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Laccase activity profiling in Trametes versicolor cultures degrading endocrine-disrupting compound Delor 103
Plačková, Martina ; Svobodová, Kateřina (advisor) ; Mikušová, Gabriela (referee)
In this work endocrine disrupting potential of Delor 103, a commercial mixture of PCB congeners, was studied along with its effect on production of laccase by the ligninolytic fungus Trametes versicolor. Using a gene-reporter yeast assay for evaluation of hormonal activity Delor 103 showed an androgenic activity with an EC50 value of 2.29. 10-2 mg/l. Chlorbenzoic acids, Delor 103 potential metabolites resulting from microbial degradation, displayed on the other hand an estrogenic activity, indicating possible changes in hormonal activity of Delor 103 during its microbial degradation. The addition of Delor 103 to mineral medium T. versicolor cultures resulted in an up to 257times higher laccase activities detected in fungal cultures. Delor 103 induced enzymes showed different pI values from those of control cultures. In a complex malt-extract glucose medium (MEG) the stimulation effect of Delor 103 was kept down. Further, the production of laccase and synthesis of different pI forms depended strongly on the growth phase of fungal cultures. Exponencially growing cultures of T. versicolor were able to produce up to 7 different pI forms of laccase in responce to Delor 103 whereas stationary cultures produced only 4 enzyme forms with higher pI values. Stimulation of laccase activities in T. versicolor,...
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delta subunit of bacterial RNA pol and its role in regulation of gene expression in B. subtilis
Dvořáček, Lukáš ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee)
Delta subunit of bacterial RNA pol and its role in regulation of gene expression in B. subtilis. In this work I focus on regulation of eubacterial gene expression. First, I describe recent knowledge about a key stage of gene expression - transcription, focusing on regulation of trancription iniciation via small effector molecules (guanosine tetraphosphate, initiating nucleoside triphosphate) that are important for the regulation of ribosomal RNA. Second, in the experimental part of my work, I focus on the role of the _ protein, a subunit of RNA polymarase in gram positive bacteria, in transcription iniciation and its effects on regulation of RNA polymerase by the concentration of initiating nucleoside triphosphates.
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The role of microRNAs in chronic lymphocytic leukemia
Moravec, Martin ; Macůrková, Marie (referee) ; Hájek, Miroslav (advisor)
2 Abstract MicroRNAs (miRs, miRNAs) are recently discovered molecules (19-25 nucleotides long) that regulate gene expression at post-transcriptional level by either blocking protein synthesis or mRNA degradation. As a part of gene silencing mechanism, miRNAs are involved in cellular processes, such as apoptosis, cell proliferation, development and viral defence. miRNAs have been intensely studied in connection to disease pathogenesis. Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western countries affecting mostly elderly people. In my work I focus on explanation of miRNA functions and their contributions to chronic lymphocytic leukemia (CLL). I describe previously published data about miRNA-15, miRNA-16, miRNA-143, miRNA-145 and miRNA-155 in connection to this disease. Based on recent reports, I also discuss the potential role of miRNA-326 in CLL pathogenesis.
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Role of selected ABC transporters in breast cancer development
Perglerová, Karolína ; Stiborová, Marie (referee) ; Souček, Pavel (advisor)
Breast cancer is a leading cause of death among women in many countries. In the treatment of the breast cancer cytotoxic drugs (chemotherapy) are often used. Interindividual differences of drug response are an important cause of treatment failures. Bioavailability also depends on a major extent from the expression and activity of drug transport across biomembranes. In particular efflux transporters of the ATP-binding cassette family such as ABCB1, ABCC1 and ABCC2 have been identified as major determinants of chemoresistance in tumor cells. It was hypothesized that variance in the gene expression of membrane transporters and their genetic variance could explain at least in part interindividual differences of pharmacokinetics and clinical outcome of a variety of drugs. This thesis focuses on the functional significance of gene expression of ABCB1, ABCC1 and ABCC2 and single nucleotide polymorphisms in ABCC1 gene.
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Ammonia production by colonies of mutants and aging of wrinkled colonies of Saccharomyces cerevisiae
Nedbálková, Jana ; Heidingsfeld, Olga (referee) ; Janderová, Blanka (advisor)
Production of ammonia by the colonies of mutants and aging of wrinkled colonies of Saccharomyces cerevisiae The aim of this diploma thesis is to observe the development, respectively the aging of cells in yeast colonies Saccharomyces cerevisiae. Yeast cells S. cerevisiea form multicellular organized structures on a solid substrate, i.e. colonies, which the intercellular interactions occur in. These interactions influence forming, morphology and aging of yeast colonies. This diploma thesis is focused partly on the changes in ammonia production by giant colonies of deletion mutants and partly on the aging of colonies with the wrinkled morphology. I characterized mutant strains of S. cerevisiae with the deletion in RTG1, RTG2, RTG3, FIS1, CIT2 genes. Their products play an important role in the colony development. The transcription of these genes changes during the transition from the acidic to alkali phase during developmental process of the colonies. I have found out that the ammonium production rate was in accordance with the results of the alkalization in giant colonies surroundings and mentioned mutants derived from the BY strain has been producing ammonia since the 15th day. The rate of the ammonia production by rtg3∆ strain was comparable to the parental strain. Compared to parental strain, lower...
