Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Martina Kobrlová Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Pharmacogenetics in rheumatoid arthritis Based on scientific progress in the research of human genome and the discovery of polymorphisms, which are involved in the interindividual differences in human population, there is also a growing interest in pharmacogenetics. It is a field combining pharmacology and genetics with the aim of identifying specific features that could explain the different responses of patients to treatment by clinically used drugs. Applying this knowledge could contribute to a simpler choice of medication for a particular patient and it could reduce the risk of side effects or poor response. In this diploma thesis I dealt with the latest scientific knowledge on pharmacogenetics in rheumatology, in particular the rheumatoid arthritis. From available studies, reviews, and meta-analyzes that have been published, I summarized current data on the relationship between polymorphisms and disease modifying drugs (DMARDs) used for the treatment of this disease. The largest amount of data was found on the most commonly used methotrexate. Further, the work examines the leflunomide and other...

The theoretical part of the thesis summarizes general information about the human TP53 gene and p53 protein, which is encoded by this gene. There is also dealt with the transcriptional variants - p53 isoforms. The experimental part is focused on the detection of isoform p53 and its shorter, yet undescribed form. The starting material is peripheral blood of patients from University Hospital Brno, from which RNA was isolated. Subsequently, RNA was transcribed by reverse transcription into cDNA which was amplified by polymerase chain reaction (PCR). Experimental measurements demonstrate that p53 occurs in all the B-lymphocytes of patients with chronic lymphocytic leukemia. It also demonstrats the existence of a shorter form of p53.

This master's thesis deals with the matter of predicting the effects of aminoacid substitutions on protein stability. The main aim is to design meta-classifier that combines the results of the selected prediction tools. An evolution strategy was used to find the best weights for each of the selected tools with the aim of achieving better prediction performance compared to that achieved by using these tools separately. Five different and obtainable prediction tools were selected and their prediction outputs were weighted. Two different approaches of evolution strategy are investigated and compared: evolution strategy with the 1/5-rule and evolution strategy with the type 2 of control parameters self-adaptation. Two independent datasets of mutations were created for training and evaluating the performance of designed meta-classifier. The performed experiments and obtained results suggest that the evolution strategy could be considered as a~beneficial approach for prediction of protein stability changes. However, the special attention must be paid to careful selection of tools for integration and compilation of training and testing datasets.

(EN) Humans and cells in their bodies are exposed to various mutagens in their lifetime that cause DNA damage and mutations, which affect the biology and physiology of the target cell, and can lead to the expansion of an immortalized cell clone. Genome-wide massively parallel sequencing allows the identification of DNA mutations in the coding sequences (whole exome sequencing, WES), or even the entire genome of a tumour. Mutational signatures of individual mutagenic processes can be extracted from these data, as well as mutations in genes potentially important for cancer development ('cancer drivers', as opposed to 'passengers', which do not confer a comparative growth advantage to a cell clone). Many known mutational signatures do not yet have an attributed cause; and many known mutagens do not have an attributed signature. Similarly, it is estimated that many cancer driver genes remain to be identified. This Thesis proposes a system based on immortalization of mouse embryonic fibroblasts (MEF) upon mutagen treatment for modelling of mutational signatures and identification and testing of cancer driver genes and mutations. The signatures extracted from WES data of 25 immortalized MEF cell lines, which arose upon treatment with a variety of mutagens, showed that the assay recapitulates the...

Staphylococcus aureus is a opportunistic pathogen that can cause severe and chronic infections. The reason of the infections relapse is often the persistence. It is about adapting to stressful conditions by inducing a dormant state, which would allow bacteria to survive exposure to antibiotics and grow again after their elimination. Bacteria that persist in the patient acquire various adaptive mutations, which are transmited creating subpopulations that have a better ability to persist. The aim of this diploma thesis was to compare individual methods of persistent study that could be used in clinical practice in the future, and at the same time to try a closer molecular characterization of the persistent state with using methods for calculating gene expression. I had chronological isolates of Staphylococcus aureus at my disposal, the initial one being the primoisolate, an isolate taken at the diagnostics of cystic fibrosis before the start of antibiotic treatment. Another was taken at a distance of three-quarters of a year and the last with a half-year interval from the previous one. Following whole genome sequencing, genes in which adaptive mutations occurred were identified. The first method determines the degree of persistence by calculating CFU (Colony Forming Units) after antibiotic treatment....

