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Pore-forming properties of Bordetella pertussis CyaA toxin and composition of the lipid bilayer.
Rädisch, Robert ; Konopásek, Ivo (advisor) ; Krůšek, Jan (referee)
Bordetella pertussis produces many virulent factors including adenylate cyclase toxin (CyaA) This toxin preferentially invades cells of immune system with integrin receptor CD11b/CD18 and weakens the immune system of the host. CyaA affects invaded cells in two ways. First, CyaA creates a cation-selective pores in the membrane of invaded cell and causes colloidal osmotic lysis. Second, CyaA converts cytosolic ATP into signal molecule cAMP, which causes a loss of physiological function of invaded cell and also leads to cellular death. The aim of my thesis was to test a suitability of a new model system composed from synthetic lipids - diphytanoyls, for a characterization of pore-forming properties of adenylate cyclase toxin. In the past, asolectin model system comprising many different lipid was used for characterization but it was found to be too complex for defining the role of individual lipids in CyaA activity. Further the effect of cholesterol for activity of CyaA was studied in a new model system because it was found recently that translocation of adenylate cyclase domain takes place at lipids rafts with high concentration of cholesterol. The last aim of my thesis was to characterize a newly discovered type of channel with the two conductance levels. Key words: Bordetella pertussis, adenylate...
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The role of RTX domain in the activity of adenylate cyclase toxin from Bordetella pertussis
Klímová, Nela ; Bumba, Ladislav (advisor) ; Konopásek, Ivo (referee)
The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein comprising an amino-terminal adenylate cyclase (AC) domain and a carboxy-terminal Repeat-in-Toxin (RTX) domain. The RTX domain is a hallmark of the family of RTX proteins, which are secreted from the cytosol of Gram-negative bacteria to the cell environment through the Type I Secretion System (T1SS). The RTX domain of CyaA consists of five blocks of RTX nonapetide repeats with a consensus sequence X-(L/I/V)-X-G-G-X-G- X-D. The aim of this work was to determine the role of the RTX domain in biological activities of CyaA and its role in the secretion of the toxin molecule from Bordetella pertussis. Systematic deletion analysis revealed that none of the prepared CyaA constructs was able to translocate its AC domain across the cytoplasmic membrane of host cells and make pores in target membranes. Moreover, deletion of individual RTX repeat blocks resulted in a very low efficacy of secretion of CyaA mutants into cell exterior. These data suggested that structural integrity of the RTX domain of CyaA is essential not only for cytotoxic activities of the toxin molecule but also for its secretion through the T1SS.
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Analysis of Memrane Proteins of Pathogenic Bacterim Francisella Tularensis
Schmidt, Monika ; Szotáková, Barbora (advisor) ; Černý, Jan (referee) ; Konopásek, Ivo (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate Mgr. Monika Schmidt Supervisor Doc. Ing. Barbora Szotáková, Ph.D. Title of Doctoral Thesis Analysis of membrane proteins of pathogenic bacterium Francisella tularensis Bacterium Francisella tularensis is highly infectious pathogen causing disease tularaemia. Due to the lack of standardization and little protection against highly virulent strains, the only vaccine developed against this pathogen is not allowed for clinical usage. Conserved hypothetical lipoprotein homological to thiol/disulfide oxidoreductase (DsbA) was recently described as essential virulence factor of Francisella tularensis. The dsbA gene deletion led to attenuation of the strain and development of immunoprotection. The DsbA protein sequence revealed the presence of carboxy-terminal DsbA_Com1-like domain harbouring the catalytic active site C-X-X-C and cis-proline and domain amino-terminal to FKBP type peptidyl-prolyl isomerase. This work was focused on functional a substrate characterization of DsbA protein. The functional analysis of this protein showed both the importance of the active site, cis-proline and the FKBP_N domain for the thiol/disulphide oxidoreductase activity. Further, this work also revealed the in...
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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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