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Mutational analysis of manumycin antibiotics biosynthetic routes
Kolek, Jan ; Petříček, Miroslav (advisor) ; Zikánová, Blanka (referee)
Mutational analysis of manumycin antibiotics biosynthetic routes The manumycin antibiotics are secondary metabolites, which come from a big group of polyketide metabolites. They are produced by bacteria from genus Streptomyces. Manumycin antibiotics are characterized by two linear polyketide chains, which are connected to a central mC7N moiety. The lower chain is often terminated by the C5N moiety. Manumycin metabolites show many biological activities. They have antimicrobial activity, especially against gram-positive bacteria. Next, they posses antifungal, insecticidal or antiinflamatory activities. Manumycins are also potentional anticancer agents. In order to prepare these compounds by the fermentation, the detailed knowledge of their biosynthetic routes is required. Mutational analysis is based on techniques of genetic engineering. Mutational analysis is a useful pool for analysis of biosynthetic pathways of secondary metabolites and the genes, which are involved in these pathways. This knowledge is essential for application of combinatorial biosynthesis for the design of new metabolites. The new hybrid compound could be used in future as new antibiotics or anticancer drugs. Keywords: manumycin, polyketide, streptomyces, asukamycin
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Preparation of cDNA of selected gene and cloning.
Bogdanovič, Igor ; Miletín, Miroslav (advisor) ; Bunček, Martin (referee)
ABC drug efflux transporters are transmembrane proteins which are known to play a crucial role in drug resistance. The ability of these proteins to actively transport a wide variety of compounds out of the cell is one of the key reasons behind the resistance of cancer cells to chemotherapeutic agents. To study the activity of efflux transporters and identify their substrates or inhibitors a number of in vitro models have been developed. These models are based on the mammalian cells genetically modified in order to express the protein of interest. For genetic manipulations of the cells various expression plasmids are used. In our study we attempted to prepare a universal expression plasmid, containing a short sequence of V5 tag. Such plasmid enables further cloning of coding sequences of selected efflux transporters so that the V5 tag is localized on their 3'-end. Once constructed, the plasmid can further be used to transfect the desired cell line, thus developing an in vitro cell model. Since the transporter is produced as a fusion protein with the V5 tag localized on their C-end, the tag could be used as a universal marker of the transporter expression detectable by immunohistochemical methods. To construct the V5 tag sequence two approaches were employed both leading to the product of the desired...
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Role of the oncogenic microRNAs miR-17-92 and miR-155 in the regulation of hematopoietic differentiation and leukemogenesis
Pospíšil, Vít ; Stopka, Tomáš (advisor) ; Pospíšek, Martin (referee) ; Machová Poláková, Kateřina (referee)
(English version): Hematopoietic differentiation is highly ordered multistep process, where generation of terminal blood cells is dependent upon coordinated regulation of gene expression by key regulators: transcription factors and mikroRNAs. PU.1 (Sfpi1) is a versatile hematopoetic transcription factor required for the proper generation of both myeloid and lymphoid lineages. MikroRNAs represent a novel class of ~22 nucleotide long non-coding posttranscriptional regulators that inhibit expression of genes by blocking protein translation or by mRNA degradation. In this PhD thesis I present research data documenting novel mechanisms of regulation and function of two oncogenic mikroRNAs, miR-17-92 cluster and miR-155 and myeloid transcriptional factors PU.1 upon macrophage differentiation of myeloid progenitors. The miR-17-92 cluster (Oncomir1) encodes seven related mikroRNAs that regulate cell proliferation, apoptosis and development and is overexpressed in number of malignancies including myeloid leukemia. Presented PhD thesis documents novel macrophage specific regulatory mechanisms involving the oncogenic cluster miR-17-92. Using transgenic PU.1-/- myeloid progenitors we show that upon macrophage differentiation, the transcription factor PU.1 induces the secondary determinant, the transcription...
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Mapping of regulatory elements within 5' region of the Disp3 locus
Oltová, Jana ; Bartůněk, Petr (advisor) ; Krásný, Libor (referee)
Dispatched 3 (Disp3), a thyroid hormone-regulated gene, is studied extensively in our laboratory. Phenotype of cells with overexpressed Disp3 and its expression pattern make it a perfect candidate for a molecular link between thyroid hormone action and cholesterol homeostasis in the brain. Moreover, we hypothesize that it might play a role in certain neurodegenerative disorders and brain tumours. This thesis is aimed at the process of regulation of this gene via thyroid hormone receptor (TR), specifically identification of responsive elements of the thyroid hormone receptor that are necessary for the regulation. Also, we searched for elements recognized by liver X receptor (LXR), as LXR binds to the same arrangement of repeats as TR and there are a number of genes regulated by both of them. We combined in silico analysis of the Disp3 locus with reporter luciferase assays. A cluster of six elements identified around the first exon with two of them being conserved among human and mice draw our attention. In order to analyze this sequence in more detail, reporter vectors of various truncations of 3 kb region around exon 1 were constructed and tested in reporter assays. Reporter assays did not reveal any substantial element activated by TR or LXR; on the other hand, region containing repressor element(s)...
