20.500.12556/RUL-185723 Detekcija dimerizacije proteina TDP-43 s presejalnima sistemoma Tox in Lex Detection of TDP-43 protein dimerization using the Tox and Lex screening systems Protein z molekulsko maso 43 kDa, ki veže transaktivacijski odzivni element molekule DNA, znan tudi kot TDP-43, je visoko ohranjen RNA- in DNA-vezavni protein, vključen v uravnavanje procesiranja RNA. Pretežno se nahaja v jedru, kopičenje agregatov v citoplazmi pa je značilno za bolnike z amiotrofično lateralno sklerozo (ALS) in frontotemporalno demenco (FTD). TDP-43 je sestavljen iz N-končne domene, dveh RNA-prepoznavnih domen, C-končne domene, jedrnega lokalizacijskega signala in signala za transport iz jedra. V tem diplomskem delu smo detektirali dimerizacijo TDP-43 s presejalnima sistemoma Tox in Lex z uporabo celotnega proteina TDP-43 in njegovih dveh delecijskih mutantov. Zapise za TDP-43 in dva delecijska mutanta, TDP-43ΔNTD in TDP-43ΔNTDΔRRM1, smo pomnožili s PCR iz obstoječih plazmidov, pri čemer smo pripravili tudi različice z dodanim fuzijskim partnerjem vezavni protein za maltozo (MBP) na C-koncu. Pripravljene konstrukte smo vstavili v sistema Tox in Lex ter spremljali fluorescenco kot posledico dimerizacije testnega proteina. Sistem Tox je aktivatorski, kar pomeni, da se v primeru homodimerizacije testnega proteina fluorescenca poviša. Nasprotno se zgodi pri sistemu Lex, ki je represorski, saj se ob homodimerizaciji testnega proteina fluorescenca zniža. TDP-43 je v obeh sistemih pokazal statistično značilen signal dimerizacije. Pri delecijskih mutantah TDP-43ΔNTD in TDP-43ΔNTDΔRRM1 rezultata med sistemoma nista bila skladna, saj smo v sistemu Tox pri obeh mutantah zaznali šibko dimerizacijo, medtem ko v sistemu Lex dimerizacije nismo zaznali. Dodatek fuzijskega partnerja MBP na C-konec delecijskih mutantov je v obeh sistemih zmanjšal signal dimerizacije pri mutantih, medtem ko je pri TDP-43 dimerizacijski signal ostal enak, oziroma višji. Rezultati kažejo, da je za dimerizacijo proteina TDP-43 pomembna N-končna domena. Transactive response DNA binding protein 43 kDa (TAR DNA-binding protein 43), a highly conserved RNA- and DNA-binding protein, is involved in the regulation of RNA processing. It is predominantly localized in the nucleus, while the accumulation of cytoplasmic aggregates is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. TDP-43 consists of an N-terminal domain, two RNA-recognition domains, a C-terminal domain, a nuclear localization signal, and a nuclear export signal. In this thesis, we detected the dimerization of TDP-43 using the Tox and Lex screening systems, employing wild-type TDP-43 and its two deletion mutants. Sequences encoding TDP-43 and two deletion mutants, TDP-43ΔNTD and TDP-43ΔNTDΔRRM1, were amplified by PCR from existing plasmids, and variants with the fusion partner maltose-binding protein (MBP) added at the C-terminus were also prepared. The prepared constructs were inserted into the Tox and Lex systems, and fluorescence was monitored as a readout of potential dimerization of the test protein. The Tox system is activator-based, meaning that homodimerization of the test protein increases fluorescence. The opposite occurs in the Lex system, which is repressor-based, where homodimerization of the test protein decreases fluorescence. Full-length TDP-43 showed a statistically significant dimerization signal in both systems. For the deletion mutants TDP-43ΔNTD and TDP-43ΔNTDΔRRM1, results were inconsistent between the two systems: in the Tox system, weak dimerization was detected for both mutants, whereas no dimerization was detected in the Lex system. The addition of the fusion partner MBP to the C-terminus of the deletion mutants reduced the dimerization signal in mutants in both systems, while for TDP-43 the dimerization signal remained unchanged or increased. These results suggest that the N-terminal domain is important for TDP-43 dimerization. TDP-43 presejalni sistem Tox Lex amiotrofična lateralna skleroza dimerizacija TDP-43 screening system Tox Lex amyotrophic lateral sclerosis dimerization true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2026-08-19 08:45:04 2026-08-19 08:45:12 2026-08-28 12:13:23 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 25952 289277699 Tepus_Taja_-_Detekcija_dimerizacije_proteina_TDP-43_s_presejalnima_sistemoma_ToxR_in_LexA.pdf Tepus_Taja_-_Detekcija_dimerizacije_proteina_TDP-43_s_presejalnima_sistemoma_ToxR_in_LexA.pdf 1 46E5CAE373D98E30CBCBCB1C21CF62BD a419728bd8fdb71ad57ad678f90873550aa0e602c0820be70325e8d5ca27eab4 281c3d6f-9b99-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=244544 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0