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Simulation of the interaction of steroid allosteric modulators of NMDA receptors with membrane
Riedlová, Kamila ; Konopásek, Ivo (advisor) ; Novák, Josef (referee)
Molecular dynamics (MD) method allows the real-time monitoring of the system composed of molecules and atoms, such as phospholipid bilayer or biomolecule. Applications of MD are very common in drug design where the real experimental procedures could be much more financially- and time-consuming or even impossible. The aim of this project is to explain the applications and advantages of MD method in case of studies of lipid membranes, with a special emphasis on a study od neurosteriod behaviour in lipid bilayer. Properly designed and synthesized neurosteroids could be used for the treatment of the serious neurological diseases. This work also included the experimental data obtained by MD simulations for two neurosteroids - pregnanolone glutamate and pregnanolone sulphate. Behaviour of this molecules in model membranes was observed and analyzed by MD simulations. Key words: molecular dynamics simulation, model membrane, lipid bilayer, NMDA receptor, neurosteroids, pregnanolone glutamate, pregnanolone sulfate
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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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Development and validation of methods for GMO detection
Hodek, Jan ; Ovesná, Jaroslava (advisor) ; Konopásek, Ivo (referee) ; Pazlarová, Jarmila (referee)
The present work is focused on the issue of genetically modified organisms (GMOs) detection in the plants and derived products. Existing legislation in the European Union regulates monitoring of GMO and lays down rules for their labeling and traceability. The aim was to contribute to solving certain issues of critical points in GMO detection. The first point was DNA extraction. The problem was adressed to the example of DNA extraction from the fruit of papaya and candied papaya. For both food products the suitability of the selected extraction methods was verified and confirmed by amplification of a specific DNA sequence by PCR. The second critical point was related to PCR inhibitors. We observed the effect of real-time PCR inhibition due to the influence of the residual EDTA and the residual amount of heavy metal ions, which were present in laboratory plastic. In both examples, the inhibition affects the PCR efficiency. Another critical point in the GMO detection is use of screening elements. That work was focused on false positive results. Development of new methods for GMO detection was another goal of this work. We have developed the method for garden pea taxon-specific gene quantification by real-time PCR. The method was tested on 13 cultivars of garden pea and several related plants of...
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Ecology of deadwood-associated fungi in the ecosystems of nature-like forests
Zrůstová, Petra ; Baldrian, Petr (advisor) ; Konopásek, Ivo (referee)
Dead wood plays an important role in forest ecosystems in the context of C dynamics, nutrient cycling, forest regeneration and biodiversity. Decaying wood sustains biodiversity by providing habitats and energy for fungi, bacteria, invertebrates, and many other organisms. Dead wood is resistant to decomposition and its decay is driven mainly by filamentous fungi. Community structure of wood- inhabiting fungi changes during decomposition, but the relationship between substrate quality and decomposer community is still poorly understood. This work studied fungal community composition with respect to tree species, stage of decay, volume and physico-chemical properties (such as pH, carbon and nitrogen content) of dead wood. Fungi were identified using next generation sequencing approaches - 454-pyrosequencing and Illumina MiSeq sequencing. Tree species, volume of dead wood (branches x logs) and stage of decay were the main variables affecting fungal community composition. Higher enzyme activities and content of fungal biomass indicate faster colonization of small branches than tree trunks by fungi. Fungal community composition, wood chemical properties and enzyme activities changed during decomposition. Both content of nitrogen and fungal biomass increased during decomposition. Enzyme activites peaked...
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Morphogenesis of bacterial colonies
Čepl, Jaroslav ; Markoš, Anton (advisor) ; Konopásek, Ivo (referee) ; Nemec, Alexandr (referee)
The topic of this thesis has been built on previous work of our group, especially Rieger et al. 2008 and Čepl et al. 2010. We examined and described the regularity of morphogenesis of S. marcescens, morphotype F colonies (from "the fountain", because of a shape it resembles). Typical colony consists of elevated red navel, low non-pigmented ring and again elevated red rim. Structured profile of the colony together with changes in pigmentation of structures during development without need of artificial dying, provides considerable advantage in observation of the morphogenesis. Aims of this thesis were (i) to find other factors that affect the morphogenesis, (ii) to characterize interactions of S. marcescens colonies with other bacterial strains (S. rubidaea and E .coli) and finally (iii) to study the phenomenon of induced resistance to the antibiotics described in (Heal and Parsons, 2002; Lu 2004; Bernier et al. 2011) in our model organisms. (i) Mutual interactions of colonies on the plate indicates that morphogenesis is affected by autocrine signals, which diffuse into the medium and the atmosphere and affect the development of surrounding colonies. We have detected changes of pH of the medium during the development of colonies in their vicinity. At ...
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Mechanism of secretion of adenylate cyclase toxin from Bordetella pertussis via Type I secretion system (TISS)
Klímová, Nela ; Bumba, Ladislav (advisor) ; Konopásek, Ivo (referee)
Type I secretion system in Gram-negative bacteria translocates proteins from the cytoplasm to the extracellular medium in a single step across both membranes. The membrane-spanning channel is made up of just three proteins - an ATPase in the inner membrane, a membrane fusion protein and a specific outer membrane protein. This work provides a summary of current knowledge concerning the structure of the secretion system, as well as the assembly of the trans-envelope complex and the mechanism of protein secretion. The role of substrate folding on secretion is highlighted. It deals to some extent with the properties of the substrates translocated by the type I secretion system, with emphasis on the adenylate cyclase toxin of Bordetella pertusis, the agent causing whooping cough.
