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Determination of the structure of pore-forming colicins
Riedlová, Kamila ; Fišer, Radovan (advisor) ; Barvík, Ivan (referee)
6 Abstract This master's thesis provides study of individual helixes from C-terminal pore-forming domain (CTD) of colicin U and their behavior in lipid bilayer on atomic level. For this purpose the all-atom molecular simulation method was used. Later the study was extended an applied on CTD of published structures of other pore-forming colicins. On the base of study extension the ability of disruption of lipid bilayer integrity by helixes H1 and H10 was successfully observed. Helix H1 was synthesized and its activity was experimentally proved on black lipid membranes. The other helixes are often too short to be able to keep position in lipid bilayer and their behavior could be affected by artificial termini, therefore they were not synthesized. The MD simulations of pairs of helixes show that structure stability and their ability to stay in the membrane depends on binding partners. The results of the thesis show the importance of H10 for colicin pore-formation, which has not been observed yet. The results also support the toroidal pore model suggested previously for colicin E1. The results prove that colicins contain specific secondary structures, which are able to disrupt the inner bacterial membrane not only in its native form but also when artificially separated from the rest of the protein. Klíčová...
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Control of cell division of Streptococcus pneumoniae by unique signaling pathway
Kubincová, Hana ; Branny, Pavel (advisor) ; Fišer, Radovan (referee)
Genome of S. pneumoniae contains only one copy of the gene coding eukaryotic type protein kinase StkP and corresponding phosphatase PhpP. These two enzymes form a functional signaling pair regulating cell division, which could be used in the future for the design of new bacteriostatic compounds. Not only kinase and phosphatase are important components of the system, but also other members of this pathway - specific substrates of these enzymes. The identification of the Ser/Thr phosphoproteom with a focus on the membrane fraction provided information not only about already known substrates such as LocZ, Jag and DivIVA but also about an unknown protein P15 with a molecular weight about 15 kDa. In this thesis the protein was identified as rhodanase (spr0595) by MS MALDI TOF. However, its subsequent deletion did not confirm it as a StkP/PhpP substrate. Therefore we investigated another substrate, protein FtsA, which has already been identified as a substrate of this kinase in a previous study (Beilharz et al., 2012). FtsA is an essential cell division protein that anchors FtsZ filaments into the membrane. Phosphorylation of this protein was detected on the Thr residue at position 404. Using phosphoablative substitution we found out, that Thr404 is indeed phosphorylated by protein kinase StkP, however, FtsA...
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The mechanism of action of phage tail-like bacteriocins on target cells and artificial membrane systems.
Hryzáková, Klára ; Fišer, Radovan (advisor) ; Ulrych, Aleš (referee)
Fonticins are phage tail-like bacteriocins produced by gram-negative bacterium Pragia fontium from the family Enterobacteriaceae. Phage tail-like bacteriocins can be divided into two different families: flexible ones (F-type) and contractile particles (R-type). Pragia fontium produces R-type particles that adsorb on the surface of sensitive bacterial cell and form pores probably during the contraction using mechanism similar to Type VI Secretion System. The pore-forming activity of fonticins was tested in vivo using bacterial cells. It was also characterized in vitro on artificial lipid membranes. On Black Lipid Membranes fonticins create large channels into the membranes; single channel conductance (G) is about two times higher than single channel conductance of well known α-hemolysine produced by Staphylococcus aureus. Further, we tested the voltage-dependent blocking of fonticin pores by native and unfolded proteins, dsDNA, ssDNA, polyethylene glycol and diamond nanoparticles. The rigid structure of fonticin nanotube in combination with constant conductivity makes it a promising device for analysing the size and shape of nanoparticles and large macromolecules. Key words: fonticin, bacteriocine, nanopore, Pragia fontium, blocking, pore-forming activity, black lipid membranes.
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Metabolic control of bacterial division.
Valtová, Aneta ; Lichá, Irena (advisor) ; Fišer, Radovan (referee)
Metabolic control of cell cycle has been study for a long time, but it is not completely known. Mechanisms of metabolic control described for a several decade has been explained on molecular level with using a modern methods. Regulation of cell cycle in consideration of metabolism and nutritional status is going on at the several level of cell replication. The most known is about assembly of bacterial cell divisiome. Changes in nutrient availability induce stress response that use low-molecular substances in signaling pathways leading to changes in the cell cycle. One of the most studied is (p)ppGpp that participates in stringent response and affect sigma factors, directly inhibits the initiation of replication by binding to the DnaG primase and indirectly inhibits the elongation of replication. Current researches has revealed that some enzymes with already known enzymatic function in the major metabolic pathways (glycolysis or TCA) also has a function as sensors that transmit the nutritional change signal directly into the cell dividing process. These signals most often inhibits FtsZ protein or affect its helper proteins and subsequent ring formation. Analogues of these enzymes were found in gram-positive (Bacillus subtilis) and gram-negative bacteria (Escherichia coli, Caulobacter crescentus)....
