20.500.12556/RUL-185975 Zaviranje poškodbe celične membrane z delovanjem na C-konec ninjurina 1 Plasma membrane rupture inhibition by acting on the C-terminus of ninjurin 1 Ninjurin 1 je transmembranski protein, ki po nastopu litične celične smrti polimerizira in povzroči poškodbo celične membrane, sprostitev znotrajceličnih molekul in s tem aktivacijo vnetja, ki pomembno vpliva na potek kroničnih vnetnih bolezni. Namen magistrskega dela je bil razviti dva pristopa k uravnavanju ninjurina 1 z delovanjem na C-konec. V prvem delu smo izvedli predstavitev na bakteriofagu z uporabo knjižnic Tomlinson I in J z namenom izolacije enoverižnih fragmentov variabilne regije protiteles, ki vežejo C-konec ninjurina 1. Z zaviranjem delovanja ninjurina 1 zaradi vezave protiteles bi namreč lahko dosegli omiljen potek kroničnega vnetja. Izvedli smo tri zaporedne stopnje elucije z zaostrujočimi pogoji spiranja, nato pa še encimsko-imunski test, s katerim smo preverili uspešnost selekcije. Rezultati so pokazali, da nam z metodo predstavitve na bakteriofagu ni uspelo pridobiti klona, ki bi izkazoval močno afiniteto do ninjurina 1. Nato smo se osredotočili na raziskovanje ali lahko poškodbo celične membrane uravnavamo tako, da na C-konec ninjurina 1 pripojimo globularno proteinsko domeno preko distančnika. V ta namen smo z metodami molekulskega kloniranja pripravili več konstruktov ninjurin 1-distančnik-globularna domena in z vnosom konstruktov v človeško celično linijo spremljali vpliv različnih komponent konstrukta na litično delovanje ninjurina 1. Delež celične smrti smo spremljali z merjenjem stopnje prevzema propidijevega jodida v celice in sproščanja laktat dehidrogenaze iz celic. Ugotovili smo, da večje globularne domene in krajši distančniki bolje zavirajo ninjurin 1. Ustvarjene konstrukte smo nato poskušali razcepiti na cepitvenem mestu, ki smo ga vnesli v distančnik in s tem reaktivirati ninjurin 1. To nam je uspelo v omejenem obsegu. Z načrtovano sprožitvijo imunogene celične smrti v tumorskem mikrookolju zaradi opisanega zaviranja in aktiviranja delovanja ninjurina 1 bi v tumorskem mikrookolju izzvali lokalno vnetje, kar bi predstavljalo inovativen pristop v protitumorski terapiji. V magistrskem delu smo poizkušali razviti dva načina uravnavanja poškodbe celične membrane, ki jo posreduje ninjurin 1. Čeprav nam z metodo predstavitve na bakteriofagu ni uspelo razviti fragmentov protiteles z močno afiniteto, smo v drugem delu raziskali inovativen način uravnavanja litične aktivnosti ninjurina 1. Pokazali smo, da lahko z delovanjem na C-konec ninjurina 1 učinkovito zavremo poškodbe celične membrane, kar bo vodilo v razvoj novih pristopov za nadzorovano sprožitev celične lize, predvsem pri imunoterapiji raka. Ninjurin-1 is a transmembrane protein that polymerizes and causes damage to the cell membrane after necrotic cell death, releasing intracellular components and thereby activating inflammation. Suppressing Ninjurin-1-mediated plasma membrane rupture could alleviate chronic inflammation. On the other hand, the regulated activation of immunogenic cell death through the action of Ninjurin-1 in the tumour microenvironment would provoke local inflammation, representing an innovative approach in antitumor therapy. This work aimed to develop two different approaches for regulating Ninjurin-1-induced plasma membrane rupture via its C-terminus. In the first part, we attempted to isolate single-chain variable fragments that bind the C-terminus of Ninjurin-1 by phage display using Tomlinson I and J libraries. We performed three successive elution steps with increasingly stringent washing conditions, followed by an enzyme-linked immunosorbent assay to verify the success of the selection. The results showed that the phage display method failed to obtain a clone that would show strong affinity for Ninjurin-1. We then focused on investigating whether cell membrane damage can be regulated by attaching a globular protein domain to the C-terminus of Ninjurin-1 via a spacer. For this purpose, we prepared several Ninjurin-1-spacer-globular domain constructs using molecular cloning methods and, by introducing the constructs into a human cell line, monitored the influence of different components of the construct on the lytic activity of Ninjurin-1. Cell death was monitored by measuring propidium iodide uptake and the release of lactate dehydrogenase from the cells. We found that larger globular domains and shorter spacers inhibit Ninjurin-1-mediated plasma membrane rupture better. We then attempted to cleave the constructs at the cleavage site that we introduced into the spacer and thereby reactivate Ninjurin-1. We succeeded in this to a limited extent, so further optimization of the cleavage would be necessary in the future. In this master's thesis, we attempted to develop two ways to regulate cell membrane damage mediated by Ninjurin-1. Although we were unable to develop antibody fragments with high affinity using phage display, in the second part, we investigated an innovative way to regulate the lytic activity of Ninjurin-1. We showed that by acting on the C-terminus of Ninjurin 1, we can effectively inhibit cell membrane damage, which will lead to the development of new approaches for the controlled induction of cell lysis, especially in cancer immunotherapy. Ninjurin 1 poškodba celične membrane vnetje protitumorska terapija Ninjurin 1 plasma membrane rupture inflammation anti-tumour therapy true false false [N. Jakop] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-08-25 08:45:34 2026-08-25 08:45:43 2026-09-04 07:22:22 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2026-09-04 577.2:576.3(043.2) 127186 288938755 32905.pdf 32905.pdf 1 A80E3EFAC26625180BD1D768FFA08277 eb6b6e11f0597999ae03a0bed949c89f9428593affe6de2584ecc9bd39c9bd94 305a2c07-a050-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=245012 Fakulteta za farmacijo 0 0 0