20.500.12556/RUL-183696 Karakterizacija Nef-vsebujočih zunajceličnih veziklov iz mikroglij in limfocitov T in njihov vpliv na živčne matične celice Characterization of Nef-containing extracellular vesicles, released by microglia and T lymphocytes, and their effect on neural stem cells Ozadje: Okužba z virusom humane imunske pomanjkljivosti tipa 1 (HIV-1) se je iz bolezni s smrtnim izidom preoblikovala v kronično stanje, saj več kot 70 % zdravljenih posameznikov dosega dolgotrajno supresijo virusa. Kljub uspešni kombinirani protiretrovirusni terapiji (ART) ostajajo pomemben klinični problem pridružene bolezni, ki povzročajo spekter kognitivnih, motoričnih in vedenjskih motenj, znanih kot s HIV povezane nevrokognitivne motnje (HAND). Eden ključnih dejavnikov njihove patogeneze je prisotnost virusnih rezervoarjev v osrednjem živčevju (OŽ), zlasti v mikroglijah, kjer se izražajo virusni proteini, kot je Nef. V okviru doktorske naloge smo želeli opredeliti vlogo proteina Nef pri sproščanju in lastnostih zunajceličnih veziklov (ZV) ter vpliv Nef-ZV na živčne predniške celice (hNPC), ki imajo tudi nekatere lastnosti matičnih celic. Hipoteze: Zastavili smo naslednje hipoteze: (i) da izražanje Nef v mikroglijah vpliva na biofizikalne in molekularne lastnosti sproščenih zunajceličnih veziklov; (ii) da izražanje Nef v limfocitih T vpliva na biofizikalne in molekularne lastnosti sproščenih zunajceličnih veziklov in (iii) da zunajcelični vezikli, ki vsebujejo Nef, vplivajo na apoptozo, proliferacijo in/ali diferenciacijo živčnih matičnih celic. Metode: V študiji smo kot vir ZV uporabili celice človeških mikroglij (h-MG) in nesmrtne limfocite T (Jurkat), kot model živčnih matičnih celic pa hNPC. Z uporabo lentivirusnih vektorjev z inducibilnim sistemom TET-ON smo vzpostavili stabilni celični liniji h-MG in Jurkat, ki izražata protein Nef.GFP (oziroma GFP kot kontrolo). Izražanje proteinov smo preverili s pretočno citometrijo in fluorescenčno mikroskopijo. ZV smo izolirali iz celičnih kultur z diferencialnim ultracentrifugiranjem in jih dodatno očistili z gostotnim jodiksanolnim gradientom. Njihove biofizikalne lastnosti smo analizirali z metodo sledenja nanodelcem (NTA), nano-pretočno citometrijo in presevno elektronsko mikroskopijo (TEM), molekularno sestavo pa s prenosom western (proteini) ter nano-pretočno citometrijo (lipidi). Prisotnost proteina Nef v ZV smo dodatno določili s TEM v kombinaciji z imunooznačevanjem, nano-Nef ELISO in nano-pretočno citometrijo. Funkcionalne učinke ZV smo ovrednotili na hNPC s testiranjem metabolne aktivnosti, proliferacije, apoptoze in diferenciacije. V ta namen smo uporabili celične teste na osnovi fluorescence (metabolna aktivnost, proliferacija, apoptoza, nekroza) in luminescence (apoptoza) ter spremljali izražanje specifičnih označevalcev s fluorescenčno mikroskopijo in pretočno citometrijo. Vsi poskusi so vključevali ustrezne kontrole in ponovitve, rezultate pa smo analizirali z osnovnimi statističnimi metodami. Rezultati: V naši raziskavi smo vzpostavili izboljšana celična modela translacijsko aktivnih rezervoarjev HIV-1, ki omogočata stabilno in nadzorovano izražanje proteina HIV-1 Nef.GFP v celicah h-MG in Jurkat. Pokazali smo, da Nef.GFP selektivno spodbuja sproščanje ZV iz celic h-MG in Jurkat s specifičnimi biofizikalnimi (večje število, majhni, s tipično morfologijo in gostoto) in molekularnimi (obogateni z Nef, vezikularnimi proteini in lipidi značilnimi za lipidne rafte) lastnostmi. Z uporabo analiz na ravni posameznih veziklov smo pokazali, da je Nef.GFP prisoten v največ polovici Nef-ZV, medtem ko smo s TEM in dodatno imunooznačitvijo določili njegovo lokalizacijo na luminalni strani ZV. V okviru naloge smo prvič preučevali