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Bacterial RNA polymerase and molecules affecting its function
Jirát Matějčková, Jitka ; Krásný, Libor (advisor) ; Vopálenský, Václav (referee) ; Staněk, David (referee)
RNA polymerase (RNAP) transcribes DNA into RNA and is the only transcriptional enzyme in bacteria. This key enzyme responds to external and internal signals from the cell, resolves the intensity of transcription of individual genes and thus regulates gene expression. RNAP is not only affected by its own subunits, but also protein factors, small molecules or small RNAs (sRNAs). The aim of this Thesis was to contribute to the understanding of the regulation of the RNAP and to add missing fragments to this broad topic. The first part of this Thesis is focused on the influence of selected proteins (δ, YdeB, GreA) on the sensitivity of RNAP to the concentration of the initiating nucleoside triphosphate ([iNTP]) during transcription initiation in Bacillus subtilis. We showed that δ affects the sensitivity of RNAP to [iNTP] at [iNTP]-sensitive promoters, but not at [iNTP]-insensitive promoters neither in vitro nor in vivo. The δ subunit is essential for cell survival during competition with other strains, because it enables the cell to react immediately to changing conditions. Further we showed that YdeB protein does not bind to RNAP in B. subtilis, and has not shown any effect on transcription in vitro. We found that both, GreA and YdeB proteins (unlike δ subunit) were unable to affect RNAP by [iNTP] at...
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Imunomodulační účinky extraktů z helminta na střevní buněčnou linii potkaního modelu
LEVÁ, Jana
In this study, we examined the immunomodulatory effect of excretory/secretory products, crude adult extracts and crude larvae extracts from Hymenolepis diminuta on the intestinal epithelilal cell line from a rat. For determination of the immunomodulation effect of all H. diminuta extracts was used relative gene expression of TNFa, IL-17re and IL-33 from epithelial cells and it was tested using real-time PCR. Our result showed that excretory/secretory products had the strongest antiinflammatory effect on the epithelial cells. We assume that crude adult extracts play an important role in increase of gene expression of IL-33 and also in the immunomodulatory ability of H. diminuta in the host organism.
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Characterization of the Caenorhabditis elegans pop-1 gene
Jakšová, Soňa ; Vacík, Tomáš (advisor) ; Cmarko, Dušan (referee)
The human proteom diversity is caused by the ability of a single gene locus to encode more protein isoforms. The TCF/LEF genes produce a broad spectrum of protein variants, which consequently leads to a great functional diversity of the TCF/LEF proteins. The TCF/LEF transcriptional factors regulate the canonical Wnt signaling target genes. In this diploma project we focused on the Caenorhabditis elegans gene pop-1, the ortholog of the TCF/LEF genes. Using the Northern blot analysis we tried to identify alternative isoforms of the pop-1 mRNA in C. elegans. Using quantitative RT-PCR we also analyzed the pop-1 mRNA levels. Key words: canonical Wnt signaling pathway, TCF/LEF transcription factors, Caenorhabditis elegans, pop-1
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GENE EXPRESSION AND IMMUNOLOGICAL RESPONSE IN MICE EXPOSED TO ZnO NANOPARTICLES
Rössner ml., Pavel ; Vrbová, Kristýna ; Strapáčová, S. ; Rössnerová, Andrea ; Ambrož, Antonín ; Brzicová, Táňa ; Líbalová, Helena ; Javorková, Eliška ; Zajícová, Alena ; Holáň, Vladimír ; Kulich, P. ; Večeřa, Zbyněk ; Mikuška, Pavel ; Coufalík, Pavel ; Křůmal, Kamil ; Čapka, Lukáš ; Dočekal, Bohumil ; Šerý, Omar ; Machala, M. ; Topinka, Jan
We analyzed gene expression changes in the lungs and the immunological response in splenocytes of mice exposed by inhalation of ZnO nanoparticles - NP. Adult female ICR mice were treated for three days and three months, respectively. Analysis of differential expression in genes involved in oxidative stress was conducted using quantitative RT-PCR. The potential immunotoxic and immunomodulatory effects of ZnO NP were analyzed by phenotyping and cytokine production by splenocytes after three months exposure. Three days exposure resulted in down-regulation of GCLC, GSR, HMOX-1, NQO-1, NF-kB2, PTGS2 and TXNRD1 mRNA expression, three months exposure increased the expression of these genes. Three months exposure caused a significant decrease in the percentage of granulocytes in the spleen cells, and affected the production of IL-10 and IL-6 by lipopolysaccharide-stimulated leukocytes. In summary, our study revealed changes in the expression of genes involved in the oxidative stress response following acute ZnO NP exposure. Subchronic ZnO NP exposure induced immunomodulatory effects in the spleen.
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Regulation of gene expression at posttranscriptional levels.
