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Topology and function of the transmembrane domain of colicin U produced by Shigella boydii
Dolejšová, Tereza ; Fišer, Radovan (advisor) ; Krůšek, Jan (referee)
Colicin U is a protein produced by strains of bacterium Shigella boydii. It exhibits antibacterial activity against some bacterial strains Shigella and Escherichia. Based on sequence homology with colicins A, B and N, the colicin U is classified as a pore-forming colicin. Interaction of colicin U with attacked bacteria is ensured by three-step mechanism: 1) First colicin U interacts with surface receptors OmpA, OmpF and core of LPS. 2) Thereafter the colicin is translocated to periplasm through interaction with Tol proteins. 3) Finally colicin U interacts with the inner membrane of the attacked bacteria causing its depolarization. In this thesis I demonstrated pore-forming features of colicin U and further observed characteristics and properties of these pores. Using methods of measuring on black lipid membranes I determined a single channel conductance (19 pS), ion selectivity, the influence of various conditions on the behaviour of the pores. These findings, in many cases, correspond to the findings on other related colicins. Furthermore, I successfully determined the pore diameter of colicin U ( ≈ 0,8 nm). The next section of the thesis focuses on creation of single cysteine mutations of colicin U. Subsequently I produced five mutant variants of colicin U and verified their functionality so that...
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Interactions of two nearby bacterial colonies - the effect of signaling molecules and nutrients on the colony growth
Dobřemyslová, Mária ; Fišer, Radovan (advisor) ; Zikánová, Blanka (referee)
Between neighbouring bacterial colonies of the same species there occur mutual interactions influencing their growth (both size and pattern). Effects of these interactions on the growth can be both negative and positive, and can change in the course of development of colonies. The primary cause of mutual influence is often competion for available sources of nutrients, production of wastes, and production and utilization of public goods. The intensity of influence depends on external factors like mutual distance of colonies, medium composition and rigidity, or possibility of mutual signal molecule exchange. In this bachelor's thesis there are described known mechanisms of intraspecies interactions that may be of some importance in communication between two colonies. In more detail there are described three particular cases of influence of two nearby colonies that have been up to now studied more intensively. Further on, there have been summarized methods of measuring the sizes of colonies and algorythms applicable to evaluation of mutual influence of nearby colonies.
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Artificial phospholipid membranes - method of prepatation, properties and their usage
Hryzáková, Klára ; Fišer, Radovan (advisor) ; Sýkora, Michal (referee)
Heterogenita biologických membrán vedla ke vzniku širokého spektra zjednodušených modelových systémů, jejichž uspořádání, velikost a tvar se dají přizpůsobovat různým aktuálním požadavkům. Existují dva rozdílné přístupy k vytváření umělých fosfolipidových membrán. První z nich je založen na vzniku membrán ve vodném prostředí. Do této skupiny patří černé lipidické membrány, dvojvrstvy na pevném podkladu, dvojvrstvy vzniklé z monovrstev na rozhraní vody a vzduchu a liposomy. Ve druhém případě vznikají dvojvrstevné membrány v množství organické fáze metodou dvojvrstev na kapičkovém rozhraní. Každý typ umělých membrán má své experimentální výhody a nevýhody, což se používá ke studiu různých problémů sahajících od chování jednotlivých fosfolipidů a proteinů až po fúze membrán. Umělé fosfolipidové membrány jsou vhodným nástrojem pro elektrickou charakterizaci dvojvrstev a nebo membránových proteinů. Tato práce je ucelený přehled nejpoužívanějších metod vhodných pro vznik umělých fosfolipidových membrán. Klíčová slova: membrána, lipid, fosfolipidová dvojvrstva, liposom, černá lipidová membrána, dvojvrstvy na pevném podkladu, dvojvrstva na kapičkovém rozhraní Abstract The heterogeneity of biological membranes has led to development of a wide spectrum of simplified model systems whose composition, size and...
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Characterization of membrane pores formed by newly discovered colicin FY from Yersinia frederiksenii
Dolejšová, Tereza ; Fišer, Radovan (advisor) ; Nunvář, Jaroslav (referee)
Colicins are toxic exocellular proteins used by Gram-negative bacteria for interspecies and intraspecies competition. The colicin FY is a pore-forming protein which was recently discovered at Masaryk university. Kolicin FY is produced by strain Yersinia frederiksenii Y27601 and is active against other strains of genus Yersinia. By comparison of aminoacid sequences of C-terminal domains of selected colicins it was proved, that colicin FY is closely related to colicin Ib (Bosák et al., 2012). In this work I was trying to create brief and integrated summary about the group of colicins from the perspective of an outer membrane traslocation mechanism, overcoming the periplasmic space up to isertion of C-terminal colicin domain into the inner membrane phospholipid bilayer. Other aim of my work was to generally summarize pore properties of known colicins and compare them with recently measured characteristics of colicin FY. Keywords: colicin, Yersinia, planar lipid membranes, membrane pore
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Factors interacting with bacterial RNA polymerase
Sudzinová, Petra ; Krásný, Libor (advisor) ; Fišer, Radovan (referee)
The bacterial cell must be able to rapidly change its gene expression to survive unstable external conditions. Transcription is the key level that affects gene expression. The pivotal enzyme of transcription is RNA polymerase (RNAP). Activity of RNAP is tightly regulated by transcription factors (TFs). These factors affect RNAP in different ways. This work presents an overview of various proteins and others factors, description of their effects on transcription and also mechanisms of their actions. TFs could be divided according to various criteria. In this work, TFs are divided according to how they interact with RNAP: TFs interacting only with RNAP; TFs binding simultaneously DNA and RNAP; TFs interacting with RNA and RNAP. This work presents a comprehensive overview of various TFs that are involved in the bacterial cell's reprogramming of gene expression that is required to withstand the changes in the environment.
