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Specific prion protein antibodies characterisation and use in diagnostic
Šafaříková, Eva ; Holada, Karel (advisor) ; Mareš, Michael (referee) ; Živný, Jan (referee)
Transmissive spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrPTSE ) in brain. PrPTSE is at present the only specific biochemical marker of human and animal TSEs. Diagnostic tests are based on the detection of PrPres after proteinase K digestion of brain homogenate using Western blot or on the immunohistochemistry of fixed brain tissue, which are both difficult and time consuming. In this work we focused on development of a new type of tests based on PrP detection without need of proteinase K digestion. As deposits of PrPTSE remain in the body for a long time, there is a substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrPTSE may have a potential to serve as a diagnostic marker. We prepared monoclonal antibodies specific for carboxymethyl lysine/arginine modified prion protein. Bacterially expressed and purified recombinant human prion protein (rhPrP) was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of laboratory mice and hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of 4...
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Remote control and visualization of bus-systems KNX and DALI for building management
Holub, Jiří ; Macháček, Jan (referee) ; Bátora, Branislav (advisor)
The thesis is focused on bus systems and their control by GUI. Basic energy management principles and the mostly known bus systems are analysed in the first – theoretical part of the paper. The theoretical part is then focused on software tool ETS 4, which is used for activation of system installation KNX. Software tool SmartServer, which is used for communication between bus system KNX and web interface, is described at the end of the first theoretical part. Two laboratory boards assembled of KNX and DALI parts were constructed in the second – practical part. Three laboratory tasks, which are prepared so they are related to theoretical description in previous part, were submitted in next step. Aim of the first task is to introduce basics of the board and PriOn interface. The second task is focused on DALI components and temperature control with PriOn regulating device. Creating of basic visualization and controlling of the board with SmartServer is described in the last task.
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Detekce prionových proteinů a jejich interakce s kovy a metalothioneinem
Cardová, Alžběta
Prion diseases are formed by a conformational change of prion-like protein (PrPC) with alfa-helix structure to the pathological isoform - prion (PrPSc) which acquires beta-sheet structure. PrPC physiological properties in the brain are insufficiently described but there is an assumption of its affinity to metal ions. Another protein with metal-binding ability is metallothionein (MT). Brain specific isoform of MT is called MT-III and it is assumed to participate in maintenance of metal ions concentration in the brain. Aim of this study was to prepare recombinant human PrPC in E. coli. Furthermore, this protein was used to detect interactions between metal ions (Cu, Zn), MT and PrPC by differential pulse voltammetry method. The final part was devoted to the MT-III determination in different genotypes of prion-infected and non-infectious mouse brain tissues.
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Synthetic scan of C-domain from prion proteins
Šebestík, Jaroslav ; Zawada, Zbigniew ; Šafařík, Martin ; Hlaváček, Jan
Prions are suspected as culprits of several neuropathogenic diseases. A combination of chemical and recombinant syntheses is a powerful tool for studying prion role in pathogenesis of neurodegenerative diseases. Due to the presence of “difficult” sequences in the prion protein molecule, chemical ligation procedure using peptide thioesters as key building blocks is a method of choice. Therefore, a synthetic scan of peptides constituting the mouse prion protein (MoPrP) C-domain 93-231 was carried out. The synthesis on chlorotritylchloride resin was the most promising for most peptides. Only the octadecapeptide MoPrP195-212 could not be synthesized by automated peptide synthesis even using the β-sheet breaking dipeptides. This difficulty was overcome by manual peptide synthesis with careful monitoring of coupling and Fmoc removal.
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