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Target genes and regulation of hypoxia inducible factors HIF1a a HIF2a
Blahová, Tereza ; Žurmanová, Jitka (advisor) ; Alánová, Petra (referee)
Oxygen supply is necessary for today's form of life on Earth. Molecular oxygen is a terminal electron acceptor in mitochondrial respiratory chain and enables the efficient production of ATP by oxidative phosphorylation. The lack of availability of oxygen decreases energy production and can endanger the processes maintaining homeostasis. Therefore the compensatory mechanisms were developed by which cells respond to hypoxia. The master regulator of cellular responses is the hypoxia inducible transcription factor, HIF. In general HIF-1 isoform supports glucose availability and glycolysis; also attenuates energy-consuming processes and thus reduces energy loss. HIF-2 isoform stimulates antioxidant mechanisms to reduce the amount of reactive oxygen species which could cause cellular damage. At the same time, both of isoforms contribute to increasing the supply of oxygen by activating erythropoiesis and angiogenesis in the affected area. HIFs provide these changes either directly, by using their target genes, or by interactions with other transcription factors and signaling pathways.
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The promoter of c-myc oncogene as a target for a novel type of heterocyclic cations stabilizing G-quadruplex.
Pohlová, Lenka ; Šmídková, Martina (advisor) ; Hájková, Hana (referee)
Targeting oncogene promoters: a novel heterocyclic cations as G-quadruplex stabilizing ligands Lenka Pohlová Abstract: The diploma thesis studies an effect of newly synthesized group of compounds - helquats - on the expression of c-myc as a major player in malignant transformation and tumorigenesis via the stabilization of G-quadruplex in c-myc promotor. The G-quadruplex c-myc stabilization ability was tested for 101 helquats using dual luciferase reporter assay. The G-quadruplex c-myc stabilization ability was found for 13 helquats by this method. 8 successful helquats was selected by a comparison of the results from dual luciferase reporter assay and FRET melting assay. Effect on cell viability of tumor (HeLa S3) and non-tumor (HUVEC) cell lines was evaluated for these 8 helquats. Three of them exhibited cytotoxic effect on tumor cells but no effect was observed on viability of non-tumor cells. Moreover, an effect of these 3 helquats on c-myc expression on both mRNA and protein level, where significant effect on c-myc mRNA expression was not found for most of incubation periods. The 30% decrease in mRNA level was observed only for 24 hours incubation period for two helquats (LS702 and MJ656). The decrease in the expression on protein level was observed for all tested helquats, and helquat LS702 had the...
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Employing an RNA interference method (RNAi) to sudy oncogenic properties of Kaposi's sarcoma-associated herpesvirus (KSHV)
Riegerová, Petra ; Lubyová, Barbora (advisor) ; Hirsch, Ivan (referee) ; Pávová, Marcela (referee)
Kaposi's sarcoma-associated herpesvirus (KSHV) is a DNA tumor virus that has been associated with all epidemiological forms of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). Like other herpesviruses, KSHV undergoes two phases of life cycle (latent and lytic replication). During latency, the viral genome persists as a circular episome in the nucleus of the host cell and only a few viral genes are expressed, namely LANA (latency- associated nuclear antigen), Kaposin, vFLIP (viral FLICE inhibitory protein), vCyclin, and vIRF3/LANA2 (viral interferon regulatory factor 3). These viral genes are responsible for regulation of host cell proliferation, prevention of apoptosis, facilitation of immune evasion, and maintenance of the extrachromosomal viral genome during cell divisions. vIRF3 is a multifunctional nuclear protein that is constitutively expressed in KSHV positive PEL cells and Castleman's disease tumors, which expression causes dramatic changes of critical host pathways that are involved in the regulation of apoptosis, cell cycle, antiviral immunity, and tumorigenesis. In our study, we have demonstrated and elucidated predicted mechanism, by which vIRF3 enhances transcription activity of c-Myc. Moreover, we have clarified the previously unappreciated...
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The promoter of c-myc oncogene as a target for a novel type of heterocyclic cations stabilizing G-quadruplex.
Pohlová, Lenka ; Šmídková, Martina (advisor) ; Hájková, Hana (referee)
Targeting oncogene promoters: a novel heterocyclic cations as G-quadruplex stabilizing ligands Lenka Pohlová Abstract: The diploma thesis studies an effect of newly synthesized group of compounds - helquats - on the expression of c-myc as a major player in malignant transformation and tumorigenesis via the stabilization of G-quadruplex in c-myc promotor. The G-quadruplex c-myc stabilization ability was tested for 101 helquats using dual luciferase reporter assay. The G-quadruplex c-myc stabilization ability was found for 13 helquats by this method. 8 successful helquats was selected by a comparison of the results from dual luciferase reporter assay and FRET melting assay. Effect on cell viability of tumor (HeLa S3) and non-tumor (HUVEC) cell lines was evaluated for these 8 helquats. Three of them exhibited cytotoxic effect on tumor cells but no effect was observed on viability of non-tumor cells. Moreover, an effect of these 3 helquats on c-myc expression on both mRNA and protein level, where significant effect on c-myc mRNA expression was not found for most of incubation periods. The 30% decrease in mRNA level was observed only for 24 hours incubation period for two helquats (LS702 and MJ656). The decrease in the expression on protein level was observed for all tested helquats, and helquat LS702 had the...
