o:31809 Transdiferencijacija alfa ćelija pankreasa miša u ćelije koje proizvode insulin ciljanom metilacijom DNK primenom Epi-CRISPR sistema doktorska disertacija Transdifferentiation of mouse pancreatic alpha to insulin-producing cells using Epi-CRISPRs directed DNA methylation : doctoral dissertation sr Ekspresija gena za Arx (engl. Aristaless related homeobox) koji ima glavnu ulogu u održavanju identiteta alfa ćelija endokrinog pankreasa regulisana je metilacijom i predstavlja glavni target za ćelijsko reprogramiranje kao jedna od strategija u terapiji dijabetesa koji u osnovi ima poremećen izvor insulina usled propadanja beta ćelija.Cilj ove doktorske disertacije je podrazumevao ispitivanje sposobnosti transdiferencijacije alfa ćelija pankreasa miša u ćelije koje proizvode insulin nakon uvođenja metilacije u promotoru Arx-a. Nakon tranzijentne transfekcije αTC1-6 ćelijske linije pomoću epigenetičkog alata za ciljanu gensku represiju ispitivani su efekti uvedene metilacije na ekspresiju gena specifičnih za beta ćelije.Optimizacijom nukleofekcije αTC1-6 ćelija uspostavljeni su uslovi pomoću kojih je dostignuta efikasnost od 71,1% pri vijabilnosti ćelija od 80%. Visoka efikasnost uvođenja metilacije dCas9-Dnmt3a3L-KRAB fuzionim (EpiCRISPR) konstruktom je pokazana targetovanim bisulfitnim sekvenciranjem. Utišavanje Arx-a praćeno pokretanjem ekspresije Ins2 na 5. i 7. danu nakon transfekcije detektovano je metodom RT-qPCR-a i analizom transkriptoma. Proteinski nivo insulina detektovan je imunocitohemijskom metodom do 12. dana, a oslobađanje iz ćelija enzimskim imunoesejem na 7. danu nakon transfekcije. Pokretanje procesa transdiferencijacije αTC1-6 ćelija ispitivano je analizom prisustva markera beta ćelija.Rezultati su pokazali da jedna tranzijentna transfekcija može da inicira transdiferencijaciju ~1% alfa ćelija pankreasa u ćelije koje proizvode 35% više insulina u odnosu na lažno transfekovane (Mock) alfa ćelije. Delujući na plastičnu prirodu epigenoma, uspešno je iniciran proces direktnog reprogramiranja alfa ćelija pankreasa u ćelije koje proizvode insulin. Aristaless-related homeobox (Arx) gene expression level is regulated by DNA methylation, plaing an important role in the maintenance of pancreatic alpha cell identity. Diabetes is characterized by a disturbed source of insulin, representing a good candidate for cell reprogramming strategy in diabetes therapy by Arx targeting.The aim of this doctoral dissertation was to examine the transdifferentiation ability of murine pancreatic alpha cells into insulin-producing cells induced by the targeted DNA methylation in the Arx promoter. The expression of beta specific marker was analiysed in transiently transfected αTC1-6 cells with a synthetic epigenetic tool for gene repression.The optimization of αTC1-6 cells nucleofection was established conditions by which was achieved an efficiency of 71.1% with an 80% of cell viability. The high efficiency of methylation induction by the dCas9-Dnmt3a3L-KRAB fusion (EpiCRISPR) construct was confirmed by targeted bisulfite sequencing. The Arx silencing followed by induction of Ins2 expression on 5 and 7 days after transfection was detected by RT-qPCR and transcriptome analysis. The insulin protein level was detected immunocytochemicaly until the 12th post-transfection day, and released insulin was detected by the enzyme immunoassay on the 7th post-transfection day. The initiation of the transdifferentiation process of αTC1-6 cells was examined by analyzing the presence of beta cell specific markers.The results showed that a single transient transfection initiate the transdifferentiation of ~1% of alpha cells into cells that produce 35% more insulin compared to mock-transfected cells. Acting on the epigenome plastic nature, the direct reprogramming of pancreatic alpha cells into insulin-producing cells was successfully initiated. Biologija - Molekularna biologija / Biology- Molecular biology Datum odbrane: 05.07.2023. Arx, CRISPR/Cas9, alfa ćelije pankreasa, dijabetes, editovanje epigenoma, ciljana metilacija DNK, transfekcija ARX, CRISPR/dCas9, pancreatic alpha cells, diabetes, epigenetic editing, targeted DNA methylation, transfection 577.2:577.175.72(043.3) 91552100 131078153 91552101 9366 2023-11-27T11:33:52.053Z 45 no 46 Marija B., 1990- Đorđević 100898569 2023 63 mentor Jelena, 1977- Arambašić-Jovanović 13686375 2023 63 mentor Melita, 1972- Vidaković 22054759 2023 63 član komisije Svetlana, 1971- Dinić 33414247 2023 63 član komisije Anja, 1988- Tolić 33400935 2023 63 član komisije Dušanka, 1972- Savić-Pavićević 12846951 2023 96 str. 6708104 http://phaidrabg.bg.ac.rs/o:31809 no yes 13 2023-11-27T11:33:52.320Z 70 11 1066841 1066867 Arx, CRISPR/Cas9, alfa ćelije pankreasa, dijabetes, editovanje epigenoma, ciljana metilacija DNK, transfekcija ARX, CRISPR/dCas9, pancreatic alpha cells, diabetes, epigenetic editing, targeted DNA methylation, transfection 577.2:577.175.72(043.3) 1738 11A02 2023