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Characterization of Cbf11 and Mga2 interactions in the fission yeast
Grulyová, Michaela ; Převorovský, Martin (advisor) ; Čáp, Michal (referee)
Transcription factor Cbf11 belongs to the CSL protein family. The CSL protein family is well known for its function in Notch signalling pathway, however representatives in Notch- less fission yeast were discovered. Mga2 protein is a transcription regulator of triacylglycerol and glycerophospholipid metabolism. A crosstalk between Cbf11 and Mga2 was found. Cbf11 and Mga2 share target genes, and both are required for mitotic fidelity. This thesis aims to validate and characterize relationship between these transcription regulators. We show here that protein levels of Cbf11 and Mga2 change in response to presence of the other protein, as well as in response of nitrogen source. We also determine phylogenetic distribution of Cbf11 and Mga2 among Fungi, pointing to its connection. Using proteomic analysis of mga2 and cbf11 deletion strains we found that there is an overlap between proteins up/downregulated in these strains. Together, these results acknowledge crosstalk between Cbf11 and Mga2 proteins, bringing a novel connection between CSL protein family member and a functional analogue of mammalian SREBP-1 protein Mga2.
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New interconnections between lipid metabolism and chromatin regulation
Princová, Jarmila ; Převorovský, Martin (advisor) ; Bágeľová Poláková, Silvia (referee) ; Malínský, Jan (referee)
Lipid metabolism has been implicated in changes of chromatin modifications resulting in altered gene expression. Such regulation is important for cellular differentiation or cancer progression, however, the mechanism of how altered metabolic flux leads to targeted changes in chromatin modifications which then regulate gene transcription or heterochromatin maintenance is still poorly understood. We describe that fission yeast cells defective in fatty acid synthesis show increased expression of a subset of stress-response genes. This altered gene expression depends on the SAGA and NuA4 histone acetyltransferases and is associated with increased acetylation of histone H3 at lysine 9 in the corresponding gene promoters. Moreover, diminished fatty acid synthesis results in increased cellular resistance to oxidative stress. Additionally, the lipid metabolism mutants display chromatin alterations in centromeres and subtelomeres, regions of constitutive heterochromatin. We propose that changes in lipid metabolism can regulate histone acetylation and transcription of specific stress-response genes, but also lead to more global changes in heterochromatin. And while we clearly see the consequences of increased stress-response genes which result in promoting redox homeostasis, the implications of altered...
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Transcriptomic analysis of Mesocestoides corti
Korená, Lucie ; Leontovyč, Roman (advisor) ; Převorovský, Martin (referee)
Some species of parasites, including helminths, can inhibit carcinogenesis in their hosts. The antitumoral effect has been discovered in the tapeworms Taenia crassiceps and Echinococcus granulosus, which genes associated with cancer regression have been identified. The effect of melanoma suppression has also been observed in tapeworm Mesocestoides corti by the Laboratory of Helminthology, Department of Parasitology, Faculty of Science, Charles University, however the mechanism-of-action, remains unknown. For the upcoming research it was essential to have the complex molecular data such as transcriptome of the developmental stage s of M. corti. This work is focused on the transcriptomic profiling of the tapeworm M. corti and the differential gene expression in two different strains of murine hosts (inbred and outbred) using the RNA-Seq. The main goal was to identify upregulated transcripts in the tapeworms from the murine hosts that could have a potential effect on cancer regression. Differential gene expression analysis was performed, and the results showed that tapeworms in murine hosts (regardless of strain) had more upregulated transcripts than tapeworms cultured in vitro. Analysis of highly upregulated transcripts in the tapeworms that were grown in the murine hosts identified several...
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Characterization of closed mitosis in the fission yeast Schizosaccharomyces pombe with perturbed lipid metabolism
Hohoš, Patrik ; Převorovský, Martin (advisor) ; Cebecauer, Marek (referee)
[EN] The division of an eukaryotic cell is mediated by the process of mitosis. It is a complex cellular process which needs to be highly regulated. In contrast to the mammalian open type of mitosis when nuclear envelope is disassembled, fission yeast Schizosaccharomyces pombe undergoes closed mitosis inside the intact nuclear compartment. Cell nucleus undergoes morphological changes as a common sphere-shaped nucleus stretches upon mitotic spindle activity forming typical dumbbell structure. Further tension results in the separation of two daughter nuclei. Such extensive changes in the nuclear envelope surface demand a sufficient supply of membrane phospholipids. Cells with perturbed lipid metabolism are unable to meet such a demand and the mitotic division in these cells usually results as a catastrophic mitotic event or CUT (Cell Untimely Torn) phenotype. Moreover, recent studies show genetic interactions between the deletions of the lipid gene regulator cbf11 and factors maintaining the centromere chromatin structure. Surprisingly, rescue of CUT phenotype has been recently reported after the deletion of several factors contributing to the centromeric H3K9 epigenetic modifications in the cells lacking the transcription factor Cbf11. Here we show no rescue of CUT phenotype after the deletion of...
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Peak identification from ChIP-nexus data.
