Tumors are one of the leading causes of death worldwide. Generally, the prognosis is better if the treatment begins at an early stage. Nowadays, the conventional chemotherapy treatment of cancer, known for its limited efficacy and side effects, is being gradually replaced by targeted biological treatment, which is used when specific genetic mutations are found. A part of the treatment is a detection of a potential progression, which is mainly based on the tumor biomarkers monitoring. Currently, further investigation of a so-called liquid biopsy method are ongoing, on which this thesis is focused. The main aim of this work was the experimental development and validation of the method for detection of the ctDNA in the plasma samples based on the somatic mutations presence. For the development and optimization of the system on the principle of denaturation capillary electrophoresis, the samples of cancer patients with KRAS mutation were used. Subsequently, a clinical part of the research was performed on a pilot set of 21 plasma samples. Finally, the method was optimized for the detection of BRAF and EGFR markers. A partial objective was to improve the detection sensitivity and increase the capture of the ctDNA in patients with advanced stage of the disease. The results of this work suggest the...

This diploma thesis deals with study of the rate-dependence of the magnitude of a current through the heart channel that conducts slowly activating component of delayed rectifier outward current (IKs). This property is very important for the IKs channel function. When other repolarizing currents are insufficient, but also when the heart rate accelerates, especially during elevated sympathetic tone, IKs provides so-called repolarization reserve, which prevents excessive lengthening of cardiac action potential repolarization. The IKs channel structure is encoded by the KCNQ1 (pore-forming -subunit) and KCNE1 (modulatory -subunit) genes. Mutations in these genes disrupt the physiological function of the IKs channel and cause inherited arrhythmogenic syndromes, especially long QT syndrome (LQTS). Such mutations include the c.926C>T (p.T309I) mutation in the KCNQ1 gene, which results in LQTS type 1 in heterozygous carriers. The theoretical part of the thesis provides basic information about the IKs channel and the patch clamp technique, this knowledge is necessary for the practical part. The experimental part is focused on cultivation of the CHO cell line and its transient transfection for subsequent electrophysiological measurements by whole-cell patch clamp technique to study the dependence of the IKs magnitude on stimulation frequency, both in the wild type channels (i.e. without mutation) and in those with cotransfected wild type and T309I subunits.

This thesis is focused on working up evolutionals and genetics algorithms issues Especially for multiobjective algorithms VEGA, SPEA and NSGA – II. Thereinafter one of FrameWork working with genetics algorithms namely WWW NIMBUS. From this mentioned algorithms was selected VEGA algorithm for implementation in JAVA to preselected problem. Thereby problem is choice thick columns of profile according to predetermined criteria. Selected algorithm works on division of population into several groups and each group evaluates the resulting fitness function. Here is a sample implementation of this algorithm. Furthermore there is a example of working with FrameWork. In the next section are compared the results of generated progam with results that were obtained by FrameWork WWW NIMBUS. As for VEGA, and the Nimbus there are shown different results. The VEGA is presented also the development of individual fitness functions. Also, there are shown graphs, that can be obtained from NIMBUS. At the end of work is introduced the comparation of the results ane propose possible improvements.

This Master´s thesis implements computer program in Java, useful for automatic model generating, specially in symbolic regression problem. Thesis includes short description of genetic programming (GP) and own implementation with advanced GP operands (non-destructive operations, elitism, exptression reduction). Mathematical model is generating by symbolic regression, exacly for choosen data set. For functioning check are used test tasks. Optimal settings is found for choosen GP parameters.

This thesis brings a new approach to the prediction of the effect of nucleotide polymorphism on human genome. The main goal is to create a new meta-classifier, which combines predictions of several already implemented software classifiers. The novelty of developed tool lies in using machine learning methods to find consensus over those tools, that would enhance accuracy and versatility of prediction. Final experiments show, that compared to the best integrated tool, the meta-classifier increases the area under ROC curve by 3,4 in average and normalized accuracy is improved by up to 7\,\%. The new classifying service is available at http://ll06.sci.muni.cz:6232/snpeffect/.

