20.500.12556/RUL-184028 Vrednotenje optičnih lastnosti strukturno raznolikih fluorescenčnih sond v prisotnosti fibril amiloida β Evaluation of the optical properties of structurally diverse fluorescent probes in the presence of β-amyloid fibrils Alzheimerjeva bolezen je najpogostejša oblika demence in predstavlja pomemben zdravstveni problem starajoče se populacije. Ena izmed njenih ključnih patoloških značilnosti je kopičenje amiloida β (Aβ), ki lahko prehaja iz topnih oblik v oligomere, fibrile in amiloidne plake. Ker se odlaganje Aβ začne že pred pojavom izrazitejših kliničnih simptomov, je razvoj občutljivih metod za zaznavanje amiloidnih agregatov pomemben za razumevanje bolezni in razvoj diagnostičnih pristopov. Pri tem so uporabne fluorescenčne sonde, pri katerih se emisija fluorescence spremeni po vezavi na amiloidne fibrile. V magistrski nalogi smo vrednotili optične in vezavne lastnosti strukturno raznolikih fluorescenčnih sond v prisotnosti fibril Aβ z dolžino 42 aminokislin – Aβ1–42. Izmerili smo absorpcijske in emisijske spektre sond v pufru ter v prisotnosti fibril Aβ1–42, neagregiranega Aβ1–42, humanega in govejega serumskega albumina ter fibril inzulina. Za izbrane sonde smo določili tudi navidezne vezavne afinitete do fibril Aβ1–42. Poseben poudarek smo namenili sondi ALZ290, ki je bila v literaturi opisana kot fluorescenčna sonda za zaznavanje agregatov Aβ z emisijo v bližnjem infrardečem območju, zato smo pri različnih pripravah fibril Aβ1–42 in drugih fibrilarnih proteinskih sistemih dodatno ovrednotili spremembo intenzitete fluorescence in lego emisijskega maksimuma. Pri večini sond dodatek fibril Aβ1–42 ni povzročil izrazitih sprememb v absorpcijskih spektrih, razlike pa so bile izrazitejše pri emisijskih spektrih. Največje povečanje intenzitete fluorescence je pokazala spojina ALZ290, izrazitejše povečanje pa so pokazale tudi nekatere druge sonde iz serije spojin ALZ. Pri večini sond neagregirani Aβ1–42 ni povzročil znatnega povečanja intenzitete fluorescence, kar kaže na povezavo spremembe emisije predvsem s fibrilarno obliko peptida. Meritve z albuminoma in fibrili inzulina so pokazale različno selektivnost sond v netarčnih proteinskih sistemih. Najugodnejše vezavne afinitete do fibril Aβ1–42 so imele sonde ALZ299, ALZ300 in ALZ293. ALZ290 sicer ni imela najnižje vrednosti disociacijske konstante, vendar je združila izrazito povečanje intenzitete fluorescence, dobro vezavno afiniteto in emisijo v bližnjem infrardečem območju. Primerljivo povečanje intenzitete fluorescence pri fibrilah Aβ1–42, pripravljenih pod različnimi pogoji, kaže na robustnost optičnih in vezavnih lastnosti ALZ290, povečanje intenzitete fluorescence pri fibrilah inzulina in alfa-sinukleina pa nakazuje, da sonda ni povsem selektivna za fibrile Aβ1–42. Alzheimer’s disease is the most common form of dementia and represents an important health problem in the ageing population. One of its key pathological features is the accumulation of amyloid β (Aβ), which can transition from soluble forms into oligomers, fibrils and amyloid plaques. Since Aβ deposition begins before pronounced clinical symptoms appear, sensitive methods for detecting amyloid aggregates are important for understanding the disease and developing diagnostic approaches. Fluorescent probes are useful in this context, as their fluorescence emission changes upon binding to Aβ fibrils. In this master’s thesis, we evaluated the optical and binding properties of structurally diverse fluorescent probes in the presence of Aβ1–42 fibrils. Absorption and emission spectra were measured in buffer and in the presence of Aβ1–42 fibrils, non-aggregated Aβ1–42, human and bovine serum albumin, and insulin fibrils. For selected probes, apparent binding affinities to Aβ1–42 fibrils were also determined. Special emphasis was placed on ALZ290, which has been described in the literature as a fluorescent probe for detecting Aβ aggregates with emission in the near-infrared region. For this probe, changes in fluorescence intensity and the position of the emission maximum were additionally evaluated using different preparations of Aβ1–42 fibrils and other fibrillar protein systems. For most probes, Aβ1–42 fibrils did not cause pronounced changes in the absorption spectra, whereas the differences were more evident in the emission spectra. The greatest increase in fluorescence intensity was observed for ALZ290, while some other probes from the ALZ series also showed a more pronounced increase. In the presence of non-aggregated Aβ1–42, most probes did not show a substantial increase in fluorescence intensity, indicating that the change in emission was mainly associated with the fibrillar form of the peptide. Measurements with albumins and insulin fibrils showed differences in probe selectivity in non-target protein systems. The most favorable binding affinities to Aβ1–42 fibrils were observed for ALZ299, ALZ300 and ALZ293. Although ALZ290 did not have the lowest dissociation constant, it combined a pronounced increase in fluorescence intensity, good binding affinity and emission in the near-infrared region. A comparable increase in fluorescence intensity in the presence of Aβ1–42 fibrils prepared under different conditions indicates the robustness of the optical and binding properties of ALZ290, whereas the increase in fluorescence intensity in the presence of insulin and alpha-synuclein fibrils suggests that the probe is not completely selective for Aβ1–42 fibrils. amiloid β fluorescenčne sonde emisijski spektri vezavna afiniteta amyloid β fluorescent probes emission spectra binding affinity true false false [M. Čadež] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-06-24 08:45:07 2026-06-24 08:45:19 2026-07-07 15:18:50 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 616.894(043.2) 125558 282898435 32308.pdf 32308.pdf 1 6D610B780B7F27D99B304C876C265EDB 1bf8b13776820c0e50821eaf2eab723596d76ea04307bb45504a45d030b7d7da 30c60791-6f98-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=236473 Fakulteta za farmacijo 0 0 0