Drahoňovská, Lucie ; Převorovský, Martin (advisor) ; Gahurová, Lenka (referee)
Proteins play a very important role in live organisms. Their roles are for example structural, transport, regulatory or catalytic. What genes will be expressed, what proteins will be made and at what rate can have a strong impact on the function or even health of the organism. Gene expression is significantly regulated by transcription factors, whose activity may cause multiple diseases or disorders (Latchman 1997). Studying those factors and their function is therefore very important. Several methods were developed to this cause, ChIP-chip, ChIP-seq, ChIP-exo and ChIP-nexus. They enable us to study the binding sites of transcription factors and other DNA-binding proteins with various degrees of resolution. In this thesis I am going to describe the above mentioned methods and peak callers, softwares used for analysis of data obtained by those methods. I will also attempt to do peak calling of ChIP-nexus data of Cbf11 protein and compare the outcomes.
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Molecular mechanism of animal cells adaptation on hyperosmotic induced stress
Novotná, Jana ; Vávra, Jiří (advisor) ; Převorovský, Martin (referee)
Various types of cells animal tissues consist of need to adapt to intracellular as well as extracellular osmotic changes in order to maintain homeostasis. Hypertonicity (increased osmolarity) is one of the factors activating complex cellular reactions. In order to manage such stress, a cell needs to incorporate regulation pathways that can either regulate ion transporters through WNK kinases or activate gene expression of transporters of compatible osmolytes through the transcription factor TonEBP/NFAT5. Physiologically exposed to hypertonic conditions are cells in renal medulla, cartilage, inner ear tissues, in specialised neurons or buccal epithelium. This work includes basic and current knowledge about the adaption of cells to increased osmolarity of outer environment. Key words: Hyperosmolarity, osmotic stress, TonEBP/NFAT5, WNK kinases, NF-κB, HSP70
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Role of Ccr4 deadenylase in cell cycle regulation in yeast colonies
Daumová, Lenka ; Čáp, Michal (advisor) ; Převorovský, Martin (referee)
Regulation of cellular processes is of key importance for survival of cells. Many regulations are mediated by the CCR4-Not complex, a highly conserved protein complex, which is present in eucaryotic cells, from yeast to mammals. In this work I study mostly the role of the Ccr4 subunit on yeast survival in the aging colony of Saccharomyces cerevisiae, mainly during cell cycle progression. Ccr4 si a deadenylase, and its main function is cleavage of poly(A) tail of mRNA molecules, and by doing so, shortening the mRNA life-time. It is very important during strictly regulated cellular processes, because it is essential that gene expression of specific genes happens only at specific time. Saccharomyces cerevisiae BY 4742 is a haploid yeast strain, which can easily be used for making deletion mutants in specific genes. In this diploma thesis I focus on studying a deletion mutant of CCR4, along with deletion of other genes, which influence yeast cell cycle. By comparing the phenotype of these mutants with wild type, it is possible to identify changes in phenotype, caused by these deletions, and their influence on yeast cell survival. Key words: cell processes regulation, CCR4-Not complex, Ccr4 subunit, cell cycle, yeast, Saccharomyces cerevisiae, gene deletion
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The role of the AGO-hook domain of histone chaperone SPT6L in regulation of gene expression
Kašpar, Tomáš ; Čermák, Vojtěch (advisor) ; Převorovský, Martin (referee)
AGO-hook domains present in some eucaryotic proteins are crucial for a binding family of ARGONAUTE proteins (AGO). These AGO proteins are essential in many biological processes regulating gene expression by small RNA (sRNA), which is complementary to the gene that is supposed to be influenced. This thesis claims to find the function of the putative AGO-hook domain of the protein SPT6L. SPT6L is an elongation factor and histone chaperon of a complex of RNA polymerase II (Pol II) where it is acting in the epigenetic marking of histones. SPT6L of Arabidopsis thaliana is one of two paralogues of SPT6 proteins, that is characteristic of the presence of the AGO-hook domain. This is a plant specificity of this protein. What's more, the function of this domain remains unknown. Despite this, it could be assumed that this domain is necessary for binding of AGO proteins in the complex of Pol II, and by these interactions, it can enable guidance of regulation of chromatin modification, or it can co-transcriptionally influence nascent transcripts of Pol II by the sRNA. This thesis casts light on the function of the AGO-hook domain of SPT6L in A. thaliana in processes of gene regulation and protein interactions. It claims to confirm the interaction of the AGO-hook domain of SPT6L protein with AGO proteins and...
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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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Chromatin immunoprecipitation of selected transcription factors
Smetanová, Jitka ; Vališ, Karel (advisor) ; Převorovský, Martin (referee)
The TEAD family of transcription factors regulates expression of genes affecting cell proliferation, differentiation and apoptosis. The activity of a particular transcription factor called TEAD1 is regulated by the Hippo signalling pathway. The Hippo pathway has been implicated to play a role in cancer suppression, however its precise mechanism remains unclear. MYC and GLUT1, genes which are coding two key regulators of glycolysis, were recently described as potential targets of the Hippo signalling pathway in human leukemia cells. In this diploma thesis, I tried to confirm the proposed interaction of the transcription factor TEAD1 with regulatory sequences of MYC and GLUT1 genes using chromatin immunoprecipitation (ChIP) analysis in human leukemic cells. However, I failed to successfully isolate TEAD1 complexes using ChIP. So, I discuss in my diploma thesis also possible reasons for this outcome, including biological and methodological issues. (In Czech) Key words: Transcriptional regulation, TEAD transcription factors, chromatin immunoprecipitation, leukemia
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