20.500.12556/RUL-172927 Priprava rekombinantnega proteasoma hipertermofilne arheje Aeropyrum pernix Preparation of recombinant proteasome of the hyperthermophylic archaeon Aeropyrumpernix Diplomsko delo obravnava celično proteostazo – homeostazo proteinov, ki je ključnega pomena za ohranjanje preživetja celic. Gre za dinamično ravnovesje med sintezo, zvijanjem in razgradnjo celičnih proteinov. Predstavljeni so glavni sestavni deli sistema proteostaze, kot so molekularni šaperoni, ubikvitin proteasomski sistem ter lizosomsko avtofagni sistem. Podrobneje je opisan proteasom, njegovo delovanje pri evkariontih in prokariontih ter vpliv staranja na učinkovitost teh sistemov. V nalogi so izpostavljene povezave med spremembami v proteostazi in pojavom bolezni, kot so Alzheimerjeva bolezen, Parkinsonova bolezen in rak. V sklopu naloge smo pripravili rekombinantne podenote proteasoma hipertermofilne arheje Aeropyrum pernix. Ustrezne gene za dve podenoti alfa in eno podenoto beta jedrnega dela proteasoma smo pomnožili iz kromosoma A. pernix in jih vstavili v ekspresijski plazmidni vektor za sintezo rekombinantnih proteinov v bakterijah Escherichia coli. Sintetizirane podenote proteasoma smo delno očistili iz bakterij s segrevanjem celičnih lizatov. Pokazali smo, da podenota alfa v kombinaciji s podenoto beta razgrajuje preprosti peptidni substrat pri temperaturi 80 °C. The thesis deals with cellular proteostasis – protein homeostasis, which is crucial for maintaining cell survival. It is a dynamic balance between the synthesis, folding, and degradation of cellular proteins. The main components of the proteostasis system are presented, such as molecular chaperones, the ubiquitin-proteasome system, and the lysosomal autophagy system. The proteasome is described in detail, its function in eukaryotes and prokaryotes, and the effect of aging on the efficiency of these systems. The paper highlights the links between changes in proteostasis and the onset of diseases such as Alzheimer's disease, Parkinson's disease, and cancer. As part of the thesis, we prepared recombinant subunits of the proteasome of the hyperthermophilic archaeon Aeropyrum pernix. The corresponding genes for two alpha subunits and one beta subunit of the core proteasome were amplified from the A. pernix chromosome and inserted into an expression plasmid vector for the synthesis of recombinant proteins in Escherichia coli bacteria. The synthesized proteasome subunits were partially purified from bacteria by heating the cell lysates. We showed that the alpha subunit in combination with the beta subunit degrades a simple peptide substrate at a temperature of 80 °C. avtofagija bolezni staranja Escherichia coli NaDS-PAGE proteasom proteostaza šaperoni age – related diseases autophagy Escherichia coli molecular chaperones proteasome proteostasis true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2025-09-12 07:19:08 2025-09-12 07:19:10 2025-10-28 03:47:58 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 263666 248966915 51055.pdf 51055.pdf 1 3CB278E49E82650E792F5427FD4ECBB5 354c76f4e5891bb28c3463680b08e3206fc479a7647b9022c210698da14e420b d927ba82-8f97-11f0-9328-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=217061 Biotehniška fakulteta 0 0 0