complex nature of uPA system regulation which is cell specific and involves various signaling pathways.

The major obstacle of successful tumor treatment with carboplatin (CBP) is the development of drug resistance. In the present study, we found that following treatment with CBP the amount of platinum which enters the human laryngeal carcinoma (HEp2)-derived CBP- resistant (7T) cells is reduced relative to the parental HEp2. As a consequence, the formation of reactive oxidative species (ROS) is reduced, the induction of endoplasmic reticulum (ER) stress is diminished, the amount of inter- and intrastrand cross-links is lower, and the induction of apoptosis is depressed. In HEp2 cells, ROS scavenger tempol, inhibitor of ER stress salubrinal, as well as gene silencing of ER stress marker CCAAT/enhancer-binding protein (CHOP) increases their survival and renders them as resistant to CBP as 7T cell subline but did not influence the survival of 7T cells. Our results suggest that in HEp2 cells CBP-induced ROS is a stimulus for ER stress. To the contrary, despite the ability of CBP to induce formation of ROS and activate ER stress in 7T cells, the cell death mechanism in 7T cells is independent of ROS induction and activation of ER stress. The novel signaling pathway of CBP- driven toxicity that was found in the HEp2 cell line, i.e. increased ROS formation and induction of ER stress, may be predictive for therapeutic response of epithelial cancer cells to CBP-based therapy.

Toll-like receptor 3, uz ostale receptore "Toll-like" (TLR, od engl. Toll-like receptors), ima ključnu ulogu u urođenoj imunosti. Nedavna istraživanja pokazala su da se TLR3, kao i ostali srodni receptori, nalazi izražen i u stanicama određenih vrsta tumora, te da njegova aktivacija potiče apotozu stanica tumora i inhibira njihovu proliferaciju. Također je otkriveno da aktivacija TLR3 u kombinaciji s protutumorskim terapeuticima povećava citotoksični učinak agonista TLR3 na stanice tumora. To dovodi do zaključka da se agonisti TLR3 mogu koristiti kao protutumorski agensi zbog svojeg citostatskog i citotoksičnog djelovanja na stanice tumora. U ovom istraživanju ispitali smo djelovanje liganda TLR3, poliriboinozilne:poliribocitidilne kiseline (poly(I:C)), u kombinaciji s kemoterapeuticima 5-fluorouracilom i metotreksatom, na stanice primarnog i metastatskog tumora ždrijela. Svrha ovog rada jest ispitati mogućnost kombinirane primjene poly(I:C) i kemoterapeutika kao protutumorske terapije. Prema dobivenim rezultatima ustanovili smo da kombinirana primjena poly(I:C) i kemoterapeutika ima značajno veći inhibitorni učinak na stanice nego zasebni tretman s poly(I:C), tj. da poly(I:C) i kemoterapeutici 5-fluorouracil i metotreksat djeluju sinergistički. Također smo utvrdili da je inhibitorni učinak kombiniranog tretmana s poly(I:C) i kemoterapeuticima posljedica poly(I:C)-aktivirane apoptoze ovisne o TLR3-signalnom putu. Toll-like receptor 3, as well as other TLRs, plays a key role in innate immunity. Recent studies have shown that TLR3 is expressed on certain types of tumor cells and that its activation stimulates apotosis of tumor cells and inhibits their proliferation. Also it has been found that activation of TLR3 in combination with antitumor therapeutics increases the cytotoxic effect of TLR3 agonist on tumor cells. This leads to the conclusion that the TLR3 agonists can be used as antitumor agents because of their citostatic and cytotoxic effects on tumor cells. In this study, we investigated the effect of TLR3 ligand, polyinosinic:polycytidylic acid (poly(I:C)), in combination with chemotherapeutics 5-fluorouracil and methotrexate on primary and metastatic pharyngeal carcinoma cell lines. The purpose of this study was to examine the possibility of combined application of poly(I:C) and chemotherapeutics as an antitumor therapy. We have found that combined application of poly(I:C) and chemotherapeutics has a significantly greater inhibitory effect on cell growth than the separate treatment with poly(I:C), i.e. poly(I:C) and chemotherapeutics act synergistically. We also found that the inhibitory effect of combined treatment with poly(I:C) and chemotherapeutics is a result of TLR3-dependent apoptosis.