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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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The role of evolutionarily conserved proteins BIR-1/Survivin and SKP-1 in the regulation of gene expression
Kostrouch, David ; Kostrouch, Zdeněk (advisor) ; Dráber, Pavel (referee) ; Pacák, Karel (referee)
SKIP and BIR/Survivin are evolutionarily conserved proteins. SKIP is a known transcription and splicing cofactor while BIR-1/Survivin regulates cell division, gene expression and development. Loss of function of C. elegans SKIP (SKP-1) and BIR-1 induces overlapping developmental phenotypes. In order to uncover the possible interactions of SKP-1 and BIR-1 on the protein level, we screened the complete C. elegans mRNA library using the yeast two-hybrid system. These experiments identified partially overlapping categories of proteins as SKP-1 and BIR-1 interactors. The interacting proteins included ribosomal proteins, transcription factors, translation factors and cytoskeletal and motor proteins suggesting involvement of the two studied proteins in multiple protein complexes. To visualize the effect of BIR-1 on the proteome of C. elegans we induced a short time pulse BIR-1 overexpression in synchronized L1 larvae. This led to a dramatic alteration of the whole proteome pattern indicating that BIR-1 alone has the capacity to alter the chromatographic profile of many target proteins including proteins found to be interactors in yeast two hybrid screens. The results were validated for ribosomal proteins RPS-3, RPL-5, non-muscle myosin and TAC-1, a transcription cofactor and a centrosome associated...
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Methods for class prediction with high-dimensional gene expression data
Šilhavá, Jana ; Matula, Petr (referee) ; Železný, Filip (referee) ; Smrž, Pavel (advisor)
Dizertační práce se zabývá predikcí vysokodimenzionálních dat genových expresí. Množství dostupných genomických dat významně vzrostlo v průběhu posledního desetiletí. Kombinování dat genových expresí s dalšími daty nachází uplatnění v mnoha oblastech. Například v klinickém řízení rakoviny (clinical cancer management) může přispět k přesnějšímu určení prognózy nemocí. Hlavní část této dizertační práce je zaměřena na kombinování dat genových expresí a klinických dat. Používáme logistické regresní modely vytvořené prostřednictvím různých regularizačních technik. Generalizované lineární modely umožňují kombinování modelů s různou strukturou dat. V dizertační práci je ukázáno, že kombinování modelu dat genových expresí a klinických dat může vést ke zpřesnění výsledku predikce oproti vytvoření modelu pouze z dat genových expresí nebo klinických dat. Navrhované postupy přitom nejsou výpočetně náročné. Testování je provedeno nejprve se simulovanými datovými sadami v různých nastaveních a následně s~reálnými srovnávacími daty. Také se zde zabýváme určením přídavné hodnoty microarray dat. Dizertační práce obsahuje porovnání příznaků vybraných pomocí klasifikátoru genových expresí na pěti různých sadách dat týkajících se rakoviny prsu. Navrhujeme také postup výběru příznaků, který kombinuje data genových expresí a znalosti z genových ontologií.
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Estrogen receptor beta (ERβ) in testicular cells and sperm
Dostálová, Pavla ; Žatecká, Eva ; Děd, Lukáš ; Dorosh, Andriy ; Postlerová, Pavla ; Jonáková, Věra ; Dvořáková-Hortová, Kateřina ; Pěknicová, Jana
Estrogen is a steroid hormone that plays an important role during sperm development in the male and female reproductive tract. Estrogen signalling is a complex process that depends on cell milieu and presence of receptors. Thanks to the steroid nature of estrogens, they can pass through the plasmatic membrane and bind to the intracellular estrogen receptors (ERs). Within the cell, there are several pools of ERs. One of them is localized to the cell nucleus and their activation leads to direct or indirect binding to DNA and ultimately to alternation in gene expression (genomic pathway). Other pools of ERs are associated with plasma membrane or are located in cytosol. Activation of membrane associated ERs leads to rapid non-genomic responses. Nowadays, two classical estrogen receptors are known – ERα and ERβ. Since ERβ is a predominant variant in testes, we focused our study on expression of ERβ variants in murine testes and sperm. We detected two variants of ERβ at mRNA level in both, testes and sperm. These variants differ in 54 nucleotids within the ligand binding domain and this variability results in different affinity to estrogen. We analyzed individual testicular cell types (spermatogonia, spermatocytes, spermatids, Sertoli cells) by RT-qPCR. Our results suggest that both ERβ variants are coexpressed in the same cell type and may therefore interact together. This may have consequences in mediating of estrogen signalling. Moreover, ERβ is expressed more in the later stages of spermatogenesis suggesting the role of ERβ in these stages or alternatively in spermatozoa alone. At the protein level, we detected ERβ in nuclear, membrane and cytosolic fraction prepared from testicular tissue suggesting the involvement of both, genomic and non-genomic, pathways of estrogen signaling in testes. In sperm, anti-ERβ antibodies localized ERβ in acrosome region and tail which is in accordance with the known role of estrogen on capacitation, acrosome reaction and motility.
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Analysis of intron sequence variability
Kukačková, Hana ; Provazník, Ivo (referee) ; Kubicová, Vladimíra (advisor)
This bachelor thesis reviews genetic expression, the main characteristics of introns, their importance, and the methodology of their recognition. Practical part focuses on intron variability analysis and conservative sequences searching. In the beginning, there is a structure of nucleic acids with their chemical and physical characteristics reviewed, follow by gene expression description with all steps – transcription, posttranscriptional splicing and translation. Afterwards, there is an analysis of introns importance, introns evolution theories and similarities among different organisms mentioned, follow by some introns recognition methods. Practical part is focused on the initial and terminal regions of introns and conservative dinucleotide sequences verifying. The thesis studies intron – exon and exon – intron boundaries. The last part tries to suggest new potentially conservative sequences – patterns, search for them and check their conservativeness.
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