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Functional analysis of phosphoglucosamine mutase GlmM in Streptococcus pneumoniae
Mühldorfová, Tereza ; Ulrych, Aleš (advisor) ; Lišková, Petra (referee)
Phosphoglucosamine mutase (GlmM), an enzyme taking part in biosynthesis of cell wall, has been recently proven to be essential for Streptococcus pneumoniae. The main goal of this thesis was to prove in vivo that GlmM serine residues S99 and S101 phosphorylation is essential while the necessity of it was already proven indirectly based on transformation efficiency. For this purpose we have prepared a strain with two copies of the glmM gene - the first one with amino acid changes on monitored serine residues located at native locus; and the second ectopic copy of the wild allele of glmM gene under control of inducible zinc promoter. We have observed morphology, growth, and GlmM expression with and without the presence of an inductor. All the observed parameters show that the cells are not viable without ectopic glmM expression, thus the essential protein GlmM is functional only when phosphorylated on S99 and S101 residues. Further, we have attempted to localize the enzyme in the S. pneumoniae cell. We have fused GlmM with fluorescent marker GFP and by using the florescent microscopy we have proved that GlmM is cytoplasmic protein. Another goal of this thesis was to find an unknown third phosforylation site of the GlmM protein which is dependent on the protein kinase StkP. From in vitro kinase assay and...
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Life strategy of facultatively intracellular pathogenic bacteria
Fejková, Kateřina ; Lišková, Petra (advisor) ; Mašín, Jiří (referee)
Facultative intracellular patogenes have the ability to grow and survive inside and also outside of cell. This is giving them a selective advatage and also better access to nutrients, protection from extracellular components of immune system and variable conditions outside of the cell. Bacteria Francisella tularensis and Listeria monocytogenes are able to resist the defence mechanisms of immune system, enter the macrophages and non-profesional phagocytes for example hepatocytes or endothelium, prevent bactericidal processes in phagosome, replicate in cytosol and resist cell death processes until they are ready to leave the infected cell and spread to another cell. For this displacement between cells is Listeria monocytogenes using actin, whereas in Francisella tularensis the mechanism is not entirely clear. The description of the mechanism of these processes in pathogens Francisella tularensis and Listeria monocytogenes is the subject of this bachelor thesis. Key words: bacterial pathogen, facultative, intracellular, Francisella tularensis, Listeria monocytogenes
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Prospective study of long-term visual sequelae of acute methanol poisonings
Nurieva, Olga ; Zacharov, Sergej (advisor) ; Lišková, Petra (referee) ; Pohanka, Miroslav (referee)
Background: Methanol poisoning is a life-threatening condition which induces acute toxic optic neuropathy with possible long-term visual sequelae in survivors. Aim: To study the prevalence, character, dynamics, and key determinants of chronic morphological and functional visual pathway changes during 4 years after methanol-induced optic neuropathy. Methods: A total of 55 patients with confirmed methanol poisoning with mean age 46.7 ± 3.6 years (46 males and 9 females), and 41 controls were included in this prospective longitudinal cohort study. The patients were examined 4.9 ± 0.6, 25.0 ± 0.6, and 49.9 ± 0.5 months after discharge. The following tests were performed: visual evoked potential (VEP), optical coherence tomography with retinal nerve fiber layer (RNFL) measurement, brain magnetic resonance imaging (MRI), complete ocular examination, biochemical tests, and apolipoprotein E (ApoE) genotyping. Results: Of 42/55 patients with all three consecutive examinations, abnormal RNFL thickness was registered in 13 (31%) and chronic axonal loss during the observation period was found in 10 (24%) patients. The risk estimate of chronic global RNFL loss for arterial blood pH<7.3 at admission was: 11.65 (1.91-71.12; 95% CI) after adjusting for age and sex. The patients with chronic axonal degeneration demonstrated...