funkcionalno vlogo Nef-ZV na hNPC, s posebnim poudarkom na vplivu na diferenciacijo v astrocite. Nakazali smo, da Nef-ZV iz h-MG in rNef pri delu populacije hNPC spodbujata diferenciacijo v astrocite, v drugem delu populacije pa povečujeta proliferacijo. Poleg tega smo rNef povezali s povečano stopnjo apoptoze in nekroze pri hNPC, ki so bili usmerjeni v diferenciacijo v astrocite, medtem ko pri Nef, pakiranem v ZV, takšnega učinka nismo zaznali. Naša študija kaže na vlogo Nef-ZV pri uravnavanju celičnega stanja hNPC, vendar so za natančnejšo opredelitev te vloge potrebni dodatni neodvisni biološki poskusi. Zaključki: V doktorski nalogi smo pokazali, da izražanje Nef vpliva na biofizikalne in molekularne lastnosti sproščenih ZV iz mikroglij in limfocitov T, in s tem potrdili prvo in drugo hipotezo. Rezultati prav tako nakazujejo, da ZV, izolirani iz mikroglij in limfocitov T, vplivajo na apoptozo, proliferacijo in diferenciacijo hNPC, uporabljenih kot model živčnih matičnih celic, s čimer smo deloma potrdili tretjo hipotezo. Prispevek k znanosti: Naši rezultati prispevajo k boljšemu razumevanju vloge Nef in Nef-vsebujočih ZV v OŽ ter nakazujejo potencialne mehanizme, povezane z razvojem HAND, ki bi jih bilo smiselno dodatno raziskati v kompleksnejših eksperimentalnih modelih. S tem naše ugotovitve odpirajo možnosti za nadaljnje raziskave medcelične komunikacije ter potencialno razvoj ciljno usmerjenih terapevtskih pristopov. Background: Infection with human immunodeficiency virus type 1 (HIV-1) has transformed from a fatal disease into a chronic condition, as more than 70% of treated individuals achieve long-term viral suppression. Despite successful combination antiretroviral therapy (ART), comorbidities remain a significant clinical issue, causing a spectrum of cognitive, motor, and behavioral impairments known as HIV-associated neurocognitive disorders (HAND). One of the key factors in their pathogenesis is the presence of viral reservoirs in the central nervous system, particularly in microglia, where viral proteins such as Nef are expressed. Within the framework of this doctoral thesis, we aimed to define the role of the Nef protein in the release and properties of extracellular vesicles (EVs) on human neural progenitor cells (hNPCs), which also possess certain stem cell-like properties. Hypotheses: We formulated the following hypotheses: (i) Nef expression in microglia affects the biophysical and molecular properties of released extracellular vesicles; (ii) Nef expression in T lymphocytes affects the biophysical and molecular properties of released extracellular vesicles; and (iii) extracellular vesicles containing Nef affect apoptosis, proliferation, and/or differentiation of neural stem cells. Methods: In this study, we used human microglial cells (h-MG) and immortalized T lymphocytes (Jurkat) as sources of EVs, while hNPCs were used as a model of neural stem cells. Using lentiviral vectors with an inducible TET-ON system, we established stable h-MG and Jurkat cell lines expressing Nef.GFP prorein (or GFP as a control). Protein expression was verified by flow cytometry and fluorescence microscopy. EVs were isolated from cell culture supernatants by differential ultracentrifugation and additionally purified using an iodixanol density gradient. Their biophysical properties were analyzed using nanoparticle tracking analysis (NTA), nano-flow cytometry, and transmission electron microscopy (TEM), while their molecular composition was analyzed by Western blot (proteins) and nano-flow cytometry (lipids). The presence of Nef.GFP protein in EVs was additionally determined by TEM combined with immunolabeling, nano-Nef ELISA, and nano-flow cytometry. Functional