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Clonning and characterization of the membrane microdomain targeted componets
Falteisek, Lukáš ; Černý, Jan (advisor) ; Brábek, Jan (referee)
3 Abstract Lipid rafts are defined as islets in the membranes of eucaryotic cells with different composition from the rest of the membrane. They appear spontaneously due to phase separation of different membrane components and are proposed to serve as platforms for concentration of selected signaling proteins. However, evidence for their existence is still indirect, despite more than decade of intense research. Some new approaches show that the fluctuations of membrane composition are more diverse and are caused more likely by presence of proteins than by lipid phase separation. We performed bioinformatical search looking for new signaling proteins targeted to putative rafts. We have identified several proteins and out of them phosphodiesterase 8a (PDE 8a) was salected for further research. To prepare the mouse monoclonal antibody we expressed and purified fragment of PDE 8a fused with GST. After immunisation we obtained one clone producing antibodies tentatively positive on western blot and by indirect immunofluorescence. We predicted that PDE 8a is targeted to putative rafts by N-terminal myristoylation and palmitoylation. To clarify, whether these modifications are present in the strusture of PDE 8a we prepared mutants of the PDE 8a N-terminal region lacking myristoylation, palmitoylation or both. These...
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Microdomains in freshwater sediment as a driving factor in the biogeochemical processes
Duchoslav, Vojtěch ; Falteisek, Lukáš (advisor) ; Beranová, Jana (referee)
A natural arsenic anomaly at Mokrsko is a site of biogenic precipitation of realgar (As4S4) in stream sediment. The extent of the phenomenon in Mokrsko is globally unique. A previous study showed the ability of microorganisms to solubilize arsenic from its secondary minerals and to produce realgar precursors, i.e. sulfides and arsenites, by anaerobic respiration. The study also raised questions about the mechanism of realgar precipitation since physicochemical conditions favoring this reaction were never detected despite significant sampling efforts. We chemically and microbiologically analyzed sedimentary profiles to the depth of ca. 120 cm in order to understand the functioning of the biogeochemical system. The profiles comprised both the unsaturated and the saturated zone. We distinguished six different domains representing environments from the surface soil to anoxic sedimnent containing realgar-encrusted wood. An analysis of phylogenetic dissimilarity revealed that microbial communities from the various domains form distinct clusters. This suggests that different conditions prevail in the various domains, and that different biogeochemical processes take place there. Incubation (microcosm) experiments showed that bioprecipitation of realgar can be conducted in vitro. It requires a suppression...
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Ecology of microbial communities in the sulfidic ore environments
Falteisek, Lukáš ; Čepička, Ivan (advisor) ; Šabacká, Marie (referee) ; Mandl, Martin (referee)
1 Abstract This thesis is focused on the diversity of microorganisms of prokaryotic type living in the environments, where microbial sulfidic mineral precipitation or decomposition occur. The relationship between the microbial community composition and geochemical processes was examined. To the best of our knowledge, we were the first to analyze microbial communities from gossan and their significance for the ecosystem of a large sulfidic ore deposit. In addition, we compared the microbial assemblies at multiple habitats associated with generation or transformation of acidic mine drainage (AMD) and described niche partitioning among closely related organisms. The unexpectedly variable communities in stalactites growing on the AMD springs were utilized as a model for assessing neutral variability of the microbial communities. They clustered almost randomly even though the environmental conditions corresponded with the localities. This is an important difference of the simple communities from stalactites and the common highly diversified microbial assemblies. The communities found in sediments, soil, and many other complex substrates usually reveal high correlation with their environment. We propose that neutral fluctuations of the community composition are suppressed by averaging multiple physically...
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Bacterial role in silicate mineral alteration in near-neutral pH conditions
Duchoslav, Vojtěch ; Falteisek, Lukáš (advisor) ; Seydlová, Gabriela (referee)
It is well established that bacteria are able to catalyze dissolution of silicate minerals. Bacteria may dissolve silicates for two different purposes. They may use certain elements that can undergo redox transitions as substrates for their energetic metabolism or they can leach nutrients, that are otherwise unaccessible in their habitat. The main mechanisms of bacterially mediated silicate leaching are acidic or basic catalysis and surface complexation. The main nutrients extracted are K, Mg, P and Fe. The only element significantly exploited as substrate for dissimilative energetic metabolism is iron. In order to leach iron as a nutrient, even extremely strong complexants (i.e. siderophores) may be employed. However, only moderate complexing agents can serve to obtain iron as terminal electron acceptor. The second possibility is to reduce iron directly in the crystal grid by means of the conductive nanofibres. The oxidative dissolution of silicates by chemoautotrophs is rare, in contrast to that of sulfides. Bacterial dissolution of silicates leaves morphological and geochemical signatures, but it is still problematic to recognize and interpret them. Although it is well-known that bacteria can dissolve most of the rock-forming minerals in diverse environments we are unable to quantify their contribution...
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Use of microbial community structure as a marker of conditions in underground biotops
Burkartová, Kateřina ; Falteisek, Lukáš (advisor) ; Drahota, Petr (referee)
The amount of data obtained by barcoding of prokaryotic 16S rDNA from natural habitats is increasing exponentially. Thus, methods enabling us to extract useful information from these data are of increasing importance. In this thesis microbial communities from water, sludge and drilling dust were analyzed by 16S rDNA sequencing in three geologically well described sedimentary aquifers in Bohemian Massif. The main goal of this research was to establish how different analytical approaches can be useful in interpretation of groundwater biogeochemical processes. Three approaches were used: First, taxonomy and metabolic traits of the most abundant microorganisms were assessed. Second, ordination methods showing metabolic and taxonomic variability between communities were used. Last the analysis of phylogenetic dissimilarity using UniFrac metrics was performed. When analyzing individual localities separately, the shift in microbial community composition corresponds with the change of environmental conditions. The unconstrained ordination method based on the variability in metabolic traits indicated, that sludge samples are more informative than water samples when asking which electron donor is used in microbial communities. On the other hand, unconstrained ordination methods were useless when the...
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Ecology of microbial communities in the sulfidic ore environments
Falteisek, Lukáš ; Čepička, Ivan (advisor) ; Šabacká, Marie (referee) ; Mandl, Martin (referee)
1 Abstract This thesis is focused on the diversity of microorganisms of prokaryotic type living in the environments, where microbial sulfidic mineral precipitation or decomposition occur. The relationship between the microbial community composition and geochemical processes was examined. To the best of our knowledge, we were the first to analyze microbial communities from gossan and their significance for the ecosystem of a large sulfidic ore deposit. In addition, we compared the microbial assemblies at multiple habitats associated with generation or transformation of acidic mine drainage (AMD) and described niche partitioning among closely related organisms. The unexpectedly variable communities in stalactites growing on the AMD springs were utilized as a model for assessing neutral variability of the microbial communities. They clustered almost randomly even though the environmental conditions corresponded with the localities. This is an important difference of the simple communities from stalactites and the common highly diversified microbial assemblies. The communities found in sediments, soil, and many other complex substrates usually reveal high correlation with their environment. We propose that neutral fluctuations of the community composition are suppressed by averaging multiple physically...
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