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Bacterial RTX toxins and their calcium-binding sites
Lišková, Petra
FrpC protein produced by Neisseria meningitidis in a human host belongs to the family of bacterial RTX toxins due to the presence of RTX domain. FrpC possesses a calcium-dependent auto-catalytic cleavage activity which is localized within its 177 amino-acids long segment Self-Processing Module (SPM). As the SPM is naturally intrinsically disordered protein without bound Ca2+, the calcium binding is crucial for SPM folding which is followed by the auto-catalytic processing. The elucidation of the SPM structure may be the key step for understanding of enzymatic and biological function. The structure of folded SPM itself can be characterized only with difficulties due to the presence of flexible loop according to preliminary NMR data. The subject of this work is the description of SPM using fluorescence methods, characterization of ions binding to SPM and structural changes occurring during Ca2+ binding. In this work, the ion binding properties of SPM segment and its ion-induced folding was characterized. It was found that the dissociation constant kD of 17 μM coincided with the folding of SPM into the native calcium-bound state which occurs in the concentration range between 1 and 20 μM Ca2+. In the attempt to characterize the structure of ion binding site, the fully active single tryptophan mutants...
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Antibacterial and antiadhesive properties of carbon nanomaterials
Budil, Jakub ; Lišková, Petra (advisor) ; Zikánová, Blanka (referee)
Increasing interest in industrial and medical applications of carbon nanomaterial leads to the need to examine its interactions with living systems. Nanocrystalline diamond (NCD) films possess high mechanical and chemical stability which, together with its biocompatibility with human cells, enables applications in human body. Some of carbon nanoparticles possess strong antibacterial activity. In this work the effects of NCD with hydrogen, oxygen and fluorine termination deposited on glass and silicone on adhesion of gram-negative bacteria Escherichia coli K-12 in mineral medium is described and the impact of cultivation medium on effects of NCD films is compared. Prior the growth of the E. coli biofilm on NCD films, the method for quantification of biofilm using crystal violet staining and the method for biofilm cultivation in mineral medium were optimised. The properties of NCD film are independent on the base substrate. Hydrogen and fluorine terminated NCD films show antiadhesive properties only in mineral medium but not in complex medium. This is explained by formation of a conditioning film on the surface of the NCD film during cultivation in complex medium. On the other hand, O-NCD film supports bacterial adhesion in both cultivation media. Second part of this thesis is dedicated to carbon...
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Bacterial RTX toxins and their calcium-binding sites
Lišková, Petra ; Konopásek, Ivo (advisor) ; Holoubek, Aleš (referee) ; Hof, Martin (referee)
FrpC protein produced by Neisseria meningitidis in a human host belongs to the family of bacterial RTX toxins due to the presence of RTX domain. FrpC possesses a calcium-dependent auto-catalytic cleavage activity which is localized within its 177 amino-acids long segment Self-Processing Module (SPM). As the SPM is naturally intrinsically disordered protein without bound Ca2+, the calcium binding is crucial for SPM folding which is followed by the auto-catalytic processing. The elucidation of the SPM structure may be the key step for understanding of enzymatic and biological function. The structure of folded SPM itself can be characterized only with difficulties due to the presence of flexible loop according to preliminary NMR data. The subject of this work is the description of SPM using fluorescence methods, characterization of ions binding to SPM and structural changes occurring during Ca2+ binding. In this work, the ion binding properties of SPM segment and its ion-induced folding was characterized. It was found that the dissociation constant kD of 17 μM coincided with the folding of SPM into the native calcium-bound state which occurs in the concentration range between 1 and 20 μM Ca2+. In the attempt to characterize the structure of ion binding site, the fully active single tryptophan mutants...
