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Elimination of acrylamide in foods
Macháčková, Kristýna ; Kolek, Emil (referee) ; Ing.Zuzana Ciesarová, CSc. (advisor)
The diploma thesis deals with the Influence of the Enzyme L-asparagine and the Inorganic salts (NaCl, CaCl2, NaHCO3 and NH4HCO3) on the elimination of the acrylamide in food-stuffs and a simulated model cereal matrix. The acrylamide belongs to the probable carcinogenic compounds which is incipient in the course of thermal processing of food, which are rich in the reducing sugars and amino acids as L-asparagine. Because of L-asparagine is the natural component of cereals and simultaneously is dominant antecedent incipient acrylamide, the way of the elimination by enzyme L-asparaginase (or the combination of L-asparaginase and salt) leads to the reduced level of acrylamide in a final product. The L- asparagine and salts were used on food-stuffs and a simulated model cereal matrix. It was found that individual used substances (except for NH4HCO3) cause the reduction of acrylamide production even about 90 % without change in the sensory properties of final product.
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The role of asparagine synthetase in leukemic cells
Šafrhansová, Lucie ; Starková, Júlia (advisor) ; Čuřík, Nikola (referee)
This thesis focuses on the detection of mutations in the enzyme asparagine synthetase on leukemia cell metabolism and the role of this enzyme in the context of L-asparaginase- based chemotherapy. The experimental part of the work is divided into two separate sections. Given the lack of asparagine synthetase gene sequencing data in leukemias, the first objective was to determine whether mutations are present in the leukemia cell line that could affect ASNS function and thus play a role in the resistance of leukemia cells to ASNase therapy. No mutations that could affect the activity of the enzyme were detected by next-generation sequencing. In the second part, a model of RS4;11 that expresses ASNS was established. The effect of ASNS on glycolysis was then studied to sensitize these cells to the effects of L-asparaginase and to the depletion of asparagine and glutamine. It was observed that ASNS expression increased the level of glycolysis and increased the resistance of these cells to asparagine and glutamine depletion and their resistance to asparaginase. Key words: ASNS, aspragine, leukemia, L-asparaginase, chemotherapy, drug resistance
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Cancer metabolism and its role in the sensitivity to ASNase of leukemic cells to L-asparaginase
Alquezar Artieda, Natividad ; Starková, Júlia (advisor) ; Procházka, Vít (referee) ; Truksa, Jaroslav (referee)
Cancer metabolism and its role in the sensitivity of leukemic cells to L- asparaginase ABSTRACT No ultimate treatment strategy exists for relapsed or non-responsive (15-20%) children with acute lymphoblastic leukemia (ALL). In this study, we aimed to elucidate the impact of metabolic rewiring in leukemic cells on poor therapy response and the emergence of resistance. This dissertation focuses on l-asparaginase (ASNase), a crucial chemotherapeutic agent and its effect on leukemia, using models of leukemic cell lines and primary cells of ALL patients. Cell metabolism was assessed by measuring metabolic pathways and nutrient influx using a Seahorse analyzer and stable isotope tracing. Main findings of the study demonstrated that the ASNase- therapy response was mitigated by the activity of the mechanistic target of rapamycin (mTOR)- regulated biosynthetic pathways. This phenomenon was induced by the bone marrow environment, which enabled the activation of the resistant mechanism in leukemic cells. We next found a correlation between the following metabolic features and lower sensitivity to ASNase: low ATP- linked respiration, high mitochondrial membrane potential and high glycolytic flux before therapy. The latter was shown to have prognostic implications. Moreover, high glycolytic flux was detected in T-ALL...
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Analysis of the pathways responsible for the resistance of leukemic cells towards L-asparaginase
Šimčíková, Markéta ; Konvalinka, Jan (advisor) ; Poljaková, Jitka (referee)
Acute lymphoblastic leukaemia (ALL) is the most frequent malignancy in childhood. Despite the very successful ALL therapy, relapses occur to 15-20 % of children. One of the possible relaps causes is the resistance to therapeutics. ALL is treated with combined chemotherapy in which cytostatic agent L-asparaginase plays the essen- tial role. L-asparaginase depletes extracellular asparagine and glutamine. Antagonist of the L-asparaginase is asparagine synthetase enzyme, which synthesizes the cellular asparagine. The specific antileukaemic effect of L-asparaginase is believed to be thanks to lower activity of the asparagine synthetase in leukaemic cells comparing to the healthy cells. The asparagine and glutamine deficiency harms the cellular proteosyn- thesis and induces apoptosis. Mechanism of the L-asparaginase cytotoxic effect and mechanism of corresponding resistance is still not fully explained. This bachelor thesis is a part of a project studying mechanisms of leukaemic cells resistance to L-aparaginase. In the model leukaemic REH cell line a deletion del(5)(q34) was discovered, which cannot be found in the resistant clone of these cells. This thesis focuses on proving different sensitivity of leukaemic cells, with or without the deletion, to L-asparaginase. The limiting dilution was used to obtain...
