National Repository of Grey Literature 64 records found  1 - 10nextend  jump to record: Search took 0.00 seconds. 
Evaluation of TD test for analysis of persistence or tolerance in clinical isolates of Staphylococcus aureus
Kotková, Hana ; Lichá, Irena (advisor) ; Fišer, Radovan (referee)
Persistence is the ability of bacteria to survive the impact of antibiotics even when the bacteria do not encode resistance genes. This is a very complex process, which is probably consequence of a reduction physiological process in subpopulation of bacteria. The aim of this study was to verify the suitability of the newly developed "Tolerance Disk Test" (TD test, Gefen et al, 2017) for detection of persistent or tolerant subpopulations of bacterial cells in clinical isolates of Staphylococcus aureus. We performed TD test for this kind of bacteria, which is a significant opportunistic pathogenic organism in humans, and compared its results with the killing curves. We have found that the ability to persist can be monitored semi-quantitatively also in this case and we consider this test suitable for introduction into clinical practice. In addition, we suggest that TD test could distinguish between persistent and tolerant subpopulations. Key words: Staphylococcus aureus, persistence, antibiotics, tolerance
Intracellular and intercellular regulation of gene expression in Gram-positive bacteria.
Pospíšil, Jiří ; Krásný, Libor (advisor) ; Lichá, Irena (referee) ; Malínský, Jan (referee)
Bacteria, the most dominant organisms on Earth, are an everyday presence in our lives. Symbiotic bacteria, which are present in the digestive tract of animals, usually have a beneficial effect on the body. On the opposite side of the spectrum are pathogenic species that cause more or less serious diseases around the world. In order to fight pathogens effectively, it is necessary to learn as much as possible about the molecular mechanisms by which bacteria respond to their environment, and also about the types of communication within bacterial populations that allow them to react to environmental changes as "multicellular" organisms. This Thesis consists of two main parts. In the first part, selected aspects of bacterial gene expression are characterized, using Bacillus subtilis and Mycobacterium smegmatis as model organisms. DNA-dependent RNA polymerase (RNAP) is the enzyme that is responsible for transcription of DNA into RNA, and thus plays a key role in gene expression. This Thesis deals with the structure of bacterial RNAP and important auxiliary factors (proteins and RNA) that associate with this enzyme and modulate its function. In the second part, the focus is on cell-to-cell communication, revealing which factors/mechanisms, including gene expression, affect this process in B. subtilis....
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....
Engineering of microbial glycosidases for modifying synthetic potential
Hovorková, Michaela ; Bojarová, Pavla (advisor) ; Lichá, Irena (referee)
Glycosidases (EC 3.2.1.) alias glycoside hydrolases are enzymes that catalyze the cleavage of a glycosidic bond between two carbohydrates or between a carbohydrate and an aglycone. Under suitable conditions (especially reduction of water activity in the reaction mixture), these enzymes are also able to synthesize a glycosidic bond. By targeted mutagenesis of the catalytic centre of the enzymes, it is possible to suppress or completely abolish their hydrolytic activity. Enzyme synthesis using glycosidases makes it possible to prepare bioactive galactosides, for example galectin ligands. The present work deals mainly with β-galactosidase from Bacillus circulans, its recombinant expression and mutagenesis. In the first part of the work, the commercially prepared plasmid of -galactosidase from B. circulans isoform A that I designed was used for recombinant expression in E. coli. It was necessary to optimize the conditions of the enzyme production. As it is a large protein (189 kDa), the expression vector pCOLD II and cold production at 15 ř C were used. The enzyme is specific for the formation of the β-1,4 glycosidic bond and has been used to synthesize complex tri- and tetrasaccharide ligands that cannot be prepared with a crude commercial preparation containing undesirable enzyme activities....
The effect of antibiotics on human gut microbiome and the influence of probiotics on its restoration
Hloucalová, Nikola ; Lichá, Irena (advisor) ; Ulrych, Aleš (referee)
Antibiotics are used for treatment of bacterial infections. They negatively affect not only the pathogens, but also other microorganisms in the gut, including the beneficial bacteria. Antibiotic treatment changes the proportion of good versus bad bacteria in the gut, causes a decrease in the number of commensal bacteria and leads to the overgrowth of opportunistic pathogens. We should consume probiotics during and after the antibiotic treatment, otherwise it results in an unhealthy stool and moreover it affects the immune system which then leads to physical and mental illnesses. This thesis summarizes the influence of probiotics on human gut during dysbiosis caused mainly by antibiotics.
The cell wall biosynthesis in gram-positive bacteria and inhibitory effect of antibiotics.
