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Prediction of effects of single point mutations on protein-nucleic acids interactions
Štěpánková, Věra Tereza ; Novotný, Marian (advisor) ; Neuwirthová, Tereza (referee)
Single point mutations are the most common type of mutations and many of them can have pathogenic effects, it is therefore useful to be able to correctly and effectively predict their impact on a given protein. Proteins interacting with nucleic acids are essential for most cellular processes. Progress in computational methods and machine learning enables the development of increasingly high-quality tools for predicting the effect of mutations on proteins. An important category of these predictors are tools for predicting the effect of single point mutations on protein interactions with nucleic acids. This thesis focuses on currently available tools for prediction of missense mutation effect on protein-NA interactions, which predict quantitative changes in protein and nucleic acid affinity and estimate the severity of the mutation based on this change. Attention is also paid to methods for evaluating the quality of predictions of individual tools. Keywords: point mutation, prediction, interaction, mechanism
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Detection of β-glucocerebrosidase gene/pseudogene recombination events leading to pathogenic alleles
Peková, Barbora ; Hřebíček, Martin (advisor) ; Schierová, Michaela (referee)
This diploma thesis provides an overview of gene conversion, its role in the pathogenesis of human diseases and the use of methods based on next-generation sequencing (NGS) for detection rare variants of DNA sequence. Labeling of target DNA molecules by random nucleotides in primer and NGS were used for detection point mutations arising de novo in the β-glucocerebrosidase gene by gene conversion between it and its pseudogene in meiotic and mitotic cells of control subjects. Primers specific for the active gene were used to selectively amplify the ninth and tenth exon of the gene where "recombinant" variants occur most frequently. Sequences generated from 20 genomic DNA samples on Illumina MiSeq platform were quality filtered, sorted by unique labels and consensus sequences were created from alignments of sequences carrying the same DNA tag. The number of potential point mutations in the samples ranged between 12 and 48. The mutations were manually re-evaluated from the alignments. The number of alignments with unique labeling was in the range of 7-15 thousand per sample. Only three samples carried possible recombinant mutations, suggesting a lower frequency of conversion in the region than reported by other techniques. Analysis of unique sequences in primer indicated possible ways to improve the...
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Functional role of cytoplasmic domains in the gating of TRPA1 channel
Vašková, Jana ; Vlachová, Viktorie (advisor) ; Zemková, Hana (referee)
The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed in a subset of primary afferent neurones where it is activated by a variety of pungent and chemically reactive compounds such as allyl isothiocyanate or cinnamaldehyde. This voltage- dependent channel is activated through covalent modification of cytoplasmic cysteines and, from the cytoplasmic side, is also critically regulated by calcium ions. Both, amino (N-) and carboxyl (C-) termini have been shown to be involved in these processes. Using electrophysiological and molecular-biology techniques, we explored the role of specific cytoplasmic domains in the activation of TRPA1. By measuring chemically-, voltage-, and calcium-activated membrane TRPA1-mediated currents, we identified highly conserved serine and threonine residues along the N-terminal ankyrin repeat domain, mutation of which strongly affected responses of the channel. In addition, using C-terminally truncated construct previously reported to be involved in calcium regulation, we present a new finding that the distal C-terminal tail contributes to voltage-dependent activation of TRPA1.
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