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Specifická izolace microRNA pomocí magnetizovatelných mikročástic
Vlahová, Veronika
MicroRNAs are small non-coding RNA molecules with length of about 22 nt. These molecules participate on regulation of gene expression at the post-transcriptional level. They represent the largest group of regulators in the cell and therefore are also involved in all key processes such as proliferation, differentiation or apoptosis. Moreover, they participate in tumor transformation. These small molecules have a great potential to be diagnostic markers or assist in the treatment and prevention of diseases. This research was focused on the development of isolation method using magnetic particles with subsequent electrochemical detection of microRNA. Optimization steps were performed and then the entire method was successfully applied to real samples of HEK293 cells expressing increased levels of miR-124. The developed method proved to be sufficiently specific and applicable to the analysis of microRNA.
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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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Diagnostické biosenzory pro encefalopatie způsobené priony
Šobrová, Pavlína
Prion diseases are fatal transmisible neurodegenerative and infectious disorders (TSEs) of humans and animals, characterized by structural transition of the host-encoded cellular prion protein (PrPC) into the aberrantly folded pathologic isoform PrPSc. The main aim of this work is to summarize present information about prion diseases and their possibilities of determination pointed to electrochemical techniques. For this purpose cyclic voltammetry (CV), differential pulse voltammetry, differential pulse voltammetry Brdicka reaction and chronopotentiometric stripping analysis (CPSA) were used. The estimated detection limits were 32 ug/ml by CV, 16 ug/ml by DPV, 16 ug/ml by DPV -- Brdicka reaction and 8 ug/ml by CPSA. Subsequently, the influence of heat denaturation was observed. It clearly follows from the obtained results that signals of prion decreased linearly depending on the duration of the heat treatment at 99°C for various time intervals 0, 15, 30, 45, and 60 min. Moreover, we aimed our attention on studying of prion protein interaction with CdTe quantum dots (QDs) using electrochemistry. Primarily, we characterized electrochemical properties of QDs and the detection limit at 100 fg/ml was estimated. Further, electrochemical study of prion and QD interactions was carried out to find the most suitable conditions for sensitive detection of prion proteins. Detection limit (3 S/N) was estimated as 1 fg in 5 ul. This makes labeling of proteins with QDs of great importance due to easy applicability and possibility to use in miniaturized devices, which can be used in situ. This should open new possibilities how to determine the presence of these proteins on surgical equipment and other types of materials, which could be contagious.
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