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National Repository of Grey Literature

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Large Extracellular Vesicles in Cell Culture and Blood: Role in Prion Transmission and Detection by Flow Cytometry Soukup, Jakub ; Holada, Karel (advisor) ; Šebestová Janoušková, Olga (referee) ; Živný, Jan (referee) Prions (PrP) are the main cause of neurodegenerative diseases such as Scrapie in sheep, bovine spongiform encephalopathy, chronic wasting disease in deer, and Creutzfeldt-Jakob disease in humans. Although the cellular PrP (PrPC ) is involved in many cellular processes, its precise function still needs to be discovered. The disease is caused by the accumulation of a pathological form of PrP (PrPTSE ), which is caused by direct contact of PrPTSE and PrPC . PrP is anchored in the membrane by GPI and can be transmitted by cell-to-cell contact, tunnelling nanotubes, or extracellular vesicles (EVs). EV factions are divided by different biogenesis into exosomes, microvesicles, and apoptotic bodies. PrPTSE was found in exosomes and microvesicles, but these fractions were never compared to each other. The first aim of the doctoral thesis is a comparison of PrP content, prion-converting activity and infectivity in these fractions on CAD5 and N2a-PK1 cellular models of infection. We isolated a fraction of large EVs (20,000× g) and small EVs (110,000× g) by centrifugation from a conditioned medium. We characterised EVs by cryo-electron microscopy and western blot with Alix, TSG-101, CD63, CD9, and HSP70 markers. The contamination from other cellular compartments was checked by calnexin. EV fractions differed... Detailed record

Nanostructured Modifcation of Material Surfaces for Controlled Cell Cultivation Kotelnikov, Ilya ; Proks, Vladimír (advisor) ; Chytil, Petr (referee) ; Šebestová Janoušková, Olga (referee) Commercial media and surfaces for cell cultivation do not promote conditions for cell cultivation and proliferation with specific interactions cell-material surface. The aim of this thesis is developing surfaces for cell cultivation which mimic conditions in living tissues. The approach introduced in this thesis is based on applying biomimetic peptide ligands on inert supporting materials with non-fouling properties. Considering that a choice of a ligand sequence and distance between peptides can dramatically influence the outcome, a few model peptides with varying parameters were synthesized and investigated. The cell adhesive peptides were synthesized by solid phase peptide synthesis and scrambled peptides were synthesized and tested as well. Spatial deposition of peptides is another highly important point of study. The 'click'- reaction was used to successfully immobilize the peptides on produced surfaces. The distance between the peptide molecules on the surfaces was controlled by their concentration in a reaction mixture. The reference samples were immobilized with radiolabeled peptides for quantitative estimation of the peptides present. Then, the materials with different types of peptides and range of concentrations were examined via cultivation of cell cultures. The experiments were focused on... Detailed record

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