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Noise and vibrations of roots blowers
Smrček, Martin ; Hájek, Petr (referee) ; Švancara, Pavel (advisor)
This thesis is concerned with the spread of noise in the housing sets blowers, blower vibration, due to the shape of the geometry of the individual parts of the blower and the design of a suitable discharge muffler in order to achieve the highest possible attenuation. Analysis blower and silencer discharge was conducted using the finite element simulation ANSYS 15.0
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Methylation profile in malignancy
Stojčeva, Nina ; Vodička, Pavel (advisor) ; Vondrejs, Vladimír (referee)
Epigenetic changes represent chemical modifications of the DNA molecule and histone proteins by which gene expression is altered. Among them, DNA methylation is a known mechanism of silencing of tumor-suppressor and DNA repair genes, with an important role in carcinogenesis. Many studies have been done in order to identify the methylation signatures of these genes in different types of cancer. In our study, we investigated the methylation status of promoter regions of eight mismatch repair genes (MLH1, MSH2, MSH3, MLH3, PMS1, PMS2, MSH6 and EXO1) in 45 sporadic colorectal cancer cases and 12 head and neck cancer patients. Two out of eight genes, MLH1 and MLH3, exhibited promoter methylation. The results from both groups of patients were concordant. We summarize that the methylation profiles of MLH1 and MLH3 promoters could be potential candidates for epigenetic biomarkers in colorectal cancer, and eventually in head and neck cancer. Further investigations, which would confirm this theory, should be carried out.
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Study of inter-variant differences in xenobiotics metabolism in flax
Hartinger, Jan ; Skálová, Lenka (advisor) ; Karlíčková, Jana (referee)
Seed root inhibition tests with 18 species of flax (Linum usitatissimum) were carried out with different concentrations of TNT (trinitrotoluene) in order to elucidate intervarietous differences regarding their abilities to degrade nitrocompounds. The curves demonstrating dependency of root lengths on TNT concentrations varied significantly. The outcomes were influenced by the fact that five of the species germinated problematically even with the zero level of TNT. Callus cultures were grown from six of the species and cell suspension cultures were derived from three of them. Cell suspension cultures were tested on the ability to degrade TNT and DNT (dinitrotoluene) present in the medium. The decrease of TNT or, respectively, the decrease of DNT concentrations in the medium was measured by HPLC. It was discovered that contrary to TNT, DNT is very toxic for the cells when its concentrations get close to the level of maximum solubility. Trinitrobenzene, 2-aminodinitrotoluene and 4- aminodinitrotoluene were identified as main degradation products of TNT. Further on, 2D electrophoresis was performed with samples from cultures of two species stressed by DNT and TNT. In case of DNT an increase of protein density was marked in a certain area while in case of TNT no similarity was found between responses...
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Secondary metabolism and its regulation in Streptomyces ambofaciens: the study of cryptic secondary metabolite biosynthetic gene clusters
Nezbedová, Šárka ; Weiser, Jaroslav (advisor) ; Petříček, Miroslav (referee) ; Mazodier, Philippe (referee)
I. ABSTRACT in English The presented work is focused on secondary metabolism and its regulation in Streptomyces ambofaciens and Streptomyces lividans with special interest in new biosynthetic pathways. The sequencing of bacterial and fungal genomes revealed that the number of their secondary metabolite biosynthetic gene clusters greatly exceeds the number of produced secondary metabolites. Further studies showed that at least some of the newly discovered clusters, called cryptic since no product had been associated to them, were expressed in certain conditions and that they directed the biosynthesis of exploitable secondary metabolites (Gottelt et al., 2010; Gross et al., 2007; Pang et al., 2004). Therefore, these cryptic clusters have been considered as one of the promising reservoirs of new bioactive molecules. Using different approaches I studied the cryptic secondary metabolite biosynthetic gene clusters of Streptomyces ambofaciens, a strain exploited industrially for the production of the antibiotic spiramycin. In the second part of this work, I was interested in the regulation of secondary metabolite biosynthesis and in manipulating regulatory proteins in order to activate the expression of cryptic clusters. The third part of this work studied the effect of the inactivation of the ppk gene, encoding...
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Studium exprese MDR pump u kvasinek Saccharomyces cerevisiae za různých růstových podmínek: metoda s fluorescenční sondou diS-C3(3)
Zahumenský, Jakub ; Gášková, Dana (advisor) ; Krůšek, Jan (referee)
In this work, we studied two yeast ABC transporters, Pdr10p and Pdr15p. At the time of assignment of this thesis, it was believed that these proteins contribute to the yeast MDR phenotype (PDR) on the grounds of their high homology to another yeast MDR protein, Pdr5p. In order to study these pumps, two sets of isogenic null-mutant strains were prepared with all possible combinations of gene deletions. We report that both of the studied proteins are very important in sus- taining the normal plasma membrane microenvironment for the most abundant, and essential, yeast plasma membrane protein, H+ -ATPase and so influence the membrane potential. Pdr10p and Pdr15p thus play an as yet unknown role in reg- ulation of the activity of this enzyme. Furthermore, we report that deletion of the genes coding for these proteins severely reduces the ability of the H+ -ATPase to be activated by the protonophore CCCP which is a weak acid. Studies performed with immunosuppressant FK506 further show that this compound reduces the viability of S. cerevisiae mutant strain PLY643 lacking genes coding for Pdr5p, Snq2p and Yor1p. Further deletion of Pdr10p and Pdr15p does not increase the lethality of this compound. Neither CCCP nor FK506 are substrates of the stud- ied pumps. 1
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Identification of Z-linked genes for the study of sex chromosome evolution in Lepidoptera
PROVAZNÍK, Jan
Moths and butterflies (Lepidoptera) have a WZ/ZZ (female/male) sex chromosome system or its numerical variants. The non-recombining W chromosome is largely formed by heterochromatin, composed of repetitive DNA and almost free of genes. Its use for evolutionary studies is thus limited due to fast molecular divergence. Whereas the Z chromosome resembles to autosomes; it is rich in genes and thus expected to be more conserved. In this study, the Z-linked genes with available DNA sequence information are summarized in order to give reference to suitable candidates for the study on evolutionary history of sex chromosomes in Lepidoptera. The use of Zlinked genes to test current hypotheses on the origin of the lepidopteran W chromosome is also discussed.
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