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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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Using of modern molecular methods for isolation and identification of ligninolytic enzymes
Řiháček, Martin
Ligninolytic enzymes are able to decay the structure of lignin. This effect can be useful in the industry (food industry, textile industry, farming, etc) because it can replace regular chemical processes. The white-rot fungus Phanerochaete chrysosporium is known for the production of these enzymes. This bachelor thesis deals with the identification and characterization of the behavior of enzymes such as lignin peroxidase, laccase and manganese peroxidase under different concentrations of copper sulfate (0, 0.1, 0.5 and 1 mM). For isolation of DNA and RNA, the fungi were grown in potato dextrose broth (PDB) during 5 days. Common PCR, reverse transcription PCR and quantitative real-time PCR were chosen for this experimental part. The PCR products were purified and sent to sequencing to confirm how their different isoforms develop under stress conditions of different concentrations of copper sulfate. Moreover, the enzymatic activity assay for the enzymes was done also under different copper sulfate environment for the experimental part. 1 mM of copper sulfate concentration influenced the transcription of the enzymatic genes resulting in the production of their isoforms. It was also observed at the level of the gene expression, with a higher expression of these 3 genes; laccase, manganese peroxidase and lignin peroxidase, compared with the control samples. On the other hand, the best conditions for carrying out their enzymatic activities were observed at 0.5 mM concentration of CuSO4 for lignin peroxidase and manganese peroxidase and 1 mM in the case of laccase. After the molecular characterization, we can conclude that production of the enzymes of Phanerochaete chrysosporium are affected by high copper concentrations.
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Comparison of soil microbial activity on sites with different forest management practices
Volánek, Jiří
Presented thesis is focused on microbial activity of forest soils and aims at characterizing some of its parameters on sites with different silvicultural management practices. Coppice, coppice-with-standard and high forest stands were compared in terms of carbon and nitrogen content, content of microbial biomass, enzymatic activity and selected physical properties of soil. Study was conducted between September 2015 and April 2016 on pre-existing TARMAG II research plot near Soběšice, Brno, Czech Republic. Samples were collected during three different calendar seasons, allowing for seasonal dynamicity assessment of the studied parameters. Statistical evaluation detected significant effect of management type on potential respiration of studied soil samples as well as significant effect of seasonality on microbial biomass content in incubated samples, phosphatase activity in fresh soil samples, catalase activity in both fresh and incubated samples and potential respiration of studied samples. Results also show that the overall potential activity of urease and catalase was at its highest during the winter season.
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