20.500.12556/RUL-185665 Vrednotenje korelativne mikroskopije za identifikacijo avtofagosomskih predelkov v rakavih urotelijskih celicah po tretiranju z izvlečkom človeške amnijske membrane Evaluation of correlative microscopy for the identification of autophagosomal compartments in cancerous urothelial cells after treatment with a human amniotic membrane preparation Avtofagija ima ključno vlogo pri ohranjanju celične homeostaze in je v rakavih celicah pogosto spremenjena. Izvlečki človeške amnijske membrane (hAM) izkazujejo protivnetne in protitumorske učinke, njihov vpliv na avtofagijo urotelijskih celic pa še ni pojasnjen. Namen diplomskega dela je bil ovrednotiti uporabnost korelativne mikroskopije (CLEM), ki združuje slikanje istega celičnega področja najprej s svetlobno in nato še z elektronsko mikroskopijo, za identifikacijo avtofagosomskih predelkov v rakavih urotelijskih celicah T24 po 24-urnem tretiranju z izvlečkom hAM. Celice smo kemijsko fiksirali, vklopili v želatino in po metodi Tokuyasu pripravili krioultratanke rezine, na katerih smo imunooznačili označevalec avtofagije LC3B (Alexa Fluor 555) in označili jedra (Hoechst). Iste regije smo zaporedoma posneli s konfokalnim in presevnim elektronskim mikroskopom ter mikrografije korelirali s programsko opremo ImageJ/Fiji in vtičnikom BigWarp. V sedmih sklopih imunooznačevanja (skupno 18 mrežic in 7 koreliranih vzorcev) je barvilo Hoechst zanesljivo označilo jedra in omogočilo korelacijo med svetlobno in elektronsko ravnino, medtem ko je bil signal LC3B šibek in pretežno nespecifičen. Elektronska mikroskopija v naših vzorcih večinoma ni omogočila nedvoumne prepoznave dvomembranskih veziklov. Zaradi teh omejitev in artefaktov pri pripravi vzorcev po metodi Tokuyasu (nespecifična vezava protiteles, gubanje in trganje rezin ter sploščena morfologija celic) formalne kvantifikacije avtofagosomov nismo izvedli. Rezultati kažejo, da metoda v trenutni obliki še ni optimizirana za kvantitativno analizo avtofagosomskih predelkov v celicah T24, hkrati pa predstavljajo pomembno metodološko izhodišče in smernice za nadaljnjo optimizacijo protokola ter njegovo kombinacijo z molekularnimi metodami (prenos western, qPCR). Autophagy plays a key role in maintaining cellular homeostasis and is frequently altered in cancer cells. Extracts of the human amniotic membrane (hAM) exhibit antiinflammatory and antitumour effects, yet their impact on autophagy in urothelial cells remains unclear. The aim of this thesis was to evaluate the suitability of correlative microscopy (CLEM), which combines light and electron microscopy of the same cellular region, for identifying autophagosomal compartments in T24 cancerous urothelial cells after 24 h of treatment with an hAM extract. Cells were chemically fixed, embedded in gelatin and processed by the Tokuyasu method into cryo-ultrathin sections, on which the autophagy marker LC3B (Alexa Fluor 555) was immunolabelled and nuclei were stained with Hoechst. The same regions were imaged sequentially by confocal and transmission electron microscopy and the micrographs correlated using ImageJ/Fiji software and the BigWarp plugin. Across seven immunolabelling runs (18 grids and 7 correlated samples in total), Hoechst reliably labelled nuclei and enabled correlation between the light and electron planes, whereas the LC3B signal was weak and largely non-specific. The electron-microscopy was mostly unsuccessful unambiguous identification of doublemembrane vesicles. Because of these limitations and the artefacts arising during Tokuyasu sample preparation (non-specific antibody binding, section folding and tearing, and flattened cell morphology), formal quantification of autophagosomes was not performed. The results indicate that, in its current form, the method is not yet optimised for quantitative analysis of autophagosomal compartments in T24 cells, but they provide a methodological basis and guidance for further protocol optimisation and its combination with molecular methods (western blot, qPCR). avtofagija rakave urotelijske celice (T24) človeška amnijska membrana (hAM) LC3B-II korelativna mikroskopija (CLEM) autophagy cancerous urothelial cells (T24) human amniotic membrane (hAM) LC3B-II correlative microscopy (CLEM) true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2026-08-17 10:55:19 2026-08-17 10:55:34 2026-08-28 08:37:12 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 26095 289200387 Robek_Tinkara_-_Vrednotenje_korelativne_mikroskopije_za_identifikacijo_avtofagosomskih_predelkov.pdf Robek_Tinkara_-_Vrednotenje_korelativne_mikroskopije_za_identifikacijo_avtofagosomskih_predelkov.pdf 1 6B0E059F2EFD4E6B716FFD0D39AF8007 912d1388ca8b8ea3b9b03aa4a28e2cbdd1c1ce26059e9a83f4e8bc2057457ccd 0f94c12c-9a19-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=244419 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0