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Mechanisms and aplications of macromolecule translocation across membranes of eukaryotic cells by bacterial toxins
Poledňák, Jan ; Fišer, Radovan (advisor) ; Žáčková Suchanová, Jiřina (referee)
Toxin translocation across the cytoplasmic membrane of the eukaryotic cell is a potent virulence factor of bacteria causing disease to eukaryotic organisms. Toxins translocate their domains responsible for the toxic activity inside the cell or create pores in cell membrane allowing the transmembrane traffic of ions, DNA, RNA or proteins. Knowledge of the toxin translocation process enables to characterize the mechanism and also the properties of the pore-forming toxin. Some of these toxins have been described in such a detail that were changed using site-directed mutagenesis and can serve as tools for characterization of the translocated molecules. One of such examples is the transfer of nucleotides or the whole nucleic acid molecules across the membrane through the pore of S. aureus α-hemolysine. Nowadays, this application is commercially used for DNA sequencing. Keywords: translocation, bacterial toxins, plasmatic membrane, nanopore sequencing
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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 - methods of prepatation, properties and their usage
Hryzáková, Klára ; Fišer, Radovan (advisor) ; Sýkora, Michal (referee)
The heterogeneity of biological membranes has led to development of a wide spectrum of simplified model systems whose composition, size and shape can be adapted to the requirements. There are two different approaches of making artificial phospholipid bilayers. One of them is based on creating bilayers in aqueous phase. This includes Black lipid membranes, Supported phospholipid bilayers, bilayers from water/air interface and liposomes. In the second approach bilayers are created in a bulk of organic phase by Droplet interface bilayer method. Each type of artificial bilayer has its experimental advantages that have been used to study many problems ranging from behaviour of single phospholipids and proteins to membrane fusion. Artificial lipid membranes are perfect tool for electrical characterisation of bilayers and embedded membrane proteins. This work is a complete review of most useful techniques of model membrane preparation. Key words: membrane, lipid, phospholipid bilayer, liposome, black lipid membrane, supported lipid bilayer, droplet interface bilayer
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Preparation and characterization of diamond-based nanocarriers for transfection of siRNA
Majer, Jan ; Cígler, Petr (advisor) ; Fišer, Radovan (referee)
Although nanodiamonds were discovered and produced tens of years ago, they have been utilized in medical and biological fields just recently, particularly in drug and gene delivery into a cell and in bioimaging methods. Nanodiamonds can be modified with specific positively charged moieties for complexation with negatively charged nucleic acids. These complexes afterwards overcome extracellular and intracellular barriers and transport the nucleic acid either into cytosol or into the nucleus. Owing to fluorescence centres nitrogen- vacancy, which can be formed in the nanodiamonds, nanodiamonds exhibit excelling optical properties, as they emit stable fluorescence without "photoblinking" or "photobleaching". This thesis reviews properties, synthesis and modifications of nanodiamonds and other selected nanoparticles and their in vitro applications. This thesis also compares their cytotoxicity and gene knockdown efficiency.
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Mechanisms and aplications of macromolecule translocation across membranes of eukaryotic cells by bacterial toxins
Poledňák, Jan ; Fišer, Radovan (advisor) ; Žáčková Suchanová, Jiřina (referee)
The bacterial protein toxins endowed with the ability to translocate across the plasmatic membrane are often crucial virulence factors of pathogenic bacteria invading eukaryotic organisms. These toxins translocate either their own protein domains carrying toxic activity or can form pores transferring other substances like small ions, DNA, RNA or proteins. By observing the translocation of these molecules together with other artificially prepared agents on synthetic membranes it allows detailed understanding of mode of action of individual pore-forming toxins. Some of the toxins were actually described in such a detail that can serve as tools for drug delivery or characterization of new translocated molecules. One of such examples is the transfer of nucleotides or the whole nucleic acid molecules across the membrane pore of S. aureus α-hemolysine. Nowadays, this application is commercially used for DNA sequencing. Keywords: translocation, bacterial toxins, plasmatic membrane, nanopore sequencing
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Ab initio prediction of the membrane protein structures
Sokol, Albert ; Fišer, Radovan (advisor) ; Plocek, Vítězslav (referee)
Knowledge of the three dimensional structure of the protein is extremely important for a full understanding of its function and molecular proteins interaction. The structure is typically determined experimentally by X-ray crystallography and NMR spectroscopy, unfortunately membrane proteins provide numerous problems for these methods. A possible solution is the computational prediction. Ab initio prediction of three-dimensional models of the membrane proteins is a complex process which cannot use any available protein structure as a general template. There are few softwares that deal with this process and selected four are described in detail in this work. These are two programs for the prediction of transmembrane helical proteins (Rosetta, EVfold_membrane) and two for the prediction of transmembrane beta barrels (EVfold_bb, 3D-SPOT). The main approaches that are used in the prediction of the three-dimensional structure of a protein are inserting short segments of amino acid sequences which are derived from the determined protein structures (Rosetta), using evolutionary information from many other protein sequences (EVfold) and formation of the beta barrel domains based on combining adjacent antiparallel beta chains (3D-SPOT). Every software uses a variety of external programs to address specific...
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