This thesis is focused on predicting the impact of amino acid substitution on protein stability. The main goal is to create a consensual predictor that uses the outputs of chosen existing tools in order to improve accuracy of prediction. The optimal consensus of theese tools was designed using evolution strategies in three variants: 1/5 success rule, self-adaptation variant and the CMA-ES method. Then, the quality of calculated weight vectors was tested on the independent dataset. Although the highest prediction performance was attained by self-adaptation method, the differences between all three variants were not significant. Compared to the individual tools, the predictions provided by consensual methods were generally more accurate - the self-adaptation variant imporved the Pearson's corelation coeficient of the predictions by 0,057 on the training dataset. On the testing dataset, the improvement of designed method was smaller (0,040). Relatively low improvement of prediction performance (both on the training and the testing dataset) were caused by the fact, that for some records of testing dataset, some individual tools vere not able to provide their results. When omitting these records, consensual method improved the Pearson's corelations coeficient by 0,118.

This thesis discusses the issue of predicting of the effect of amino acid substitutions on protein funkcion, based on phylogenetic analysis method, inspired by tool MAPP. Significant number of genetic diseases is caused by nonsynonymous SNPs manifested as single point mutations on the protein level. The ability to identify deleterious substitutions could be useful for protein engineering to test whether the proposed mutations do not damage protein function same as for targeting disease causing harmful mutations. However the experimental validation is costly and the need of predictive computation methods has risen. This thesis describes desing and implementation of a new in silico predictor based on the principles of evolutionary analysis and dissimilarity between original and substituting amino acid physico-chemical properties. Developed algorithm was tested on four datasets with 74,192 mutations from 16,256 sequences in total. The predictor yields up to 72 % accuracy and in the comparison with the most existing tools, it is substantially less time consuming. In order to achieve the highest possible efficiency, the optimization process was focused on selection of the most suitable (a) third-party software for calculation of a multiple sequence alignment, (b) overall decision threshold and (c) a set of physico-chemical properties.

Circulating tumor DNA (ctDNA) in peripheral blood of patients with metastatic colorectal cancer appears to be a promising molecular marker that provides various applications. ctDNA levels vary depending on the presence, alternatively on the volume of tumor mass within patient's body, which can be used primarily for early detection of disease progression or recurrence and moreover for evaluating radicality of surgical treatment, all within long-term postoperative follow-up of the patient. Due to minimal invasivity of ctDNA analysis from peripheral blood (so-called liquid biopsy), it is possible to perform it repeatedly at relatively short time intervals. On account of very low fraction of ctDNA in total cell-free DNA (cfDNA) ranging between units and hundreds of percent, the key factor is optimal methodology covering all steps from the isolation process to a sufficiently sensitive detection technology. In this thesis I focus on an optimization of isolation process and analysis of ctDNA obtained from tumor tissue and plasma of selected patients with metastatic colorectal cancer in connection with surgical radicality and correlation with a clinical status of the patients.

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.

In Introduction, the author of this dissertation deals with postoperative venous thromboembolism (VTE), hereditary and acquired risk factors, prophylaxis regimens and recent recommendation of VTE prevention in surgery. In Practical part of this work the author assesses the risk of VTE in surgical patients according to risk assessment model. Genetic testing is carried out in all patients to determine the incidence of hereditary thrombophilia and coagulation markers are measured in 28-days postoperative period. Prevalence of VTE in 1-year postoperative period is observed. The results are analysed in group of patients with benign disease (hernia and gallstone disease) and group of patients with malignancy (colorectal cancer and pancreatic cancer) separately. The objective of this work is to determine the incidence of the most frequent thrombophilic mutations (factor V Leiden mutation and protrombin G20210A mutation) and assess the impact of hereditary thrombophilia on incidence of postoperative venous thromboembolism in general surgery. Validation of venous thrombosis risk assessment model recommended by Czech Society for Thrombosis and Hemostasis is further objective.