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Preparation of organelle markers in the yeast Saccharomyces cerevisiae
Dekha, Daniella ; Vopálenská, Irena (advisor) ; Papoušková, Klára (referee)
The aim of the work was to prepare a set of strains with fluorescently labelled organelles. The organism used is a haploid strain with a high frequency of homologous recombination Saccharomyces cerevisiae BY4742. The yeast S. cerevisiae is a unicellular organism and is one of the most studied experimental living systems. Its generation time tends to be 1.5-2 hours, which allows rapid monitoring of culture development on both solid and liquid media. For each organelle, specific proteins were selected, namely proteins localized on the vacuole membrane (Vph1, Vtc3), peroxisomes (Pex3), nucleus (Nvj1), endoplasmic reticulum (Sec63), mitochondria (Cox4) and in the vacuole lumen (Prc1). Selected proteins were labeled by attaching fluorescent proteins (yEGFP, yomCherry or yomRuby2) to their C-termini. The prepared strains were analyzed in liquid and solid media. Growth curves were constructed, fluorescence at different growth stages was compared and the effect of two different carbon sources on the expression of selected genes was determined. A total of 12 strains with fluorescent organelle markers for the vacuolar membrane, peroxisomes, nucleus, endoplasmic reticulum, mitochondria, and vacuolar lumen and 5 strains with a pair of fluorescent markers for vacuolar membrane and selected other organelle...
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Dynamics of yeast plasma membrane domains
Oláhová, Dominika ; Zahumenský, Jakub (advisor) ; Vopálenská, Irena (referee)
Plasmatic cellular mebrane consists of specialized domains which differ in shape, function and size. This compartmentalization allows cells to coordinate and separate a large number of diverse processes. Disorders of cell organization at the level of membrane domains are increasingly associated with the expansion of pathological disorders. Microdomains also play a role in the virulence of pathogenic organisms, e.g. some types of yeast. One of the best described microdomains of the yeast plasma membrane is the MCC (membrane compartment of arginine permease Can1), which is stabilized by a protein complex called the eisosome. The composition of the MCC/eisosome is described in detail, the dynamics of the individual components in the basal state (i.e. without stress) has not yet been described. The aim of this thesis is to summarize current knowledge about the dynamics of the proteins of this microdomain and its relationship with other domains of cell membranes through a detailed review of the available literature. And at the same time by using the advanced fluorescence method FRAP (fluorescence recovery after photobleaching) to show in my own experiment that in the basal state there is an exchange of individual Nce102 molecules in the MCC/eisosome microdomain. Key words: yeast, plasma membrane,...
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Virulence factors of Bordetella pertussis
Držmíšek, Jakub ; Večerek, Branislav (advisor) ; Vopálenská, Irena (referee)
Bordetella pertusis is a Gram-negative, aerobic, non-spore-forming coccobacillus. Although it's strictly human pathogen, it's possible to infect other mammals at laboratory conditions. Transmission among hosts is mediated via respiratory tract droplets. Infection could be direct, host to host, alternatively by contaminated environment. Bordetella colonizes upper respiratory tract, wherefrom descends into lungs and causes disease known as whooping cough or pertussis leading to 195 000 deaths of 16 mil. incidences per year (according to WHO report from 2010). More than twenty years before, respectively to found pertussis toxin, that time intensively under examination, pertussis was marked as toxin-mediated disease. In the course of time, more other virulence factors were revealed, that could be divided into groups of adhesins, toxins and others. Adhesins are filamentous haemagglutinin, pertactin and fimbriae. Toxins include pertussis toxin, adenylate cyclase, tracheal cytotoxin, dermonecrotic toxin and lipopolysaccharide. Most of virulence factors are regulated by two component system Bvg. However, it is needed lots of other factors for successful infection as for example autotransporters or so called siderophores serving as iron acquisition from environment. Secretion of virulence factors is mediated by its...
