National Repository of Grey Literature 102 records found  1 - 10nextend  jump to record: Search took 0.00 seconds. 
Influence of Staphylococcus aureus Quorum sensing system to antibiotic tolerance
Šaňková, Michaela ; Lichá, Irena (advisor) ; Buriánková, Karolina (referee)
Staphylococcus aureus is a gram-positive opportunistic pathogenic bacterium that causes a range of chronic infections especially to weakened individuals. Recently, the most serious problem in clinical medicine has been infections caused by so-called persisters. They form a subpopulation of cells that, thanks to adaptations to stressful conditions, are able to withstand lethal doses of antibiotics and then cause an infection again. Agr system, which is responsible for the mechanism of quorum-sensing, plays the main role in switching between the active virulent form and the persistent form associated with biofilm formation. During chronic infections, adaptive mutations accumulate in this Agr system and affect the ability to persist. For the purpose of this thesis, clinical isolates were obtained from patients diagnosed with cystic fibrosis in chronological order within the first three years of their lives. The aim of this thesis was to confirm the influence of adaptive mutations on the persistence ability of clinical isolates and also to observe how the membrane potential and metabolic activity change depending on the on the action of selected antibiotics. Additional aim was to create a deletion of the response regulator AgrA gene using the CRISPR/Cas9 system. In order to characterize the persistence...
Effect of adaptive mutations on persister formation in Staphylococcus aureus
Hloucalová, Nikola ; Lichá, Irena (advisor) ; Buriánková, Karolina (referee)
Persistence represents a transient state during which are bacterial cells able to survive antibiotic treatment. Only a small subpopulation of cells enter this state, these cells are then capable of causing disease recurrence. The size of a persister subpopulation is influenced by adaptive mutations formed during a chronic disease in genes related to transition of cells between a virulent form and biofilm formation. Regulation of entering and exiting persistence is also influenced by intercellular communication and by molecules produced into the cell surroundings. It is assumed that this effect is caused by PSM modulines and AIP molecules which take part in Quorum sensing mechanism. The main aim of this thesis was to clarify how adaptive mutations of clinical isolates obtained from two patients diagnosed with cystic fibrosis influence the ability to persist. Another aim was to determine changes in membrane potential and metabolic aktivity while entering persistence and to establish the effect of extracellular molecules produced into the culture medium on growth parameters of the studied isolates. By using techniques of TD test and establishment of persistence rate by CFU determination, it was found that the isolates obtained from patient 2 did not acquire adaptive mutations affecting persistence...
Methods for characterization of persistent state after exposure to selected antibiotics in Staphylococcus aureus
Valtová, Aneta ; Lichá, Irena (advisor) ; Tkadlec, Jan (referee)
Staphylococcus aureus is a opportunistic pathogen that can cause severe and chronic infections. The reason of the infections relapse is often the persistence. It is about adapting to stressful conditions by inducing a dormant state, which would allow bacteria to survive exposure to antibiotics and grow again after their elimination. Bacteria that persist in the patient acquire various adaptive mutations, which are transmited creating subpopulations that have a better ability to persist. The aim of this diploma thesis was to compare individual methods of persistent study that could be used in clinical practice in the future, and at the same time to try a closer molecular characterization of the persistent state with using methods for calculating gene expression. I had chronological isolates of Staphylococcus aureus at my disposal, the initial one being the primoisolate, an isolate taken at the diagnostics of cystic fibrosis before the start of antibiotic treatment. Another was taken at a distance of three-quarters of a year and the last with a half-year interval from the previous one. Following whole genome sequencing, genes in which adaptive mutations occurred were identified. The first method determines the degree of persistence by calculating CFU (Colony Forming Units) after antibiotic treatment....
Structure and evolution of efflux pump in gramnegative bacteria.
Gálová, Diana ; Lichá, Irena (advisor) ; Matyska Lišková, Petra (referee)
Drug resistence in microorganisms is of major concern and there is an increasing number of pathogenic bacteria resistent to clinically used antibiotics. The major mechanism of resistence in Gram-negative is active efflux that prevent the intracellular accumulation of antibiotics to toxic levels. We recognise five families of efflux pumps, mainly using proton motive force to translocate the substrate, less common driven by ATP hydrolysis. The multidrug pumps with broad substrate specifity are of a key role in drug resistance of pathogens, typically represented by RND superfamily. The source of efflux pumps are probably the producers of antibiotics and organisms exposed to toxic compounds in their natural habitat. The genes for resistance can then spread out via horizontal gene transfer to other bacteria. It is presumed that eflux pumps have developed from transporters serving physiological functions like transport of endogenous substrates.