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The relationship between circadian system and cell cycle
Vrtílková, Andrea ; Bendová, Zdeňka (advisor) ; Fárková, Eva (referee)
The circadian system is able to oscillate by itself owing to the transcriptional-translation feedback loop. Components of this loop do not affect just their own run, but they also have an impact on some other functions of the cell, for example cell cycle. This interaction is made by clock proteins (PER, CRY etc.) and by clock-controlled proteins (WEE1, TIM, XPA etc.). These proteins participate in the cell cycle run and have an impact on check-points. Disruption of the circadian clock can cause faults in cell cycle check-points, storing of DNA damages and increased cell apoptosis or tumor progression. Key words: circadian systém, cell cycle, WEE1, XPA, P21, C-Myc, TIM, PER
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Regulation of C-MYC oncoprotein by natural drugs.
Filandr, František ; Novák, Petr (advisor) ; Flieger, Miroslav (referee)
Sesqiterpene lactones, a group of plant secondary metabolites which include Cnicin from Cnicus benedictus plant, have an anti-proliferative and anti-tumor effect on mammalian cells by activating specific signaling pathways while also generating oxidative stress. These factors combined drive tumor cell apoptosis. A few of these compounds have reached clinical trials and seem to be a promising chemotherapeutics. The focus of this work is to elucidate the effect of cnicin on C-MYC transcription factor and oncoprotein which is overexpressed in majority of tumor tissues, the effect of cnicin on DEAD-box RNAhelicase DDX3 and on the expression levels of several metabolic genes is also studied. Through the use of western blotting, immunodetection and qPCR it was found out, that cnicin is regulating the expression of C-MYC oncoprotein on both transcriptional and translational levels, while also lowering C-MYC protein stability probably through the effect on PIM-2 kinase. Cnicin is not affecting the total amount of DDX3 protein in cells, but it seems it is lowering its degradation rate. The possible transcriptional regulation of DDX3 by cnicin is still not clear and requires further research. With the use of LC-MS quantitative analysis and qPCR, it was found out that cnicin does not affect the metabolism of...
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The role of Hippo Signalling pathway in tumor cell metabolism
Lettlová, Sandra ; Stiborová, Marie (advisor) ; Dračínská, Helena (referee)
Vitamine E analogues α-tocopheryl succinate (α-TOS) and mitochondrially targeted vitamine E succinate (MitoVES) are anti-cancer agents from the group of "mitocans", the compounds acting via mitochondria which present a promising invariant target for cancer cell therapy. α-TOS and MitoVES induce apoptosis selectively in various cancer cell types involving generation of reactive oxygen species (ROS). Generated superoxid anion radicals in response to α-TOS and MitoVES are believed to be converted into hydrogen peroxide that is known to activate Mammalian sterile 20-like kinase (Mst1), the central component of Hippo signalling pathway, that presents an universal size control mechanism in all metazoans and its deregulation is linked to tumourigenesis. MitoVES and α-TOS were both reported to activate Mst1 that phosphorylates Forkhead box O1 (FoxO1) transcription factor resulting in its transport to nucleus where induce the expression of pro-apoptotic genes, including NOXA, and thus promote apoptosis. The target of Hippo signalling pathway is transcriptional co- activator Yes-associated protein (Yap) which was found in Drosophila melanogaster to regulate the expression of transcription factor c-Myc which is known as the most prominent human oncogene. This thesis focused on involvement of Hippo signalling...
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Autoimmune and lymphoproliferative diseases: associations and common mechanisms
Dobiášová, Alena ; Daňková, Pavlína (advisor) ; Hušáková, Markéta (referee)
Autoimmune and lymphoproliferative diseases share some etiologic mechanisms. The origin of the diseases is complicated process that involves an accumulation of hereditary and somatic mutations in a hematopoetic cell, which thanks to changed activity overcomes different growth and survival control checkpoints. Such mutations are for example those located in genes coding for transcription factors, apoptotic signaling molecules, costimulatory molecules and secreted exctracellular molecules. All these molecules influence the balance between survival and programmed cell death. Their dysregulated expression enables the cell to overcome defensive mechanisms of the immune system. Therefore, autoimmune and malignant cells are able to survive though, under usual circumstances, they would be selected. The main aim of this work is to shed the light on the influence of the dysregulated expression of the particular molecules on the origin of autoimmune and lymphoproliferative diseases. Key words: autoimmune ilnesess, lymphoproliferative diseases, etiology, AIRE, c-MYC, TP53, FOXP3, Fas, PTEN, Bim, CTLA-4, CD5, CD30, CD40/CD40L, BAFF, α-taxilin, IL- 10.
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Target genes and regulation of hypoxia inducible factors HIF1a a HIF2a
Blahová, Tereza ; Žurmanová, Jitka (advisor) ; Alánová, Petra (referee)
Oxygen supply is necessary for today's form of life on Earth. Molecular oxygen is a terminal electron acceptor in mitochondrial respiratory chain and enables the efficient production of ATP by oxidative phosphorylation. The lack of availability of oxygen decreases energy production and can endanger the processes maintaining homeostasis. Therefore the compensatory mechanisms were developed by which cells respond to hypoxia. The master regulator of cellular responses is the hypoxia inducible transcription factor, HIF. In general HIF-1 isoform supports glucose availability and glycolysis; also attenuates energy-consuming processes and thus reduces energy loss. HIF-2 isoform stimulates antioxidant mechanisms to reduce the amount of reactive oxygen species which could cause cellular damage. At the same time, both of isoforms contribute to increasing the supply of oxygen by activating erythropoiesis and angiogenesis in the affected area. HIFs provide these changes either directly, by using their target genes, or by interactions with other transcription factors and signaling pathways.
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