20.500.12556/RUL-187499 Identifikacija epitopov in peptidnih mimetikov epitopov avtoantigena ADAMTS13 pri trombotični trombocitopenični purpuri Identification of autoantigen ADAMTS13 epitopes and peptide epitope mimetics in thrombotic thrombocytopenic purpura Trombotična trombocitopenična purpura (TTP) je življenjsko ogrožajoča avtoimunska bolezen, ki v arteriolah povzroči nastanek majhnih krvnih strdkov ali mikrotrombov. Nastanejo zaradi avtoprotiteles proti encimu ADAMTS13, ki se v telesu zdravega posameznika veže na multimere von Willebrandovega faktorja (vWF) in jih razgradi. Ob zmanjšani aktivnosti ADAMTS13 pa se multimeri vWF kopičijo v krvnem obtoku in tvorijo s trombociti bogate strdke, kar lahko pri bolnikih s TTP povzroči ishemijo in odpoved organa. Možnosti zdravljenja TTP so omejene in zaradi neželenih učinkov mnogokrat bolniku neprijazne, zato smo se v magistrskem delu osredotočili na iskanje novih terapevtskih pristopov za zdravljenje TTP. Naš cilj je bil identificirati kratke peptide, ki bi preprečili interakcijo med avtoprotitelesi anti-ADAMTS13 in antigenom ADAMTS13 ter tako onemogočili nadaljnje imunske reakcije pri bolnikih s TTP. Za odkrivanje peptidov smo izbrali metodo predstavitve na bakteriofagu. Izvedli smo afinitetno selekcijo iz dveh komercialnih bakteriofagnih knjižnic; knjižnice linearnih peptidov Ph.D.™-7 in knjižnice cikličnih peptidov Ph.D.™-C7C. Naša tarča so bila anti-ADAMTS13 protitelesa, izolirana iz seruma imuniziranih živali. Tarčo smo imobilizirali na magnetne delce, nanjo vezali knjižnico fagov in fage z visoko afiniteto vezave eluirali na dva načina; specifično z antigenom ADAMTS13 in nespecifično s pufrom z nizkim pH. Pridobljen eluat fagov smo amplificirali v kulturi E.coli in iz bakterijskih celic izolirali fagno DNA, ki smo jo poslali na sekvenciranje. Prav tako smo izvedli test ELISA, kjer smo vrednotili specifičnost vezave izbranih bakteriofagnih klonov na tarčo anti-ADAMTS13. Rezultate sekvenciranja smo bioinformatično obdelali in dobljena zaporedja glede na njihove motive razporedili v peptidne gruče. Znotraj gruč smo identificirali glavne epitope in mimetike epitopov antigena ADAMTS13 ter v končni fazi načrtovali peptidne vabe, za katere bi bilo v prihodnjih raziskavah smiselno preiskusiti, ali lahko preprečijo interakcijo med avtoprotitelesi in avtoantigenom, ter oceniti njihov potencial pri zdravljenju TTP. Thrombotic thrombocyopenic purpura (TTP) is a life-threatening autoimmune disease in which small blood clots or microthrombi form in the arterioles. The formation of these is caused by autoantibodies against the ADAMTS13 enzyme, which, in healthy individuals, binds to von Willebrand factor (vWF) multimers and degrades them. With reduced ADAMTS13 activity, vWF multimers accumulate in the bloodstream and form platelet-rich clots, which can cause ischemia and organ failure in patients with TTP. Treatment options for TTP are limited and can many times be unpleasant for the patient due to possible side effects, which is why we focused on finding new therapeutic approaches for treating TTP in this master's thesis. Our initial goal was to identify short peptides that would prevent the interaction between anti-ADAMTS13 autoantibodies and ADAMTS13 antigen, thus preventing further immune reactions in patients with TTP. For detection of peptides we chose the phage display method, where we performed an affinity selection from two commercial bacteriophage libraries: the Ph. D.TM – 7AK linear peptide library and the Ph. D.TM – C7C cyclic peptide library. Our target were anti-ADAMTS13 antibodies, isolated from the serum of immunized animals. We immobilized the target onto magnetic particles, bound the phage library to it and eluted the phages with high binding affinity. We used two different elution techniques; specific with antigen ADAMTS13 and non-specific with a low pH buffer. We amplified the phage eluate in E.coli and isolated the phage DNA from bacterial cells, which we sent to sequencing. We also performed an ELISA test, where we evaluated the binding specifity of isolated bacteriophage clones to the anti-ADAMTS13 autobodies. We processed the results with bioinformatics software and arranged the obtained sequences into peptide clusters based on their motifs. Within the clusters we identified major epitopes and epitope mimetics of the ADAMTS13 antigen, as well as designed peptide decoys. In future research, it would be interesting to investigate whether those decoys can prevent the interaction between autoantibodies and the autoantigen, and evaluate their therapeutic potential in the treatment of TTP. trombotična trombocitopenična purpura anti-ADAMTS13 avtoprotitelesa antigen ADAMTS13 predstavitev na bakteriofagu afinitetna selekcija sekvenciranje epitop mimetik epitopa peptidne vabe thrombotic thrombocytopenic purpura anti-ADAMTS13 autoantibodies ADAMTS13 antigen phage display affinity selection sequencing epitope epitope mimetic peptide decoys true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-09-11 08:45:39 2026-09-11 08:45:42 2026-09-13 03:30:30 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2027-09-10 128691 33782.pdf 33782.pdf 1 A297EB95752481901D520D06E5544701 f71a08a35ecf783dbbd7018a0fe913dd0483ec26b63c2c4c4ce99b935e765c58 62fc0d9f-adac-11f1-8bc5-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=247143 Fakulteta za farmacijo 0 0 0