Introduction: About 5 - 10% of breast carcinomas are caused by genetic mutations. The most common genetic mutation that is involved in the development of this malignancy is a mutation in the tumor suppressor genes BRCA1/2 whose carriers have approximately a 70% lifetime risk of developing breast cancer. The prognosis of patients with BRCA1/2-asociated breast carcinoma, compared to patients with sporadic breast carcinoma is the subject of many studies with ambiguous results. Aim: The aim of the theoretical part of this work was to approach the issue of breast cancer and the most common genetic syndromes associated with it. In the practical part of this work a retrospective study was carried out in order to compare BRCA1/2 mutated breast cancer patients with non-mutated breast cancer patients in the tumor profile, methods of treatment and prognosis. Methods: We retrospectively analyzed the data of 134 patients who were tested for the presence of BRCA1/2 mutation at the Institute of Medical Genetics, University Hospital in Pilsen during the years 2013-2018 and at the same time were treated for early breast cancer at the University Hospital in Pilsen during the years 2000-2020. 32 patients were BRCA1 positive (24%), 10 BRCA2 positive (7%) and 92 without BRCA1/2 mutation (69%). The follow- up time was...

Notch pathway plays a critical role during the development and life of metazoan organisms. CSL proteins are the component of the Notch pathway that mediates the regulation of target genes. The discovery of CSL-like proteins in yeast raised the question of their function in unicellular organisms which did not utilize the canonical Notch pathway. CSL-homologues in yeast are conserved in parts that are important for DNA binding and for fission yeast proteins it was shown that they bind to CSL recognition elements in vitro. In fission yeast, CSL paralogues Cbf11 and Cbf12 play antagonistic roles in cell adhesion and the coordination of cell and nuclear division. Yeast CSL proteins have long and intrinsically unstructured N- terminal domains compared to metazoan CSL proteins. In this study, we investigated the functional significance of these extended N-termini of CSL proteins by their complete removal. For newly constructed truncated variants of proteins Cbf11 and Cbf12 in Schizosaccharomyces pombe we observed the lack of ability to bind CSL recognition RBP probe. The removal of N-terminal parts of CSL proteins in fission yeast led to the change in their cellular localization. Once strongly preferred nuclear localization changed by the removal of N-terminal domains to cytoplasmic localization with a...

This dissertation shows various concepts of the term gene that have appeared since the birth of genetics in 1900 up to the first half of the 50s. It focuses especially on the 40s and the beginning of the 50s. Scientific papers from that period were the main source of information. The author tried to capture not only generally accepted notions about genes and genic action but also those that had not pushed through in those days, nevertheless, that had offered a non- standard point of view which could have later become inspirative for molecular genetics. The work documents searching for links between genes and enzymes or ideas of potential divisibility of the gene. The dissertation assigns a very important role to those phenomena that emphasized the significance of the gene order or the order of genic parts. In Goldschmidt's interpretation of pseudoallelism the author sees the thought that the essence of a gene is its position and considers this the beginnings of "digital" thinking about the gene. The dissertation pays attention to "analogue" thinking about the gene, as well. This thinking took account of molecular shaping and represented a blind alley for the early molecular genetics. The work confirms to a certain extent the Kuhnian vision of the development of scientific disciplines. It finds the...

Genetic causes of medullary thyroid carcinoma and Hirschsprung's disease Abstract Medullary thyroid carcinoma (MTC) and Hirschsprung's disease (HSCR) are classified as simple neurocristopathies, i.e. diseases linked to neural crest-derived cells. MTC is derived from parafollicular cells of the thyroid and HSCR is characterized by absence of enteric ganglia in the gastrointestinal tract. The RET proto-oncogene is only expressed in neural crest-derived cells, including parafollicular cells and enteric neurons. The RET encodes a transmembrane tyrosinekinase receptor that plays an important role during proliferation, differentiation and cell survival, and activates many signaling pathways. If the strictly regulated activation fails, e.g. due to mutations in the specific gene locations, the RET becomes a highly effective oncogene. Activating germline mutations in the RET proto- oncogene lead to hereditary forms of MTC, whereas sporadic forms of MTC are caused by somatic mutations in the tumor tissue. On the contrary, inactivating mutations induce migration failure of ganglion cell precursors during the development of enteric nervous system and result in the development of HSCR. In rare cases, the coexistence of both diseases is caused by mutations with a dual gain-of-function and loss-of-function character....

Translation initiation in the hepatitis C virus (HCV) occurs through a cap- independent mechanism that involves an internal ribosome entry site (IRES) capable of interaction with and utilization of the eukaryotic translational machinery. We focused on the structural configuration of the different HCV-IRES domains and the impact of IRES primary sequence variations on secondary structure conservation and function. For this purpose we introduced into our laboratory, methods such as denaturing gradient and temperature gradient gel electrophoresis for screening the degree of heterogeneity and total amount of HCV-IRES variability accumulated in HCV infected patients over a period of time. The selected samples showed variable migration pattern of the HCV-IRES (from all the patients) visualized in DGGE and TGGE, were sequenced and evaluated for translation efficiency using flow cytometry. In some cases, we discovered that multiple mutations, even those scattered across different domains of HCV-IRES, led to restoration of the HCV-IRES translational activity, although the individual occurrences of these mutations were found to be deleterious. We propose that such observation may be attributed to probable long- range inter- and/or intra-domain functional interactions. We established a large-scale HCV-IRES...