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INTRODUCTION: Ochratoxin A (OTA) is a worldwide spread mycotoxin that contaminates our food. Detrimental effects of OTA on human health are evident by damage to renal cells, causing nephropathies. Long non-coding RNAs (lncRNAs) are also implicated in the pathogenesis of renal nephropathies and cancers. Herein I report on a novel lncRNA, WISP1-AS1, an antisense transcript in the WISP1 locus. OTA is the only known inducer of WISP1-AS1. However, WISP1-AS1 functions in OTA-treated cells were undiscovered. This research was set to investigate WISP1-AS1 characteristics and functions in OTA toxicity and renal cancer cells and to understand the molecular mechanisms behind it. METHODS: WISP1-AS1 expression in normal and cancer renal cells was investigated by PCR methods and Northern blot. WISP1-AS1 knockdown was established by LNA™ GapmeR antisense oligonucleotides (ASOs). By RNAsequencing, influence of WISP1-AS1 on transcriptome was evaluated. Changes in transcription factor (TF) activities were analyzed by promoter activity assays. Glucose consumption and lactate production were determined by specific enzymatic assays. The effect of WISP1-AS1 on cell death (apoptosis and necrosis) was assessed by measuring caspase activities and lactate dehydrogenase (LDH) release. Nuclear fragmentation and mitochondrial membrane potential were analyzed by Cytation 3 Cell Imaging Multi-Mode Reader. RESULTS: WISP1-AS1 transcribes in antisense in comparison with WISP1 mRNA. Expression in antisense was confirmed by Northern blot. WISP1-AS1 transcription is completely inside the WISP1 locus and does not suppress WISP1 mRNA. WISP1-AS1 influences the activity of certain TFs. Promoter activity assays confirmed that the activity of TF EGR-1 was increased and E2F activity was suppressed in a WISP1-AS1-dependent manner. WISP1-AS1 expression was elevated in renal cancer cells and OTA further upregulated it. High WISP1-AS1 expression in renal cancer cells without OTA treatment indicates its implication in carcinogenesis. Functional experiments demonstrate that WISP1-AS1 might direct glucose towards mitochondrial respiration to obtain ATP. In WISP1-AS1 presence, caspase-3 activity was significantly low and the GAS6 gene, involved in caspase-3 suppression, was upregulated. CONCLUSIONS: WISP1-AS1 is an antisense noncoding transcript. WISP1-AS1 upregulates the activity of TF EGR-1 and suppresses the activity of E2F. It lowers caspase-3 activity and acts antiapoptotic, possibly by upregulating GAS6. In WISP1-AS1 presence, OTA-treated cells maintain normal energy metabolism.

Metilacija igra važnu ulogu u regulaciji bioloških procesa. Nepravilnosti u DNA metilaciji su uključene u nastanak različitih poremećaja zdravlja u ljudi. Nedostatnost S-adenozil homocistein hidrolaze (AHCY) je nova bolest, otkrivena u Hrvatskoj populaciji 2004. godine. Bolest rezultira u upečatljivom povišenju S-adenozil homocisteina (adoHcy) i S-adenozil metionina (adoMet) u krvi i različitim tkivima. AHCY uspješno uklanja adoHcy, nusprodukt transmetilacijskih reakcija i moćan inhibitor DNA metiltransferaza. Dakle, AHCY igra ključnu ulogu u regulaciji biološke metilacije. Cilj mog istraživanja bio je procijeniti promijene u DNA metilaciji nastale zbog nedostatnosti AHCY. Zato je DNA izolirana iz stanične linije ljudskog tumora jetre, HepG2, koja je utišana u AHCY ekspresiji korištenjem RNA interferencijske tehnologije. DNA metilacijski status je proučen na odabranim utisnutim lokusima te utvrđen bisulfitnim sekvenciranjem. Lokusi sa značajnim promjenama u metilacijskom statusu se mogu koristiti kao biomarkeri za detekciju bolesti. Methylation plays an important role in regulating biological processes. Abnormalities in DNA methylation are involved in the etiology of various human disorders. S-adenosylhomocysteine hydrolase (AHCY) deficiency is a novel human disease, discovered in Croatian population in 2004. The disease results in striking elevations of S-adenosylhomocysteine (adoHcy) and S-adenosylmethionine (adoMet) in blood and various tissues. AHCY efficiently removes adoHcy, the by-product of transmethylation reactions and potent DNA methyltransferase inhibitor. Thus, AHCY plays a critical role in regulation of biological methylation. The goal of my research was to evaluate AHCY deficiency derived changes in DNA methylation. To do so, DNA was isolated from human liver tumor cell line, HepG2, which has been silenced for AHCY expression using RNA interference technology. DNA methylation status was studied on certain imprinted loci and confirmed by bisulfite sequencing. Loci with significant changes in methylation status may serve as biomarkers for disease detection.

Najvažnija komponenta stanice odgovorna za sastavljanje mikrotubula je gama tubulin. On potiče dimerizaciju alfa i beta tubulina i tako nastanak mikrotubula. Lokaliziran je naročito na površini jezgre biljnih stanica koja je otkrivena kao glavna mjesta sastavljanja mikrotubula. Također, kortikalna membrana i fragmoplast su mjesta na kojima se mikrotubuli sastavljaju. Visoka dinamika sastavljanja i rastavljanja mikrotubula vrlo je bitna kod diobe stanice kada se mikrotubuli intenzivno premještaju. Proteini povezani sa mikrotubulima imaju važnu ulogu u njihovoj stabilizaciji te pomažu sastavljanje mikrotubula. Buduća istraživanja bi trebala razjasniti molekularnu funkciju proteina koji potiču izduživanje mikrotubula na acentrosomalnim polovima i unutar diobenog vretena. Također, kontrolni mehanizmi koji reguliraju funkciju mikrotubulskih organizacijskih centara su još nedovoljno otkriveni. The most improtant component of the cell, responsable for microtubule assembly is gammatubulin. Gamma-tubulin induce polimerisation of alpha and beta tubulin so as microtubule assembly. Its localized mostly on the nuclei surface, and this site is discovered as most active nucleating site of microtubules. Cortical membrane and phragmoplast are also sites of microtubule assembly. Highly dynamic assembly and dissasembly of microtubules is very important in mitosis when microtubules fast move in the cell. Proteins associated with microtubules are very important in stabilization of micortubules and for their assembly. Further researches should explain molecular function of proteins which induce polimerization of microtubules on acentrosomal poles and in mitotic spindle. Also, control points of regulation microtubule`s organization centers are still insufficient explained.