Recent knowledge suggests that the onset of cancer is modulated by the interplay of internal and external environmental factors along with numerous gene variants. Structural chromsomal aberrations in peripheral blood lymphocytes are considered as biomarkers of effect of genotoxic carcinogens and reflect elevated risk of cancer. Incomplete or deficient repair of double-strand breaks in DNA underlie chromosomal aberrations and the measurement of cytogenetic alterations may reflect interindividual differences in the response towards the mutagen. In this study the expected deficiences in the DNA repair capacity have been determined in incident oncological patients with breast, colorectal and urogenital cancers. The determination of chromosomal aberrations have been supplemented by the measurement of variants in genes involved in double-strand breaks repair (XRCC3, rs861539; RAD54L, rs1048771). Methodologically, we employed conventional cytogenetic analysis, cytogenetic analysis following the induction of chromocomal damage by bleomycin ("Challenge assay"), TaqMan discrimination analysis for the detection of allelic variants and statistical analyses. By using these methods we did not observe statistically signifiant differences either in chromosomal breaks (p=0,354) or in a percentage of cells with...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Šárka Strachoňová Supervisor: PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: Studium of gene regulation of nucleoside transporters in BeWo cell line Nucleoside transporters (NTs) localized in syncytiotrophoblast control placental uptake of nucleosides. Dysregulation of NTs can disrupt nucleoside homeostasis with a negative consequences on placental and fetal development and can lead to a change in placental pharmacokinetics of nucleoside-derived drugs. Therefore, understanding the expression and function of NTs is necessary for effective and safe pharmacotherapy during pregnancy. The aim of this diploma thesis was to study the adenylate cyclase (AC) activated regulatory pathways of gene expression of concentrative nukleoside transporter 2 (CNT2). For this purpose, qRT-PCR and in vitro accumulation assays using the model substrate [3 H]-adenosine were employed. The human placental choriocarcinoma-derived BeWo cell line has been exposed to an AC activator, forskolin (50 µM), and/or inhibitors of AC/cAMP/PKA, AC/cAMP/MAPK (MEK1/2, p38 MAPK) signaling pathways, PKA inhibitor, KT 5720 (5 μM), an inhibitor of MEK1/2, U0126 (10 μM) and an inhibitor of p38 MAPK, SB202190 (10 μM). The...

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Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. It is a molecular and prognostic heterogeneous disease. Three main genetic subtypes are called germinal center-like DLBCL (GC-like DLBCL), non-germinal center-like DLBCL (nonGC-like DLBCL) and primary mediastinal B-cell lymphoma (PMBL). These subtypes can be reliably distinguished only with usage of gene expression profiling (GEP). The GEP method can be applied only when fresh frozen tissue is available. The method is technically difficult and expensive. Thus, it is not used routinely. Since the DLBCL subtypes differ in prognosis, it is extremely important to be able to distinguish them. The presented thesis is focused on distinguishing of PMBL diagnosis in the group of DLBCL. Easily stored formalin-fixed, paraffin-embedded tissue (FFPE) and gene expression analysis using real-time quantitative polymerase chain reaction (RTqPCR) are used. In the first step, PMBL and DLBCL cases were distinguished with an internationally accepted clinical-pathological method. The agreement between clinical-pathological diagnosis and GEP is only 76%. In the presented text a genetic algorithm for PMBL/DLBCL distinguishing is suggested. It uses three carefully chosen genes and their expression is measured with RTqPCR. Both, the...

The topic of the presented thesis is the regulation of gene expression level of the three HSP70 genes in mononuclear cells. We investigated the dependence of expression regulation (induction) abiliy on selected point mutations, so-called SNPs (single nucleotide polymorphism) in the observed genes. The mononuclear cells were obtained from peripheral blood samples of healthy individuals. In order to analyze their gene expression, we selected individuals who were homozygous for at least one of the monitored point mutations. Taking into account the chosen criteria for healthy individuals we based on interviews with these individuals and their personal history. We determined the polymorphisms observed in two cell stress-inducible HSP70-1 (HSPA1A) and HSP70-2 (HSPA1B) genes and in one constitutively expressed gene HSP70-Hom (HSPA1L). Further, we have analyzed HSP70s gene expression regulation and the relation between the expression regulation and studied polymorphisms. We determined the degree of regulation of a gene expression in the studied genes in relation to two SNPs -110A/C (rs1008438), +190G /C (rs1043618) gene HSP70-1, and two SNPs +1267A/G (rs1061581), +2074G /C (rs539689 ) of the HSP70-2 gene, and the mutation of one five-nucleotide (rs9281590) HSP70-2 gene, and one SNP +2437T/C (rs2227956) of...

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. It is a molecular and prognostic heterogeneous disease. Three main genetic subtypes are called germinal center-like DLBCL (GC-like DLBCL), non-germinal center-like DLBCL (nonGC-like DLBCL) and primary mediastinal B-cell lymphoma (PMBL). These subtypes can be reliably distinguished only with usage of gene expression profiling (GEP). The GEP method can be applied only when fresh frozen tissue is available. The method is technically difficult and expensive. Thus, it is not used routinely. Since the DLBCL subtypes differ in prognosis, it is extremely important to be able to distinguish them. The presented thesis is focused on distinguishing of PMBL diagnosis in the group of DLBCL. Easily stored formalin-fixed, paraffin-embedded tissue (FFPE) and gene expression analysis using real-time quantitative polymerase chain reaction (RTqPCR) are used. In the first step, PMBL and DLBCL cases were distinguished with an internationally accepted clinical-pathological method. The agreement between clinical-pathological diagnosis and GEP is only 76%. In the presented text a genetic algorithm for PMBL/DLBCL distinguishing is suggested. It uses three carefully chosen genes and their expression is measured with RTqPCR. Both, the...

In animals, some of the most critical regulators of gene expression are nuclear hormone receptors (NRs) and their coregulators, specifically the Mediator complex. Of particular interest are the NRs implicated in metabolic and developmental regulation and in carcinogenesis: thyroid hormone receptors (TRs) and retinoid X receptors (RXRs). In this work, I venture to elucidate some aspects of gene expression regulation by these NRs: the degree of evolutionary conservation of signalling based on NRs and their coregulators; the mechanisms of negative regulation by NRs; and possible implications of these findings for clinical medicine. State-of-the-art bioinformatical, genome editing and microscopic techniques are applied at three levels of animal evolution to study NRs and Mediator. Reverse genomics in human patients suffering from the syndrome of resistance to thyroid hormones β are used to infer the structure and function of TRβ subdomains. Alignments, binding studies and in vivo experiments in Trichoplax adhaerens allow identification of a close orthologue of human RXR at the basis of metazoan evolution. Employing database queries, genome editing and microscopy, we describe a correct orthologue of the Mediator subunit 28 in Caenorhabditis elegans, indicating a complete homology of the Mediator complex...