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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 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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Study of leukemic cells' metabolism in association with response to the therapy
Šimčíková, Markéta ; Starková, Júlia (advisor) ; Čuřík, Nikola (referee)
Acute lymphoblastic leukemia (ALL) is the most common malignant dise- ase in children. Despite great advancements in treatment of this disease, around 15-20 % of patients suffer a relapse. One of the possible reasons for relapse is developed resistance to cytostatic drugs. L-asparaginase is an im- portant chemotherapy component for childhood ALL and resistance to this drug often complicates treatment. To date, causes of developing resistance have not been sufficiently described. This thesis is a part of a greater research project focusing on mechanisms of L-asparaginase's activity and reasons for developing resistance to this chemotherapeutic agent. Differential metabolic requirements of cancerous cells have been described as early as 1924 by O. H. Warburg and they have been subject to scientific inquiry since. This study aimed to describe the relationship between basal metabolic determinants of leukemia cells and their sensitivity to L-asparaginase. For this reason, two metabolic pathways, glycolysis and oxidative phosphorylati- on, were studied in detail using a Seahorse Bioanalyzer. Further, expression of specific genes involved in glycolysis was detected. Content of mitochon- drial reticulum in cells, expression of the asparagine synthetase gene, and cell size were also studied. Experiments were...
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Analysis of the pathways responsible for the resistance of leukemic cells towards L-asparaginase
Šimčíková, Markéta ; Konvalinka, Jan (advisor) ; Poljaková, Jitka (referee)
Acute lymphoblastic leukaemia (ALL) is the most frequent malignancy in childhood. Despite the very successful ALL therapy, relapses occur to 15-20 % of children. One of the possible relaps causes is the resistance to therapeutics. ALL is treated with combined chemotherapy in which cytostatic agent L-asparaginase plays the essen- tial role. L-asparaginase depletes extracellular asparagine and glutamine. Antagonist of the L-asparaginase is asparagine synthetase enzyme, which synthesizes the cellular asparagine. The specific antileukaemic effect of L-asparaginase is believed to be thanks to lower activity of the asparagine synthetase in leukaemic cells comparing to the healthy cells. The asparagine and glutamine deficiency harms the cellular proteosyn- thesis and induces apoptosis. Mechanism of the L-asparaginase cytotoxic effect and mechanism of corresponding resistance is still not fully explained. This bachelor thesis is a part of a project studying mechanisms of leukaemic cells resistance to L-aparaginase. In the model leukaemic REH cell line a deletion del(5)(q34) was discovered, which cannot be found in the resistant clone of these cells. This thesis focuses on proving different sensitivity of leukaemic cells, with or without the deletion, to L-asparaginase. The limiting dilution was used to obtain...
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Elimination of acrylamide in foods
Macháčková, Kristýna ; Kolek, Emil (referee) ; Ing.Zuzana Ciesarová, CSc. (advisor)
The diploma thesis deals with the Influence of the Enzyme L-asparagine and the Inorganic salts (NaCl, CaCl2, NaHCO3 and NH4HCO3) on the elimination of the acrylamide in food-stuffs and a simulated model cereal matrix. The acrylamide belongs to the probable carcinogenic compounds which is incipient in the course of thermal processing of food, which are rich in the reducing sugars and amino acids as L-asparagine. Because of L-asparagine is the natural component of cereals and simultaneously is dominant antecedent incipient acrylamide, the way of the elimination by enzyme L-asparaginase (or the combination of L-asparaginase and salt) leads to the reduced level of acrylamide in a final product. The L- asparagine and salts were used on food-stuffs and a simulated model cereal matrix. It was found that individual used substances (except for NH4HCO3) cause the reduction of acrylamide production even about 90 % without change in the sensory properties of final product.
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