The variety of bacteria and their genomes sometimes causes conservation of homologue molecules to be displayed not in sequence but in secondary and tertiary structures. In the case of the regulatory 6S RNA, sequence homologues have been found in over 100 bacterial species so far. However, none were found in the genus Streptomyces. The unique genome of these soil- dwelling bacteria, known for their capacity to produce antibiotics, has a high G/C content and diverges substantially from distantly related bacteria. Yet in the non-coding 6S RNA it is the secondary structure that is crucial for its function. The 6S RNAs trap sigma factors by mimicking target promoter sequences in order to help with switching sets of expressed genes during developmental transitions. 6S-like RNA genes in Streptomyces coelicolor have been computationally predicted by comparison of in silico modelled secondary structures of known 6S RNAs. The aim of this thesis was the verification of these 6S-like RNA predictions. The experimental approach was based on RNA co-immunoprecipitation (RNA CoIP), as well as RT- PCR from RNA samples. The outcomes of this project are the detection of six novel ncRNA transcripts with possible 6S-like RNA functions, which also served as the wet-lab verification of the in silico prediction technique...

Chronic myeloid leukemia is a clonal haematopoietic disease, with characteristic BCR-ABL1 fusion gene. Despite the significant improvement in patient treated with tyrosine kinase inhibitors (TKI), 20-30 % of patients develop resistance. One of the main causes of treatment failure are mutations in the BCR-ABL1 kinase domain (KD). The aim of this work was to elucidate the molecular mechanisms of resistance and mutagenesis development in CML using an in vitro CML model KCL-22. The main part of this work was focused on the identification of genes involved in DNA damage response and repair, that could play a role in the process of mutagenesis of BCR-ABL1. We used the RT2 Profiler PCR Arrays method for the group of selected genes regulating DNA damage response and repair. We identified the genes XRCC6 and PARP1 whose gene expression was significantly and specifically decreased during KD BCR-ABL1 mutagenesis. Products of these genes are involved in repairing DNA double-strand breaks through non-homologous end joining (NHEJ). During study of the KD BCR-ABL1 mutagenesis we also found that clones, which developed mutations, did not show the increased BCR-ABL1 expression in the beginning of the culture compared to the clones in which mutations have not evolved. Key words: myeloid leukemia, mutation,...

Dermatophytes are a group of fungi, some of which can cause skin diseases in humans and animals due to their ability to degrade keratinized tissue. Representatives of this group also include strains from the Trichophyton benhamiae complex, known to cause dermatophytosis especially of small rodents and rabbits. In the last decade, one of four populations of this complex has spread epidemically across Europe among guinea pigs and their breeders. To answer the question what stands behind the successful spread of this population, the gene expression and production of volatile organic compounds of epidemic and non-epidemic populations of T. benhamiae was investigated. Gene expression of three strains from each population was studied during growth in liquid medium and on ex vivo mouse skin models prepared according to a newly optimized protocol. RNAseq and RT-qPCR methods were chosen for the gene expression analysis. Based on the literature and the results of RNAseq preliminary analysis, several genes were selected for which specific primers were designed. The spectra of the produced volatile organic compounds of the same strains growing on sheep wool in vials were analyzed by GC-MS. While non-epidemic populations did not differ in gene expression and production of volatile organic compounds, the...

6 Abstract Lipopolysaccharides are composed of covalently bound saccharides. They are a characteristic component of the cell wall of gram-negative bacteria. They are the cause of severe sepsis in humans and complications in human medicine. Lipopolysaccharides are a constant part of the infections of gram-negative bacteria. We expect an evolutionarily conserved non-specific immune response and protection. The question is whether there is an immune response in the model organism Caenorhabditis elegans. If so, what mechanism is controlled and regulated. We submitted lipopolysaccharides from the bacteria Pseudomona aeruginosa with the bacteria Escherichia coli OP50 and observed the influence of lipopolysaccharides on the expression of selected genes. We examined metabolism and development. We have shown the influence of lipopolysaccharides on gene expression of C-type lectine clec-60 a clec-71, nextna lys-5, hsp-60 a F44G.3.2.1 genes. We incubated Caenorhabditis elegans on some components of lipopolysaccharide. We found regulation of these selected genes with hydrophobic components of lipopolysacharide, lipid A. We did not observe regulation with saccharide components of lipopolysaccharide, glucose and galatose. The metabolism of lipids had changed. We demonstrated a reduction of neutral lipids and changes in...