Factors interacting with bacterial RNA polymerase and their effect on the regulation of transcription initiation
Ramaniuk, Volha ; Krásný, Libor (advisor) ; Lichá, Irena (referee) ; Valášek, Leoš (referee)
(ENGLISH) The bacterial cell needs to regulate its gene expression in response to changing environmental conditions. RNA polymerase (RNAP) is the pivotal enzyme of this process and its activity is controlled by a number of auxiliary factors. Here I focus on RNAP-associating factors involved in regulation of transcription in G+ bacteria:  factors, initiating nucleoside triphosphates (iNTPs), HelD, δ and small RNA Ms1. The main emphasis is on σ factors from Bacillus subtilis. σ factors allow RNAP to specifically recognize promoter DNA. In my first project I set up in vitro transcription systems with purified alternative σ factors, σB , σD , σH , σI from B. subtilis. Using these systems, I studied the effect of initiating NTP concentration ([iNTP]) on transcription initiation. I showed that promoters of alternative  factors are often regulated by [iNTP]. In the next project I comprehensively characterized one of the least explored alternative  factors from B. subtilis, I . I identified ~130 genes affected by I , though only 16 of them were directly affected. Moreover, I discovered that I is involved in iron metabolism. Finally, I showed that I binding requires not only the conserved -35 and -10 hexamers, but also extended -35 and -10 elements located in the spacer region. In collaboration with...
Study of effect of Bacillus subtilis yxkO gene on motility during stress response to osmotic upshift.
Streitová, Eliška ; Lichá, Irena (advisor) ; Krásný, Libor (referee)
Bacillus subtilis is gram-positive soil bacteria. In its natural environment it is constantly exposed to changes of chemical and physical conditons, including changes of osmolality. It responds to high osmolality by transporting of potassium ions and afterthat transporting and/or synthetising of compatible solutes. In last years the mutant strain Bacillus subtilis L-42 was isolated with non-specific insertional mutagenesis (mini Tn10) in our laboratory. This strain displays limited growth and inability to cope with hyperosmotic shock in a defined medium with potassium concentration of < 1 mmol/l. Insertion of transposon was located in yxkO gene which encodes a protein of unknown biological function. Some other data also indicate a possible role of disruption of yxkO gene in regulation of expression of hag gene, which encodes flagelin - a pivotal protein of bacterial flagellum. The goal of this thesis was to clarify if the disruption of yxkO gene influences motility and whether is affected the transcription of hag gene. With integrative vector pMUTIN4 a mutant strain with specific mutation of yxkO gene was prepared. Vector was pasted into chromosome of Bacillus subtilis strain 1A839 - genotype of this strain allows to extrude the known transcriptional regulation of hag gene. Cell's motility was...
Comparison of specific expression in bacterial and yeast biofilms
Kicko, Peter ; Palková, Zdena (advisor) ; Lichá, Irena (referee)
The development and maintenance of biofilm is a complex process that is based on a change in genetic expression. The biofilm formation is observed in some prokaryotic and eukaryotic cells. During its formation, cell aggregation occurs, extracellular matrix is created and we observe the formation of metabolically differentiable cells, often with increased resistance to antimicrobial drugs. This work focuses on important steps leading to biofilm formation associated with specific gene expression and highlights the similar and different processes between bacterial and yeast cells. The work begins by comparison of cell signalling, it continues by comparing the expression of the adhesive proteins and extracellular enzymes, synthesis of exopolysaccharides, formation of extracellular nucleic acid, and in the last chapter we focused on the formation of persistors. The aim of this work is to connect the acquired information and to contribute to the understanding the complexity of this process. Key words: biofilm, signalling, adhesins, exopolysaccharides, extracellular nucleic acid, persistor
Regulation of cell cycle in Bacillus subtilis.
Zelenka, Tomáš ; Lichá, Irena (advisor) ; Harant, Karel (referee)
2 Abstract Relations between several events running in bacterial cell during cell cycle were the subject of many studies during last years. More advanced techniques showed, that bacterial cell life has much more variable factors, than we supposed before. Relatively recent researches managed to reveal function and in few events molecular principle of several mechanisms coordinating those events such as progression of replication and its initiation, segregation of newly replicated chromosomes and after all synchronization of complex cell division machinery. Furthermore it showed variability of those events during changing living conditions of the cell. Keywords: Cell cycle, regulation, initiation, replication, segregation of chromosome, cytokinesis, Bacillus subtilis
Mutant glycosidases with a high substrate specificity and their analysis
Nekvasilová, Pavlína ; Bojarová, Pavla (advisor) ; Lichá, Irena (referee)
β-N-acetylhexosaminidases (EC, GH 20) are retaining exo-glycosidases that in vivo cleavage both β-N-acetylglucosamine (GlcNAc) or β-N-acetylgalactosamine (GalNAc) residues fom glycostructures. Under suitable reaction conditions, these enzymes are able to synthesize the glycosidic bond in good yields. Substitution of selected amino acid(s) in the emzyme active site by site-directed mutagenesis may change the enzyme's substrate specificity or suppress the hydrolytic activity of the enzyme in favor of synthesis. The present thesis deals with three mutant β-N-acetylhexosaminidases from Talaromyces flavus, in which the amino acid residues responsible for binding to C-4 hydroxyl of the substrate (Arg218, Glu546) were exchanged for amino acids proposed on the basis of molecular modeling. The effect of introduced single point mutations on substrate specificity of prepared enzymes was studied. Mutant β-N-acetylhexosaminidases were heterologously expressed in Pichia pastoris and characterized. Furthermore, transglycosylation reactions with these enzymes were performed. The prepared carbohydrate products were characterized by NMR.

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