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Development and genetic basis of glycopeptide resistance in coagulase-negative staphylococci
Prášilová, Jana ; Balíková Novotná, Gabriela (advisor) ; Lišková, Petra (referee)
Glycopeptides are the so-called last-resort antibiotics in clinical practice used to treat heavier, predominantly nosocomial infections caused by multi-resistant coagulase-negative staphylococci. The origin and genetic basis of resistance to glycopeptide antibiotics has not yet been elucidated within coagulase-negative staphylococci. Research on Staphylococcus aureus has shown, that intermediate resistance to glycopeptide antibiotics is associated with the presence of one or more mutations, rather than being conditioned by the support of a particular genetic element, such as in enterococci. By using various types of in vitro resistant mutant selection, we were able to obtain isogenic pairs of glycopeptide sensitive and resistant strains of Staphylococcus epidermidis and Staphylococcus haemolyticus. By sequencing the genomes of these pairs, one nucleotide polymorphisms were identified and predominantly found in metabolic and cell wall control systems. Phenotypic analysis did not reveal a direct association of glycopeptide resistance with increased biofilm formation. In clinical practice, the cross-resistance of glycopeptides and other antibiotics is problematic. For the non-glycopeptide antibiotics imipenem and rifampicin, the incidence of cross-resistance with glycopeptide antibiotics in S. aureus...
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The role of PIP5K family kinases in plasma membrane remodeling
Apolínová, Kateřina ; Macůrková, Marie (advisor) ; Lišková, Petra (referee)
Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is the enzyme responsible for the production of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which has long been known as a precursor of two important second messengers, diacylglycerol and inositol trisphosphate. However, PI(4,5)P2 also acts as a second messenger in its own right and regulates many processes occurring on the plasma membrane such as endo- and exocytosis, actin cytoskeleton remodeling, and the formation of cell-cell contacts. The action of PIP5K is carefully spatially and temporally regulated in order to form localized pools of PI(4,5)P2 crucial for its many roles in a wide variety of cell processes. This bachelor's thesis focuses on the description of regulatory mechanisms that control PIP5K activity in vivo and on its physiological functions at the plasma membrane.
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Influence of size and geometry of nanoparticles on cellular internalization pathways
Číhařová, Barbora ; Španielová, Hana (advisor) ; Lišková, Petra (referee)
Nanoparticles can be used in biomedical disciplines as carriers for transport of diagnostic as well as therapeutic substances into cells. Variety of different shapes, sizes and different compositions are used experimentally. Despite the discoveries already made in this area, the exact nature of the interaction between a nanoparticle and a cell has not been fully understood yet. The objective of this thesis is to provide the knowledge about possibilities of utilisation and aspects influencing the interaction between the cell membrane and several types of nanoparticles: liposomes, gold nanoparticles and virus-like nanoparticles. The comparison shows that generalisation of the mechanism of nanoparticle entry into the cell is problematic, although it seems that the spherical nanoparticles with the diameter of 50 nm provide the most efficient entry.
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Anterior segment dysgenesis disorders and their molecular genetic cause
Moravíková, Jana ; Lišková, Petra (advisor) ; Krulová, Magdaléna (referee)
Proper eye development depends on expression and mutual regulation of many genes. Anterior segment dysgenesis (ASD) are a highly heterogeneous group of diseases exhibiting all types of Mendelian inheritance, which manifest as combination of congenital abnormalities of the cornea, iris, anterior chamber angle or lens. Screening of genes associated with ASD does not often lead to the identification of the underlying genetic cause implying that there are still novel variants or genes to be discovered. Molecular genetic analysis in 12 probands with ASD using Sanger and whole-exome sequencing were performed. Functional analysis by Exon trapping assay was provided in variants predicted to effect pre-mRNA splicing. Four PAX6 mutations evaluated as pathogenic or likely pathogenic in a heterozygous state were found in four probands c.183C˃G; p.(Tyr61*), c.1032+1G>A, c.1183+1G>T and c.622C>T; p.(Arg208Trp). One proband was found to be a compound heterozygote for c.244A>G; p.(Met82Val) and c.541delG; p.(Glu181Lysfs*26) mutations in FOXE3. In 7 probands, no potentially pathogenic variants were identified. Exon trapping assay confirmed that mutations c.1032+1G>A and c.1183+1G>T have an effect on pre-mRNA splicing of the PAX6 gene. Detailed molecular-genetic analysis in patients with ASD may contribute to...
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