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Fat tissue plays a main role in the development of the metabolic syndrome. Many components of the metabolic syndrome may be improved by dietary arrangements, including an increased intake of n-3 PUFA. In addition to the positive effect of n-3 PUFA, a possible effect of a higher calcium intake on influencing weight loss and energy metabolism has also been discussed for a long time. In addition to nutritional factors, genetic factors significantly contribute to influencing weight and lipid and glucose metabolism. In study A, 40 obese women were observed during three weeks of weight reduction management. Women were randomly divided into two groups. The first group received yogurt enriched with n-3 PUFA, and the second group consumed yoghurt without the supplementation. The results show that low-dose supplementation with n-3 PUFA in yogurt in combination with a reduced energy intake increases n-3 PUFA content in serum lipids and prevents adverse changes in the composition of FA in serum after a short-term low-calorie diet. In study B, we monitored the influence of n-3 PUFA supplementation on short-term weight management with VLCD in 20 women with severe obesity, who were randomly divided into two groups. The first group received VLCD enriched with n-3 PUFA, the second group VLCD with a placebo. The addition of...

The "omics" is a concept of biological disciplines that globally characterizes and quantifies biomolecules involved in the key functions of an organism. The "omics" methods are used e.g. in molecular epidemiology, where they help to evaluate potential biomarkers that identify the impact of environmental factors for human health. In this thesis, the "omics" methods were applied in samples collected from newborns born in localities of the Czech Republic mostly differing by pollution levels from industrial sources. The principal aim was to determine whether environmental changes during prenatal development can affect gene expression and its regulation in newborns. The thesis further aimed to evaluate the level of air pollution at the time of biological samples collection. Using the whole genome approach, differentially expressed genes (DEGs) in newborns from districts Karvina and Ceske Budejovice (CB) were identified. In a pilot study of a small group of newborns from districts Most and CB, differentially methylated CpG sites in DNA were assessed. These sites attenuate gene activity and could be responsible for long-term changes at the genetic level. Finally, the aim was to find differentially expressed small non-coding RNA (DE miRNA) in newborns from Most and CB. Samples of umbilical cord blood from...

Burkholderia cenocepcia is one of the serious infectious agents of respiratory tract among cystic fibrosis patients. There are problems mainly with strains which are capable of epidemic spread. The known epidemic in the Czech Republic was caused by ST32 strain in the past. In this work, there was completed whole genome sequence of referential isolate 1232 of B. cenocepacia ST32 in cooperation with bioinformatics by new generation sequencing techniques and by determining the problematic areas by a combination of Sanger sequencing bioinformatics approaches and manual assembling of sequence reads localized in these areas. The final version of the genome sequence was annotated by PGAAP and at the present time it is finalized. Second part of this work is dedicated to looking for a prognostic marker of infection caused by ST32 strain in patients with cystic fibrosis. We analysed the results of ST32 trancriptomic experiment and chose genes possibly connected with the cepacia syndrome - serious, mostly fatal state of infection. By quantitative PCR we compared their expression in isolates from 4 patients from time of cepacia syndrome and month before that. We checked the possibility of direct detection of the expression of these genes in clinical material. We identified genes for type III secretion system as...

Diplomová práce Abstract - Iveta Brabcová Abstract Ischemia-reperfusion heart injury is one of the most significant diseases affecting mankind and therefore current research pays more attention to its prevention and knowledge of the possible mechanisms which protect the heart. Adaptation to hypoxia has been known for several decades as a cardioprotective intervention but the main issues of protective mechanisms which are induced by the adaptation are still not completely understood. An important role of mitochondria as the main producers of energy and reactive oxygen species which can play a signalizing role in these mechanisms is confirmed in many studies. For this reason a special conplastic strain SHR/OlaIpcv-mtBN/Crl was created. This strain carries the nuclear genome of spontaneously hypertensive rat (SHR) and the mitochondrial genome of normotensive, highly resistant strain Brown Norway (BN). The aim of this study was to compare the expression of selected gene transcripts in the area of energy metabolism, of genes which are related to mitochondrial biogenesis and signaling and antioxidant systems. Comparing the expression was analyzed between strains and after chronic hypoxia adaptation, which cause cardioprotective phenotype in both of these strains. Our results showed a different expression HIF-1α...

Adipocytokines released by the adipose tissue play an important role in the regulation of immune and inflammatory responses. In obesity their production is dysregulated, which is one of the major factors contributing to the onset of a chronic low-grade systemic inflammation representing a risk factor for the progression of other diseases, such as atherosclerosis or type-2 diabetes. The main goal of this thesis was to analyze the secretion of selected adipocytokines (adiponectin, IL6 and MCP1) by in-vitro differentiated adipocytes, isolated from the adipose tissue prior to and after a dietary intervention, and this under basal conditions and during stimulated lipolysis. In case of adiponectin, the secretion of its isoforms was analyzed too. The concentration of adiponectin, IL6 and MCP1 was determined by the ELISA method, the Western Blot method was used to determine the distribution of the adiponectin isoforms. The thesis also concentrates on the gene expression of ATF3, ATF4 and HSPA5, factors engaged in the ER stress in the course of the differentiation of adipocytes. The changes in the gene expression were measured by the quantitative Real Time PCR method. At the same time the development of the endoplasmic reticulum (ER) in the course of adipogenesis was monitored by indirect...