Mašková, Kateřina ; Ulrych, Aleš (advisor) ; Lichá, Irena (referee)
The cell wall of Gram-positive bacteria includes, in addition to the core peptidoglycan molecule, unique polysaccharides such as teichoic acids, capsular polysaccharides and exopolysaccharides, and covalently bound surface proteins. Together, they create a strong and durable layer that provides protection but also communication with the external environment. Peptidoglycan biosynthesis in Gram-positive bacteria can be divided into three phases: cytoplasmic, membrane and extracytoplasmic phase. The individual phases consist of specific reactions that are catalyzed by often conserved bacterial enzymes, which are potential targets for antibiotic molecules. Most known antibiotics effective against Gram-positive bacteria are aimed at inhibiting the process of cell wall synthesis. The mechanisms of action of individual antibiotics are described with varying degrees of detail. Some are known and widely used in medicine or veterinary practice, and some have so far only shown the potential to become drugs. Another use of antibiotics is in the basic research itself, especially in the study of cell wall biosynthesis and bacterial division. In this work, I have compiled a summary of knowledge about cell wall biosynthesis of Gram-positive bacteria and a list of antibiotics and a description of the mechanisms of...
Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae
Holečková, Nela ; Doubravová, Linda (advisor) ; Lichá, Irena (referee) ; Petříčková, Kateřina (referee)
Analysis of signaling cascade of protein kinase StkP in Streptococcus pneumoniae Streptococcus pneumoniae is not only an important human pathogen but also an appropriate model organism to investigate cell division in ovoid bacteria. This bacterium lacks both, NO and Min systems for selection of cell division site. Thus, the mechanism which determines the site of cell division is unknown. Additionally, the genome of S. pneumoniae encodes a single gene for eukaryotic-like serine/threonine protein kinase StkP and a single gene for eukaryotic-like serine/threonine protein phosphatase of PP2C type called PhpP. StkP is one of the main regulators of cell division. Cell division is probably affected by the phosphorylation of its substrates, which include, among others, cell division proteins FtsZ, FtsA, DivIVA, MacP, Jag/KhpB/EloR, and LocZ/MapZ. The aim of the first project of this dissertation thesis is determination of the function of protein LocZ in the cell division. In summary, locZ is not essential, however, it is involved in proper septum placement in S. pneumoniae and our data suggest that it is a positive regulator of Z-ring placement. Cells lacking LocZ are able to form Z-ring, but the Z-ring is spatially misplaced resulting in cell division defects, shape deformation, and generation of unequally sized,...
Metabolic control of the cell cycle in bacteria
Valtová, Aneta ; Lichá, Irena (advisor) ; Fišer, Radovan (referee)
Metabolic control of cell cycle has been studied for a long time in bacteria, but it is not still fully elucitated. The mechanisms described for several decades have been described in more detail and find new connections between basic metabolism and the cell division process itself. Cell cycle regulation, depending on metabolism and nutritional conditions, takes place over all steps of the cycle. The most mechanisms are studied at the level of bacterial division formation. Nutritional deprivation induces stress responses that use low-molecular substances which are involved in signaling pathways and which regulate the cell cycle. One of the most studying is the molecule of guanosine (penta)tetraphosphate (p)ppGpp, which affects cell cycle at the level of genes expression, at the level of proteins involved in the process of creating divisome, even at the level of replication. Recent research revealed that some enzymes with their already known enzymatic function in major metabolic pathways (glycolysis and TCA), also have a function as sensors that transmit a signal about the nutritional change directly to the division apparatus of the cell. These enzymes regulate the formation of the Z ring through the protein FtsZ or its auxiliary proteins most often and have been found in Gram-positive (Bacillus...
Applications of flow cytometry in the study of microbial subpopulations.
Hřebíček, Ondřej ; Lichá, Irena (advisor) ; Sudzinová, Petra (referee)
This work reviews common flow cytometric methods and applications for the study of bacterial organisms. Flow cytometry is fluorescent method capable of both quantitative and qualitative analysis at the single cell level. It can offer insights about bacterial population dynamics, phenotypic heterogeneity and more. This work features a basic introduction to flow cytometry and presents some of the commonly measured variables, such as viability or membrane potential with an emphasis on the fluorescent probes used to visualize them. The difficulties of adapting flow cytometry to bacterial physiology are discussed, as well as the advantages and disadvantages of the particular probes and methods. Finally, this work seeks to demonstrate the flexibility as well as the shortcomings of flow cytometry using examples of practical applications in basic research, environmental microbiology, biotechnology, clinical practice. Keywords: flow cytometry, microbial subpopulations, fluorescent labelling, bacterial physiology, bacterial viability
Mutant glycosidases with a high substrate specificity and their analysis
Nekvasilová, Pavlína ; Bojarová, Pavla (advisor) ; Lichá, Irena (referee)
β-N-acetylhexosaminidases (EC 3.2.1.52, 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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