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The use of magnetic microparticles for bacterial DNA isolation
Hrudíková, Radka ; Horák, Daniel (referee) ; Španová, Alena (advisor)
The aim of the work was testing of two types of magnetic mikrosheres functionalised with –COOH groups for the isolation of bacterial DNA. Isolation of DNA was carried out from crude lysates of cells prepared from pure culture of Lactobacillus paracassei RL-10 in the presence of binding buffer with 2 M NaCl and 16% PEG 6000. The influence of RNA degradation by enzyme RNase A on the amount of isolated DNA was investigated. It was estimated that RNA degradation affects the amount of DNA isolated. The amount of DNA depended on the type of microparticles. Higher amounts of DNA were isolated using particles with higher content of carboxyl groups. DNA applicable in PCR was isolated using both types of microsheres. In next part of the work, microparticles functionalised with –NH2 groups were used to DNA isolation using electrostatic forces. It was shown that buffer with lower pH is suitable for DNA adsorption onto magnetic microparticles.
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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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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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Exosome and its role in RNA metabolism of budding yeast S. cerevisiae
Holická, Eliška ; Půta, František (advisor) ; Groušl, Tomáš (referee)
Exosome is a protein complex present in the yeast nucleus and cytoplasm, which participates in RNA degradation, processing and turnover. The core of exosome consists of nine catalytically inactive subunits, which physically associate with RNA nuclease Rrp44. The function of exosome is dependent on many cofactors or facultatively associated enzymes, and these associations provide high versatility of the complex. In different compartments the complex works by other means and plays a role in distinct processes. In nucleus, exosome acts mainly in pre-RNA processing, whereas in cytoplasm its major role is to degrade native mRNA. Nevertheless, in all of these processes, its general role is the 3' exonucleolytic cleavage of single-stranded RNA. Exosome has homologs in many various kinds of organisms - e. g. different types of bacterial nucleases, archeal exosome, human PM-Scl complex (or exosome), which implicates high conservation of this degradation machinery. Thus, it is very likely that some exosome components lost their original function over the evolution, more than that the yeast exosome is an evolutionary innovation.
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The use of magnetic microparticles for bacterial DNA isolation
Hrudíková, Radka ; Horák, Daniel (referee) ; Španová, Alena (advisor)
The aim of the work was testing of two types of magnetic mikrosheres functionalised with –COOH groups for the isolation of bacterial DNA. Isolation of DNA was carried out from crude lysates of cells prepared from pure culture of Lactobacillus paracassei RL-10 in the presence of binding buffer with 2 M NaCl and 16% PEG 6000. The influence of RNA degradation by enzyme RNase A on the amount of isolated DNA was investigated. It was estimated that RNA degradation affects the amount of DNA isolated. The amount of DNA depended on the type of microparticles. Higher amounts of DNA were isolated using particles with higher content of carboxyl groups. DNA applicable in PCR was isolated using both types of microsheres. In next part of the work, microparticles functionalised with –NH2 groups were used to DNA isolation using electrostatic forces. It was shown that buffer with lower pH is suitable for DNA adsorption onto magnetic microparticles.
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