effects of EVs were evaluated in hNPC by assessing metabolic activity, proliferation, apoptosis, and differentiation. For this purpose, fluorescence-based cell assays (metabolic activity, proliferation, apoptosis, necrosis) and luminescence-based assays (apoptosis) were employed, and the expression of specific markers was monitored using fluorescence microscopy and flow cytometry. All experiments included appropriate controls and replicates, and the results were analyzed using basic statistical methods. Results: In our study, we established improved cellular models of translationally active HIV-1 reservoirs, enabling stable and controlled expression of the HIV-1 Nef.GFP protein in h-MG and Jurkat cells. We demonstrated that Nef.GFP selectively promotes the release of EVs from h-MG and Jurkat cells with distinct biophysical (higher abundance, small size, typical morphology, and density) and molecular (enriched with Nef, vesicular proteins, and lipids characteristic of lipid rafts) properties. Using single-vesicle analysis approaches, we showed that Nef.GFP is present in up to half of Nef-EVs, while immuno-TEM analysis revealed its localization on the luminal side of the vesicles. Within the framework of this dissertation, we investigated for the first time the functional role of Nef-EVs in hNPCs, with particular emphasis on their effects on differentiation into astrocytes. We indicated that Nef-EVs derived from h-MG and recombinant Nef (rNef) promote differentiation into astrocytes in one subset of the hNPC population, while increasing proliferation in another subset. In addition, we associated rNef with an increased level of apoptosis and necrosis in hNPCs directed toward astrocytic differentiation, whereas no such effect was observed for Nef packaged within EVs. Our study suggests a role for Nef-EVs in regulating the cellular state of hNPCs; however, additional independent biological experiments are required to more precisely define this role. Conclusions: In this doctoral thesis, we demonstrated that Nef expression affects the biophysical and molecular properties of EVs released from microglia and T lymphocytes, thereby confirming the first and second hypotheses. The results also suggest that EVs isolated from microglia and T lymphocytes influence the apoptosis, proliferation, and differentiation of hNPCs, which were used as a model of neural stem cells, thereby partially confirming the third hypothesis. Contribution to science: Our results contribute to a better understanding of the role of Nef and Nef-containing EVs in the central nervous system and suggest potential mechanisms associated with the development of HAND that should be further investigated in more complex experimental models. Furthermore, our findings open new possibilities for future research into intercellular communication and the potential development of targeted therapeutic approaches. Virus humane imunske pomanjkljivosti (HIV) mikroglija limfociti T protein Nef zunajcelični vezikli (ZV) človeške živčne predniške celice (hNPC) diferenciacija s HIV povezane nevrokognitivne motnje (HAND) Human immunodeficiency virus (HIV) mikroglia T lymphocytes protein Nef extracellular vesicles (EVs) human neural progenotor cells (hNPC) differentiation HIV-associated neurocognitive disorder (HAND) true false false Slovenski jezik Angleški jezik Doktorsko delo/naloga 2026-06-17 14:05:17 2026-06-17 14:05:29 2026-06-18 04:34:04 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2027-12-31 39353 9063.pdf 9063.pdf 1 DDC3EDF0D1A2F9D06DE257FB4942E07D ff836302e0d37f70de9a0dd00750f8d5739fc050754bc769dbc32db7e670fa38 c631c0a1-6a44-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=235846 Medicinska fakulteta 0 0 0