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The role of ETV6-RUNX1 fusion protein in the sensitivity of leukemic cells to L-asparaginase
Staněk, Petr ; Starková, Júlia (advisor) ; Burjanivová, Tatiana (referee)
Translocation t(12;21) with the presence of the fusion gene ETV6-RUNX1 (TEL-AML1) is the most common chromosomal aberration found in acute lymphoblastic leukemia in childhood. The occurrence of the ETV6-RUNX1 is associated with excellent prognosis and high sensitivity to the treatment with the enzyme L-asparaginase (ASNase). Resistance to the drug aggravates the outlook of the patient and increases the risk of treatment failure, therefore, the CLIP working group has been for a long time involved in the identification of the mechanism of action of ASNase and the origin of the resistance to it. This thesis follows previous findings of the group and is devoted to the analysis of the importance of ETV6-RUNX1 and signalization and metabolic changes accompanying shifts in the L-asparaginase resistance. In the first part of the thesis, the knockout clones with stable increased resistance to ASNase have been established thanks to the CRISPR/Cas9 system, which created frameshift in the fusion gene. The accomplishment in this regard and removal of the fusion protein was confirmed on the level of DNA, mRNA a protein expression. The presence of other significant chromosomal aberrations affection the sensitivity to ASNase was ruled out by the means of SNP analysis. In the second part of the project, the signalization...
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The family of 4E translation factors explored in human cell lines
Čečmanová, Vendula ; Mašek, Tomáš (advisor) ; Herrmannová, Anna (referee)
The eIF4E is an important eukaryotic translation initiation factor, because of its ability to bind cap at 5'end of mRNA. There are three members of this protein family found in humans: eIF4E1, eIF4E2 and eIF4E3. eIF4E1 also plays role in in export of some mRNA from nucleus to cytoplasm. This protein is mostly regulated by mTOR signaling pathway and malfunctions in regulation leads to increased cell proliferation and thus tumorogenesis. eIF4E2 plays a role in regulating of translation during embryogenesis and it is known to mediate translation in terms of hypoxia. Role of eIF4E3 is so far shrouded in mystery. Some studies suggest it might be able to suppress tumor growth, but no studies have been done on human eIF4E3. Big potential of our work is, that all proteins we work with, are human. Based on our results, the endogenous amount of eIF4E3 protein is higher than it was thought. This is one of the reasons, why this protein should not escape our attention. In my diploma thesis, I have studied physiological characteristics of cell cultures overexpressing eIF4E proteins after mTOR inhibition treatment. I have realized that the most efficient inhibitor in all tested cell cultures is PP-242, which binds directly into active site of mTOR kinase. I have cloned 3xC FLAG tagged eIF4Es constructs and used...
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Renal carcinoma bological therapy and the role of cell signaling checkpoints
Černá, Kristýna ; Otavová, Katarína (advisor) ; Tolde, Ondřej (referee)
Principles of targeted biological treatment of metastatic renal cell carcinoma include mainly inhibitors of the tyrosine kinase receptors VEGFR and inhibitors of intracellular mTOR kinase. Across the new healing regimes there are the blockades of immune checkpoints of the immune system cell. Detailed molecular characterization of tumor is necessary not for only aplication of medicaments, but also for the development of drugs that target specific molecular pathway of cell signalization of the carcinoma cells. The work is focused on the description of the signaling pathway mTOR and VEGF in metastatic renal cell carcinoma. It summarizes all validated clinical biomarkers which are used to diagnose and stratify patients for the treatment of mRCC. It also offers insight into the present experiments that are finding new specific molecular markers. That may be the future solution for customized approach in the treatment of renal carcinoma an tumors in general.