Kollárová, Johana ; Kostrouch, Zdeněk (advisor) ; Macůrková, Marie (referee) ; Jindra, Marek (referee)
Regulation of gene expression in response to cellular and organismal needs is essential for sustaining organisms' survival and successful competition in the evolution of life forms. This regulation is executed at multiple levels starting with regulation of gene transcription, followed by regulation at multiple posttranscriptional levels. In this thesis, I focused on posttranscriptional mechanisms that contribute to gene expression regulation in the model organism Caenorhabditis elegans which enables powerful genetic and genomic techniques and allows the visualization of experimental genetic manipulations in toto, on the level of the complete organism during its life span. For this, we analysed the function of the orthologue of mammalian transcriptional corepressor NCOR, GEI-8. We used a functionally defective mutant gei-8(ok1671). I analysed the whole genome expression of homozygous gei- 8(ok1671) mutant and its link with observed mutant phenotype that includes defective gonad development and sterility and performed experiments leading to the proposition that disbalances in 21-U RNAs of piRNA class present in the most derepressed gene, the predicted mitochondrial sulfide:quinine reductase encoded by Y9C9A.16, are associated with the gonadal phenotype. In the second part of the thesis, I focused on...
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Molecular mechanisms of mutagenesis and resistence in CML cell lineages
Karasová, Dominika ; Čuřík, Nikola (advisor) ; Savvulidi Vargová, Karina (referee)
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,...
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The inhibitors of tyrosine kinases and their effect on the expression of biotransformation enzymes
Dvořák, Josef ; Dračínská, Helena (advisor) ; Moserová, Michaela (referee)
Tumor diseases are one of the most common causes of death in the human population. One of the many possible causes of tumor growth is abnormal function of tyrosine kinases, which are involved in signal transfer and regulation of the most important cell processes. These processes include the control of cell growth, division and cell differentiation and apoptosis. For the therapy of tumor diseases caused by the abnormal function of tyrosine kinases, their specific inhibitors are developed. For the targeted treatment of thyroid tumors, the tyrosine kinases vandetanib and lenvatinib are newly used. In this bachelor thesis, the effect of vandetanib and lenvatinib on the gene expression of the cytochrome P450 family of 2 (CYP2A2, CYP2B1, CYP2C11, CYP2D1, CYP2E1) biotransformation enzymes has been investigated as they are involved in the biotransformation reactions of a large portion of the available drugs on the market. To examine the relative gene expression of CYP2, quantitative PCR of samples of cDNA, synthesized from isolated RNA from rat liver and kidney exposed to the above-mentioned tyrosine kinase inhibitors, was used. The results suggest, that vandetanib and lenvatinib do not have a significant effect on the gene expression of cytochrome P450 family 2 in rat liver and kidney tissue. KEY WORDS:...
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Function of nuclear phosphoinositides and their binding partners in gene expression
Uličná, Lívia ; Hozák, Pavel (advisor) ; Šolc, Petr (referee) ; Macůrková, Marie (referee)
(ENGLISH) Phosphoinositides (PIs) are negatively charged glycerol-based phospholipids with inositol head (ring) which can be phosphorylated. Inositol ring phosphorylation yields in seven different PIs species which can be mono-, bis,- or tris-phosphorylated. Roles of cytoplasmic PIs have been extensively studied in for membrane and cytoskeletal dynamics, vesicular trafficking, ion channels and transporters and generating of second messengers. Nuclear PIs have been implicated in posttranscriptional processing of pre-mRNA, DNA transcription and chromatin remodelling. While cytoplasmic functions are very well described, the molecular mechanism of their nuclear functions are still poorly understood. In this study we focus on description of localization of nuclear PIs in particular functional nuclear compartments, which enable us to reveal PIs involvement in nuclear processes. We also focused on identification of nuclear PIs involved in the regulation of genes transcription and revealed detailed mechanism of PI(4,5)P2 a PHF8 interaction in the regulation of ribosomal genes transcription. By two independent approaches, we have described PIs localization to the nuclear membrane, nuclear speckles, small foci in the nucleoplasm, and the nucleolus. This spread nuclear localization suggests and confirms PI's...
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The importance and role of reverse transcriptases in gene expression analysis
Žucha, Daniel ; Valihrach, Lukáš (advisor) ; Španielová, Hana (referee)
The continuously advancing field of gene expression analysis enables the evaluation of even the slightest changes that occur in the cell transcriptome. In order to ensure accuracy of the observed biological variances, it is fundamentally important to be aware of the possible biases introduced during sample processing. In gene expression research, the methods of reverse transcription−quantitative PCR (RT−qPCR) and RNA- Sequencing (RNA-Seq) are often the primary choice, mostly because of their high precision and reproducibility. Since these both methods require DNA template, they are coupled with the same initial step - reverse transcription (RT), a reaction producing DNA complementary to its RNA template. It is well known that RT introduces bias. As a result, it is therefore of importance to thoroughly evaluate the effects of these biases. One such annotated source of artifacts is the reverse transcriptase (RTase) itself. However, it has been shown that the enzyme does not account for most of the variance alone. Surprisingly, choice of primers or RNA template may influence the reaction outcome even more than the bias introduced from the enzyme. This is especially the case with recent advances in protein engineering. Production of highly efficient RTases may pronounce the variation originating from...
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