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The delta subunit of RNA polymerase from gram positive bacteria
Matějčková, Jitka ; Beranová, Jana (referee) ; Krásný, Libor (advisor)
1 Abstrakt Aby bakteriální buňka přežila neustále se měnící podmínky, musí se na ně adaptovat. Tato adaptace je podmíněna změnou genové exprese. Klíčovým krokem genové exprese je transkripce. Hlavním enzymem bakteriální transkripce je RNA polymerasa (RNAP), což je esenciální vícepodjednotkový enzym. RNAP je nejvíce prostudována u Escherichia coli, modelového organismu gram negativních bakterií. Porovnala jsem E. coli a Bacillus subtilis (zástupce gram pozitivních bakterií) a shrnula jsem rozdíly v RNAP a transkripci. Jejich RNA polymerasy se liší přítomností podjednotky δ u gram pozitivních bakterií. Tato podjednotka zvyšuje promotorovou selektivitu, recykluje jádro RNAP a celkově stimuluje syntézu RNA. Podjednotka δ ovlivňuje sporulaci a virulenci některých bakterií. V této práci jsem shromáždila současné poznatky o jednotlivých částech genové exprese, zejména o regulaci iniciace transkripce a o podjednotce δ RNAP.
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The effect of cancerogenic azo dye Sudan I on expression of biotransformation enzymes
Hejduková, Žaneta ; Dračínská, Helena (referee) ; Svášková, Dagmar (advisor)
Sudan I is a widely used azo dye which has the ability to cause carcinomas in organs and tissues of experimental animals. During reactions catalyzed by microsomal monooxygenase enzyme systems or cytoplasmic biotransformation enzymes, Sudan I is oxidized to reactive metabolites that covalently bind to nucleic acids and cause their damage. Sudan I can also be metabolized by reduction, e. g. by a DT-diaphorase enzyme (NQO1). Reduction of Sudan I is considered to be a detoxification reaction. In this work, the in vivo action of Sudan I is examined in terms of its ability to induce an expression of the biotransformation enzyme DT-diaphorase in tissues of rats treated with the azo dye. The aim of this work was to quantify the degree of NQO1 induction at mRNA level. After the isolation of total RNA from organs of rats treated with Sudan I, the RNA was converted to cDNA by reverse transcription using random hexamers as primers. Using specific probes, the abundance of mRNA for the enzyme NQO1 in the organs of treated rats was quantified by "real-time" PCR, relatively to the control gene with a constant expression (β-actin). Through comparing thus determined amounts of mRNA in individual organs of treated and untreated rats, it has been found that Sudan I had caused a significant increase in the expression...
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The effect of Vps34p in yeast colony
Červenka, Jakub ; Schierová, Michaela (advisor) ; Převorovský, Martin (referee)
The phosphatidylinositol-3-kinase (PI3K) signalling pathway is evolutionarily conserved in all eukaryotes and its main function is the regulation of autophagy and protein sorting to the vacuole/lysosome. In the pathogenic yeast species Candida albicans and Cryptococcus neoformans the PI3K signalling pathway is required for virulence. In the yeast Saccharomyces cerevisiae the PI3K signalling pathway consists of two proteins - phosphatidylinositol-3-kinase, Vps34p and its regulator Vps15p. In this diploma thesis I analyse the role of the PI3K signalling pathway in the growth and development of colonies of natural and laboratory strains. I proved that VPS34 or VPS15 deletion in haploid laboratory strains has a significant influence on colony size and invasive growth (in strain ΣSh vps15Δ). Deletion of VPS34 or VPS15 also increases sensitivity of cells to oxidative stress and detergents. Attempts to delete VPS34 in natural strains were unsuccesful, probably because VPS34 is essential in these strains. Constitutive expression of VPS34 does not affect cell resistance in inhibitory tests, the size and differentiation of colonies or ammonia signalling but differences are notable in giant colony morphology and in patterns of invasiveness of the medium. Tagging of the C-terminal of Vps34p with GFP affects...
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Laboratory diagnostics of micrometastases in breast cancer patients
Mikulová, Veronika ; Zima, Tomáš (advisor) ; Svoboda, Marek (referee) ; Průša, Richard (referee)
Introduction: The presence of circulating tumor cells (CTC) in the peripheral blood has been associated with worse prognosis and early relapse in breast cancer patients. CTC determination in the peripheral blood has been considered as a liquid biopsy. The aim of this project was to analyze the presence of CTC followed by their molecular characterization with the potential use not only as a new biomarker for real-time monitoring of therapy efficacy but also as a suitable tool for patient's stratification and individualization of treatment for breast cancer. Methods: A total of 54 patients with diagnosed early breast cancer were enrolled into a prospective study. Ten millilitres of peripheral blood were sequentially collected to test for the presence and characterization of CTC during the follow-up of patients. CTC isolation and detection was performed by AdnaTest BreastCancer™ (AdnaGen AG, Germany), which is based on the detection of EpCAM, HER2 and MUC1 specific transcripts in enriched CTC- lysates. cDNA from isolated CTC has been further used for newly optimized qPCR assays for breast tumor and therapy resistance associated genes: TOP1, TOP2A, CSTD, ST6GAL, KRT19 and reference gene actin. qPCR results have been analyzed by Genex software (MultiD Analysis). Results: 195 blood samples have been...
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