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Role of the Erv14 protein in the maintenance of alkali-metal-cation homeostasis in the yeast Saccharomyces cerevisiae
Hrášková, Michaela ; Papoušková, Klára (advisor) ; Vopálenská, Irena (referee)
The Erv14 protein works as a cargo receptor in the COPII vesicles. Many proteins, including Na+, K+/H+ antiporter Nha1, which participates in the maintenance of cell alkali-metal-cation homeostasis, need Erv14 for their trafficking from the ER. When Erv14 is missing, the Nha1 antiporter is partially retained in the ER and its overall transport activity is affected. Although Erv14 interacts with the antiporter through Nha1's transmembrane domains, a shortened version of Nha1 lacking its long hydrophilic C-terminus does not require Erv14 for its efficient trafficking to plasma membrane. This thesis contributes to the understanding of the role of the Erv14 protein in the maintenance of alkali-metal-cation homeostasis in S. cerevisiae cells. Two S. cerevisiae strains lacking ERV14 gene were prepared and the effect of this deletion on the growth and salt tolerance of cells was studied. Using heterologous expression of NHA antiporters from various yeast species with variable lengths of their hydrophilic C-termini we studied localisation and function of these antiporters in S. cerevisiae cells in the presence and in the absence of Erv14 protein. Our results suggested that the length of the antiporter's C-terminus might play a role in its requirement of Erv14 presence for its trafficking through the...
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Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane
Doudová, Lenka ; Malínský, Jan (advisor) ; Vopálenská, Irena (referee)
Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane Yeast plasma membrane is divided into several different compartments. Membrane compartment of Can1 is specific for its protein and lipid composition, furthermore it creates furrow-like invaginations on the plasma membrane. These invaginations are made by multiprotein complexes called eisosomes, which are located in the cytosolic side of MCCs. It was established that this domain plays an important role in response to various environmental stresses. Sur7 and Nce102 are transmembrane MCC proteins of unknown function. Sur7 is most likely a structural protein within MCC, Nce102 is probably important in regulation of kinases associated with MCC/eisosomes. Key words: biological membrane, membrane microdomains, eisosome, S. cerevisiae, Sur7, Nce102
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Structure and function of bacterial pore-forming toxins
Suková, Anna ; Mašín, Jiří (advisor) ; Vopálenská, Irena (referee)
The pore-forming bacterial toxins are crutial virulence factors of pathogenic bacteria. Their essential feature is the capacity to form hydrophilic transmembrane pores in cytoplasmic membrane of target cells. It leads to impairment of osmotic balance which can result in cell lysis. These toxins use different mechanism of cell attack. However, there is few similar steps - membrane binding, insertion into membrane, oligomerization and forming of the transmembrane pore. Keywords: bacterial pore-forming toxins, mechanism of membrane insertion, cholesterol-dependent cytolysins, α-toxin, aerolysin, α-hemolysin, adenylate-cyclase toxin
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Role of cytoplasmic membrane and cell wall in bacterial resistance to cationic antimicrobial peptides
Havlová, Noemi ; Seydlová, Gabriela (advisor) ; Vopálenská, Irena (referee)
Cationic antimicrobial peptides are part of the innate immune system of all organisms. Their properties such as structure, charge, amphipathicity and hydrophobicity make them promising agents with the potential for use not only in treatment of bacterial infections but also against some viruses, parasites, fungi and cancer cells. One of their possible targets is the cytoplasmic membrane, which they permeabilize. This mode of action has several advantages. The important feature of antimicrobial peptides is their selectivity for bacterial membranes, which makes them harmless to eukaryotic host cells. Another advantage is that the development of bacterial resistance against these peptides is more difficult since the action of antimicrobial peptides is rapid. Nevertheless, there appeared some bacterial strains that are insensitive to already used antimicrobial peptides. By using target modification resistant bacteria are able to prevent the bactericidal effects of the antimicrobial peptides. At the level of cytoplasmic membrane bacteria can alter its electrostatic or structural properties of membrane lipids and thus the antimicrobial peptides lose their ability to interact with the membrane and permeabilize it. Understanding the mode of action of antimicrobial peptides and mechanisms by which bacteria...
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Intracellular life of pathogenic bacterium Francisella tularensis in the host.
Rädisch, Robert ; Konopásek, Ivo (advisor) ; Vopálenská, Irena (referee)
Francisella tularensis is a facultative intracellular pathogenic bacterium, which causes disease named tularemia. For the entrance to the host cells Francisella uses host's cell mechanisms by which it is incorporated into cell phagosome. Subsequently, it escapes from phagosome to cytosole where bacterial growth takes place. Some of bacteria are cleared from cytosol by autophagy, from another ones dsDNA is released. This DNA is recognized by cytosolic receptors, which form inflammasome complex. Inflammasome sets off pathway leading to the death of infected cell. Since the penetration to the cell Francisella modulates cell signallization in its own benefit to ensure enough time and nutrients for its growth. Francisella do not act only in the infected cells, where it reduces recognition of itself and clearance from cytosol, but it also induces secretion of factors, which moderate activation of adaptive immunity of the host. Key words: Francisella, tularemia, fagosome, inflammasome, autophagy, adaptive immunity
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