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Automated monitoring of behaviour as a new paradigm in the research of depressive disorder
Revayová, Anna ; Valeš, Karel (advisor) ; Kubik-Zahorodna, Agnieszka (referee)
The rapid antidepressant effect of ketamine changed the direction of the research of potential antidepressants and its effect was also evaluated in this thesis. However, the main focus of this thesis is a new methodological approach to the research of depressive disorder. The main interest lies with the evaluation of automated monitoring of behaviour in this research. The first aim of this thesis was to evaluate the antidepressant effect of ketamine in the forced swimming test using software enabling automated monitoring of behaviour. The second aim was to meassure the change in phosphorylated Mammalian target of rapamycin (mTOR), using Enzyme-Linked ImmunoSorbent Assay (ELISA). The last, but the most important aim of this thesis was to implement the utilization of Phenotyper boxes in the automated behavioural evaluation of the olfactory bulbectomy model of depressive disorder and also evaluate the effect of ketamine in this model. Ketamine did not show an antidepressant effect in forced swimming test, however this observation could be influenced by chosen dose and mouse strain. Sensitivity of the test to chosen experimental protocol shows insufficient validity of this test. Observed change in level of phosphorylated mTOR corresponded with the behavioural results. Data collected from Phenotyper...
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Autophagy in the heart
Šprláková, Katarína ; Hlaváčková, Markéta (advisor) ; Tomšů, Eva (referee)
Currently, it is growing evidence that autophagy is involved in the prevention of various diseases, which of course also includes heart diseases. This thesis is therefore aimed at clarifying the role of autophagy in the heart, especially during ischemia and subsequent reperfusion. Autophagy is a physiological cellular process by which the cell maintains homeostasis by eliminating long-lived proteins and damaged organelles. The role of autophagy during ischemia/reperfusion in the heart is complex. Predominantly it functions as a pro-survival pathway, because it protects the heart from ischemia or hypoxia. However, when triggered over, which happens during reperfusion, it may lead to cell death. In the heart autophagy is activated in response to various stimuli, such as decrease in ATP and subsequent activation of AMPK, protein Bnip3, reactive oxygen and nitrogen species, the opening of mitochondrial permeability transition pore, endoplasmic reticulum stress or unfolded protein response.
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mTORC1 complex function in regulation of translation initiation
Holásková, Lucie ; Feketová, Zuzana (advisor) ; Čáp, Michal (referee)
My bachelor thesis deals with the effect of mTOR pathway to different processes in the cell. In particular, it focuses on the influence of translation initiation. mTOR protein is part of two complexes, which occur in different organisms - mTORC1 and mTORC2. Eukaryotic initiation factor 4E (eIF4E) plays an important role in controlling translation initiation. The activity of eIF4E protein is regulated by family of repressor 4E-binding proteins (4E-BPs). Linking these proteins to eIF4E is regulated by their phosphorylation state. For the release of 4E-BP1 from eIF4E, phosphorylation must occur at four phosphorylation sites (Thr37, Thr46, Ser65 and Thr70). The study also covers some of the other events that occur in the mTOR pathway.
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Crosstalk of integrin and mTOR signaling
Teglová, Lucie ; Libus, Jiří (referee) ; Rösel, Daniel (advisor)
iv Abstract Crosstalk of integrin and mTOR signalling is an essential process that monitors cellular interaction with extracellular matrix and transmits these inputs to cell growth signalling. Although adhesion status of the cell monitored by integrin signalling is clearly important for regulation of cellular growth, a little is known about the crosstalk of integrin and mTOR signalling. In this study, we employed two different approaches to describe and elucidate character of this crosstalk. p130Cas is an adaptor protein phosphorylated by Src kinase and focal adhesion kinase upon integrin ligand binding and implicated in cell adhesion, motility and survival in both Src-transformed and untransformed cells. Recently, p130Cas was also described in cellular pathology, mainly by its ability to stimulate cell invasion and metastasis. In this study, we described that p130Cas affects mTOR signalling in Src-transformed cells. Substrate domain of p130Cas was found to be indispensable for this effect and is also responsible for serum-induced activation of mTOR signalling. In addition, we prepared cell lines overexpressing various Rheb protein versions and characterized them in context of mTOR signalling, integrin signalling and cell cycle progression. Interestingly, a cell line overexpressing constitutively active...
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