20.500.12556/RUL-145220 Razvoj fluorescenčnega testa za določanje količine ceramid fosfoetanolamina vezanega s holesterolom v lipidnih membranah Development of a fluorescent method for measuring the quantity of ceramide phosphoethanolamine complexed with cholesterol in lipid membranes Parodontalna bolezen je najpogostejša kronična vnetna bolezen pri ljudeh. Najpogostejši obliki sta gingivitis in parodontitis. Gingivitis je zgolj blažja oblika vnetja dlesni, ki pa se lahko razvije v hujšo vnetno obliko, parodontitis. Slednjega spremljajo globoko vnetje, nastanek obzobnih žepov, poškodbe kostnega tkiva in navsezadnje tudi izguba zob. Vnetje in poškodbe pripisujemo predvsem parodontalnim patogenom kot so različne po Gramu negativne bakterije. Te ustvarjajo zobne obloge, imenovane biofilmi, in znatno doprinesejo k razvoju in slabšanju bolezni. V membranah tovrstnih patogenov najdemo sfingolipid ceramid fosfoetanolamin (CPE), ki predstavlja potencialni biomarker (i) za odkrivanje parodontalnih bolezni, (ii) za spremljanje poteka razvoja bolezni in (iii) za spremljanje uspešnosti zdravljenja posameznika. V povezavi z egerolizini, edinstvenimi proteini, ki imajo sposobnost vezave na lipide, predstavlja CPE odlično možnost za razvoj novega načina diagnostike parodontalnih bolezni, saj je v znatni meri prisoten v membranah parodontalnih patogenov, v membranah sesalskih celic pa je skorajda nezaznaven. V magistrski nalogi smo zato poskušali vzpostaviti metodo določanja količine CPE v vzorcih multilamelarnih lipidnih veziklov z namenom razvoja diagnostične metode za spremljanje parodontalne bolezni. Diagnostični test temelji na meritvah fluorescence fluorescenčno označenega proteina egerolizina EryA-mCherry, ki se ob pomoči rekombinantnega proteina PlyB ireverzibilno veže na CPE v membranah umetno pripravljenih veziklov. Periodontal diseases are classified as the most common chronical diseases of nowdays. Among them, the most common are gingivits and periodontitis. Gingivitis is a mild inflammatory disease of gingiva, which can develop into a more severe form named periodontitis. Latter is known for deep inflammation of the tissue, emergence of gingival pockets, decomposition of bone tissue and finally loss of teeth. Inflammation and tissue breakdown is mainly caused by periodontal pathogens which mostly belong to Gram negative bacteria. They produce biofilms which are the main contributors for developement and progression of the disease. The membranes of these pathogens contain a unique sphingolipd ceramid phosphoethanolamine (CPE), which could be a potential biomarker for assesing the developement of periodontal diseases over time. Aegerolysins are unique proteins, that have the abillity to bind membrane lipids, particularly the CPE. Ceramide phosphoethanolamine could represents a great periodontal diseases-associated biomarker, since is scarsly present in mammalian cells. In the thesis we have focused on development of a method for measuring the quantity of CPE in lipid membranes of multilamelar vesicles in order to set a new diagnostic tool for periodontal diseases. The test is based on measuring the level of fluorescence of fluorescently tagged egerolysin EryA-mCherry, which binds irrevesibly to CPE in membranes of lipid vesicles when combined with its protein partner, pleurotolysin B. parodontalne bolezni ceramid fosfoetanolamin biomarker fluorescenca periodontal diseases ceramid phosphoethanolamine biomarker fluorescence true false false [N. Pirnat] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2023-04-13 11:42:39 2023-04-13 11:42:42 2023-04-18 14:18:28 0000-00-00 00:00:00 2023 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 616.31 217657 149597955 MAG_Pirnat_Nika_2023.pdf MAG_Pirnat_Nika_2023.pdf 1 F78525BCC5E0EF11A6AB37719FBEED46 4dd895dc6c0f624b207d0913f0d4d318191c7127748e44dd3df044debb677916 62e26ac5-d9df-11ed-a314-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=167936 Biotehniška fakulteta 0 0 0