20.500.12556/RUL-180327 Pomen izražanja γ-enolaze v glioblastomskih matičnih celicah in uravnavanja njene aktivnosti s katepsinom X The importance of γ-enolase expression in glioblastoma stem cells and the regulation of its activity by cathepsin X Glioblastom (GBM) je najpogostejši in najagresivnejši primarni možganski tumor, za katerega so značilne visoka heterogenost, presnovna plastičnost in odpornost na zdravljenje. Ključno vlogo pri teh lastnostih imajo glioblastomske matične celice (angl. glioblastoma stem-like cells, GSC), ki ohranjajo sposobnost samoobnavljanja, multipotentnost in odpornost na stresne pogoje. Njihovo vedenje je tesno povezano z mikrookoljem, presnovnim stanjem in signalnimi potmi, ki uravnavajo proliferacijo, diferenciacijo in preživetje. Ena izmed molekul, ki pomembno vpliva na te procese, je γ-enolaza – encim glikolitične poti, ki ima poleg presnovne tudi signalno vlogo. Njeno delovanje uravnava katepsin X, cisteinska peptidaza, ki cepi C-končni del γ-enolaze in s tem vpliva na njeno funkcionalnost. Spremembe v delovanju obeh proteinov so bile že povezane z nevrodegenerativnimi in tumorskimi procesi, njuno medsebojno delovanje pa še ni popolnoma pojasnjeno. V okviru magistrske naloge smo s celično linijo U87 raziskali vpliv zaviranja katepsina X na izražanje in delovanje γ-enolaze ter na lastnosti, povezane z matičnostjo GSC. Celice smo gojili v treh gojiščih, in sicer kompletnem gojišču (CM), gojišču z dodanimi rastnimi dejavniki (NSM) in gojišču z odsotnostjo seruma (SFM). Uporabili smo kombinacijo morfoloških, kemijskih in celičnih analiz, s katerimi smo preučevali tvrobo sferoidov, izražanje površinskega označevalca CD133 in proteinsko raven encimov α- ter γ-enolaze. S proučevanjem vpliva zaviranja katepsina X smo celice, izpostavljene različnim gojiščem, dodatno tretirali z zaviralcem katepsina X AMS36. Rezultati so pokazali, da mikrookolje močno vpliva na rast in fenotip celic U87. Razlike med gojišči odražajo presnovno prilagodljivost celic in poudarjajo pomen aktivne oblike γ enolaze za njihovo preživetje ter maligni potencial. Zaviranje katepsina X je povečalo tvorbo sferoidov in izražanje CD133, kar nakazuje ohranjanje matičnega fenotipa. Analize so razkrile povečano raven celokupne in zmanjšano raven aktivne oblike γ-enolaze, kar potrjuje neposredno vlogo katepsina X v njeni proteolitični cepitvi. Ugotovitve nakazujejo, da katepsin X in γ-enolaza tvorita regulatorni sistem, ki vpliva na presnovo in matičnost GSC ter predstavlja novo terapevtsko strategijo pri zdravljenju glioblastoma. Glioblastoma (GBM) is the most common and most agressive primary brain tumor, characterized by high heterogenity, metabolic plasticity and resistance to therapy. A key role in these properties is played by glioblastoma stem cells (GSCs), which mantain the ability for self-renewal, multipotency and resistance to stress conditions. Their behaviour is closely regulated by microenvironment, metabolic state and signaling pathways that control proliferation, differentiation and survival. One of the molecules critically involved in these processes is γ-enolase, a glycolytic enzyme that also exerts non-metabolic signaling functions. Its activity is regulated by cathepsin X, a cysteine protease that cleaves the C terminal region of γ-enolase and thereby modulates its functionality. Altered activity of both proteins has been associated with neurodegenerative and tumor-related processes; however, their mutual interaction is not yet fully understood. In this master's thesis we investigated the effect of cathepsin X inhibiton on the expression and function of γ-enolase and on GSC-associated stemness properties using the U87 cell line. Cells were cultured in three different media: complete medium (CM), medium supplemented with growth factors (NSM), and serum-free medium (SFM). A combination of morphological, biochemical and cellular analyses was used to study spheroid formation, expression of the surface marker CD133 and protein levels of α- and γ-enolase. To evaluate the impact of cathepsin X inhibition, cells grown under different culture conditions were additionally treated with the cathepsin X inhibitor AMS36. The results showed that the microenvironment strongly influences cell growth and phenotype. Differences between culture conditions reflect the metabolic adaptability of the cells and highlight the importance of the active form of γ-enolase for GSC survival and malignant potential. Inhibition of cathepsin X increased spheroid formation and CD133 expression, indicating the maintenance of a stem-like phenotype. Protein analyses revealed increased levels of total γ-enolase and reduced levels of its active form, confirming the direct role of cathepsin X in its proteolytic processing. Overall, the findings suggest that cathepsin X and γ-enolase form a regulatory system that affects GSC metabolism and stemness and may represent a novel therapeutic strategy in glioblastoma treatment. glioblastom glioblastomske matične celice sferoidi presnova γ-enolaza katepsin X glioblastoma glioblastoma stem cells spheroids metabolism γ-enolase cathepsin X true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-03-06 08:45:06 2026-03-06 08:45:17 2026-03-07 04:40:07 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 124852 31845.pdf 31845.pdf 1 7DEAD24676FC206963448012A04CB0F9 2e51622d9e78c1ec9539f8a991609f076ca77e23d5dfd6ee4b4adfb285622aef 0768a52f-1930-11f1-a1ba-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=229604 Fakulteta za farmacijo 0 0 0