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Development of a novel method for nucleotide pool analysis in bacterial cells
Zborníková, Eva ; Rejman, Dominik (advisor) ; Pacáková, Věra (referee) ; Kozlík, Petr (referee)
(EN) This thesis deals with the determination of nucleotides in bacterial cells. Nucleotides play crucial role in most of the metabolic pathway. Determining their concentrations could give us important clues about the influence of internal and external conditions on the bacterial metabolism. Previously published papers dealing with the analysis of nucleotides and other intracellular metabolites can be divided into two groups according to the analytical approach: a) metabolomic approach and b) targeted approach dealing with narrow group of target analytes. In the case a) most authors use the state-of-the-art LC-MS/MS technique, whereas in the case b) robust UV detection coupled mainly to IP-LC is widely used. The aim of this study was to combine both approaches to obtain a method for routine analysis that would take advantages of mass detection, such as sensitivity and selectivity, while avoiding the need of demanding optimization of MS/MS transitions and expert service. The main purpose of the newly developed HILIC-MS method is its universal applicability in most biological and biochemical laboratories.
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Molecular mechanisms of bacterial persistence to antibiotics
Jirsová, Anežka ; Lichá, Irena (advisor) ; Branny, Pavel (referee)
The ability to persist is inherent in the vast majority of bacterial species. Persisters represent a small heterogenous fraction of the bacterial population that can tolerate antibiotics. Unlike resistant cells, which are genetically distinct from the rest of the antibiotic-sensitive population, persister cells form a genetically indistinguishable subpopulation. Persisters arise either as a result of stress caused by adverse environmental conditions, or persister subpopulation arise as a result of stochastic induction independent of the presence of stressful conditions. There are various mechanisms by which bacterial cells have been shown to induce a persistent state. Toxin-antitoxin systems and their interaction with stringent response effectors play an important role during the development of persistence. The persistent state is also affected by changes in proton-motive force (PMF) and the fluctuations in the gene expression level of energy generating enzymes of the Krebs cycle (TCA). In this thesis, persister cells are characterized and the current knowledge about the molecular mechanisms leading to the induction of persistence in bacteria is summarized. Key words: bacteria, persistence, antibiotics, stringent response, PMF, TCA
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Development of a novel method for nucleotide pool analysis in bacterial cells
Zborníková, Eva ; Rejman, Dominik (advisor) ; Pacáková, Věra (referee) ; Kozlík, Petr (referee)
(EN) This thesis deals with the determination of nucleotides in bacterial cells. Nucleotides play crucial role in most of the metabolic pathway. Determining their concentrations could give us important clues about the influence of internal and external conditions on the bacterial metabolism. Previously published papers dealing with the analysis of nucleotides and other intracellular metabolites can be divided into two groups according to the analytical approach: a) metabolomic approach and b) targeted approach dealing with narrow group of target analytes. In the case a) most authors use the state-of-the-art LC-MS/MS technique, whereas in the case b) robust UV detection coupled mainly to IP-LC is widely used. The aim of this study was to combine both approaches to obtain a method for routine analysis that would take advantages of mass detection, such as sensitivity and selectivity, while avoiding the need of demanding optimization of MS/MS transitions and expert service. The main purpose of the newly developed HILIC-MS method is its universal applicability in most biological and biochemical laboratories.
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