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Conformational changes of the membrane protein sensors
Sklenářová, Petra ; Konopásek, Ivo (advisor) ; Fraiberk, Martin (referee)
This work focuses on a structure and a function of prokaryotic signal systems. There are three types of signal systems in bacteria: one-component, two-component and three-component systems. These three systems differ with respect to their structure and number of protein components and domain. I have focused mainly on two-component systems in bacteria despite their occurrence has been confirmed also in lower eukaryotes. Prokaryotic two-component system are the main instruments which help bacteria to differentiate between different stimuli from the environment and to react to them. So far, there were described two types of two-component systems. The first one is the basic system and the second one is the phosphorelay system. Both types of two-component systems contain two indispensable proteins: histidine kinase and response regulator. They are different with respect to their domain structure of proteins and in a mechanism by which they transfer the signal. The signal transmission system consists of two or four steps in case of the basis or phosphorelay systems, respectively. The principle of the signal transmission is the same for both of them, a phosphate group from the histidine kinase is transferred to response regulator. In such manner, response regulator is activated by and is able to regulate the...
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Adenylate cyclase toxin of Bordetella pertussis, its conformation and ion balance in host cell.
Motlová, Lucia ; Konopásek, Ivo (advisor) ; Krůšek, Jan (referee)
Adenylate cyclase (CyaA, ACT) toxin is one of the major virulence factors of Bordetella pertussis. Although CyaA binds to many types of membranes, it is assumed that the integrin CD11b/CD18 is its receptor which is expressed on the surface of myeloid cells. CyaA belongs to the family of RTX toxin-hemolysins. CyaA acts on the host cells by two independent activities. One of them is the conversion of ATP to cyclic AMP, which is catalyzed by adenylate cyclase (AC) domain after its translocation into the cytosol of the host cell, which leads to the entry of calcium cations into the host cell. Translocation is probably initiated by interaction of CyaA monomer with the target membrane. The second activity is the formation of CyaA channel selective for cations, which probably causes colloid osmotic lysis of target cells. The channel forming activity is provided by RTX hemolysin domain which most probably forms oligomers, although it was found that CyaA as a monomer causes leakage of potassium cations from the host cell. It is also not clear whether the oligomerization of CyaA would occur in solution, or after interaction with the host membrane. The aim of this study was to examine the flow of sodium ions on the membrane of murine macrophages J774A.1, which express integrin CD11b/CD18 on their surface....
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Supported Phospholipid Bilayers and their Interactions with Proteins Studied by Ellipsometry, Atomic Force Microscopy and Confocal Fluorescence Microscopy
Macháň, Radek ; Hof, Martin (advisor) ; Fidler, Vlastimil (referee) ; Konopásek, Ivo (referee)
Supported lipid bilayers have been used as an artificial model of biological membranes and their interaction with 5 selected antimicrobial peptides was studied by several experimental techniques, mainly ellipsometry, laser scanning microscopy and fluorescence correlation spectroscopy. The thesis explains basic principles of the applied techniques focusing on their aspects relevant to characterization of lipid bilayers. The biological significance of antimicrobial peptides, their modes of interaction with membranes and the basic characteristics of the selected peptides are briefly discussed. The following text describes the main types of experimental studies performed and the interpretation of their results. Peptide-induced changes in lipid bilayer morphology were characterized by ellipsometry and laser scanning microscopy. Most interesting effects were observed in the case of melittin, which induced formation of long lipid tubules protruding from the bilayer. Lipid lateral diffusion measured by fluorescence correlation spectroscopy can provide information on bilayer organization on length-scales below resolution of optical microscopy.
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Structural and functional characterization of yeast plasma membrane domains
Strádalová, Vendula ; Malínský, Jan (advisor) ; Palková, Zdena (referee) ; Konopásek, Ivo (referee)
Plasma membrane (PM) of living cells hosts variety of important cellular functions that must be precisely coordinated in space and time. Recent research shows that the plasma membrane is organized into specific domains to accomplish all these tasks. Our laboratory is focused on the organization of the plasma membrane in Saccharomyces cerevisiae where several distinct lateral compartments were identified at the fluorescence microscopy level. One of them is the Membrane Compartment occupied by arginine transporter Can1 (MCC) which consists of isolated, highly stable, ergosterol enriched, 300nm patches containing specific proton symporters and proteins of unknown function (Sur7- and Nce102-like). These membrane domains are organized by cytosolic protein complexes called eisosomes, composed mainly of proteins Pil1 and Lsp1. This work is a continuation of studies that tried to elucidate the composition, structure and function of MCC. In the first section of this work we concentrated on ultrastructural characterization of MCC domains. Foremost, we developed a protocol preserving the plasma membrane ultrastructure. The comparison of cryofixed and chemically crosslinked cells clearly showed that cryofixation by high pressure freezing together with freeze substitution and low temperature resin embedding...
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