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Dynamics of modified diamond nanocrystals in living cells
Majer, Jan ; Libusová, Lenka (advisor) ; Fišer, Radovan (referee)
Nanodiamonds (NDs) are an interesting platform in biological applications and disease treatment. Because of their photoluminescence properties and modifiable surface, they have been investigated as potential carriers for drugs and nucleic acids as well as fluorescent probes. In order to design NDs meeting specifically desired parameters, which would succeed in clinical trials and in medicinal therapy, understanding the mechanism of uptake and intracellular fate of NDs is crucial. The diploma thesis is focused on mechanistic investigation of ND-based nanoparticles delivering nucleic acids to human cells. First, NDs coated with a novel cationic co-polymer were prepared. NDs were then complexed with siRNA in order to transfect siRNA inside U-2 OS cells. NDs proved to be biocompatible and effective transfection particles as observed by qPCR and colorimetric cytotoxicity and cell viability tests. To examine ND uptake by cells, we inhibited endocytosis by specific inhibitors. Obtained results implicated that ND uptake was clathrin- and caveolin dependent. Nonetheless, more than half of NDs was internalized by cells in a different fashion. Some NDs colocalized with early endosomes, lysosomes and caveolin-derived endosomes after internalization. Other NDs resided either in unknown cell structures or escaped from...
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Conformation of the adenylate cyclase toxin of Bordetella pertussis.
Motlová, Lucia ; Fišer, Radovan (advisor) ; Obšil, Tomáš (referee) ; Holoubek, Aleš (referee)
This work is focused on the RTX (Repeats in ToXin) domains structure of selected RTX toxins and its impact on secretion and protein folding. The structural analysis included RTX domains of ApxI (Actinobacillus pleuropneumoniae-RTX-toxin I) from Actinobacillus pleuropneumoniae, HlyA (Alfa-hemolysin) from Escherichia coli and LtxA (Leukotoxin A) from Aggregatibacter actinomycetemcomitans and blocs 4 a 5 RTX domain CyaA (adenylate cyclase toxin) from Bordetella pertussis. The structures of LtxA RTX domain and CyaA RTX blocs 4 and 5 were obtained and characterized. Two models of CyaA RTX domain were built based on SAXS (Small Angle X-ray Scattering) model, previously solved RTX structures and RTX structures presented here.
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Evaluation of TD test for analysis of persistence or tolerance in clinical isolates of Staphylococcus aureus
Kotková, Hana ; Lichá, Irena (advisor) ; Fišer, Radovan (referee)
Persistence is the ability of bacteria to survive the impact of antibiotics even when the bacteria do not encode resistance genes. This is a very complex process, which is probably consequence of a reduction physiological process in subpopulation of bacteria. The aim of this study was to verify the suitability of the newly developed "Tolerance Disk Test" (TD test, Gefen et al, 2017) for detection of persistent or tolerant subpopulations of bacterial cells in clinical isolates of Staphylococcus aureus. We performed TD test for this kind of bacteria, which is a significant opportunistic pathogenic organism in humans, and compared its results with the killing curves. We have found that the ability to persist can be monitored semi-quantitatively also in this case and we consider this test suitable for introduction into clinical practice. In addition, we suggest that TD test could distinguish between persistent and tolerant subpopulations. Key words: Staphylococcus aureus, persistence, antibiotics, tolerance
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Biosynthesis of propylproline building unit of lincomycin
Jirásková, Petra ; Janata, Jiří (advisor) ; Čejková, Alena (referee) ; Fišer, Radovan (referee)
The clinically used antibiotic lincomycin consists of an amino-sugar and an amino-acid moiety. The incorporated amino-acid 4-propyl-L-prolin (PPL) is very important for the linomycin bioactivity, as evidenced by the lower activity of the related antibiotic celesticetin, which incorporates proteinogenic L-prolin instead. Gene clusters for the biosynthesis of both lincosamides are published and reflect a common basis - biosynthesis of amino-sugar precursor and condensation reactions. Additionally, in the biosynthetic gene cluster for lincomycin there is a sub-cluster of genes encoding the biosynthesis of PPL, the alkylated proline derivative (APD). PPL has a common biosynthetic origin with other APDs that are part of the structures of antitumor pyrrolobenzodiazepines and the signal molecule hormaomycin, which is also reflected in the presence of homologous genes in their gene clusters. The acquired knowledge on PPL biosynthesis thus can be applied to a larger group of natural products. The first overall concept of APD biosynthesis was published forty years ago. The milestone was the year 1995 when the gene cluster for lincomycin biosynthesis was published and specific gene products have been proposed for individual biosynthetic steps. The functional proof of proteins has been performed so far just...
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