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Inducible expression systems and their use in the study of parasitic organisms.
Horáčková, Vendula ; Doležal, Pavel (advisor) ; Fišer, Radovan (referee)
1 Abstract Inducible expression systems are systems with ability to switch expression of genes of interest on and off. Therefore, they are useful molecular tools for analysis of gene function. Nowadays, there are tens of various inducible expression systems available that differ from each other in level of regulation of gene expression, time of induction, possibilities of use, etc. This work is focused on three of them to illustrate common features of the inducible expression systems which regulate gene expression at the level of transcription. Firstly, systems based on regulation of lactose operon of Escherichia coli are mentioned. Secondly, systems which use regulatory elements of tetracycline resistance-encoding transposon Tn10 of E. coli are described. Third chapter is focused on systems regulated by agonists of ecdysone receptor. In the last chapter cases of use of inducible expression systems in the study of parasitic organisms are summarized.
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Effect of promoter sequence on utilization of NAD+ as a substrate for transcription initiation by RNA polymerase
Pinkas, Daniel ; Krásný, Libor (advisor) ; Fišer, Radovan (referee)
For a long time, 5' cap has been thought to be privilege only for eukaryotic organisms in form of 7-methylguanosine cap at the end of mRNA. This was changed only a few years ago. By using methods liquid chromatography and mass spectrometry a new molecule associated with RNA of Escherichia coli has been found. This molecule turned out to be nicotinamide adenine dinucleotide (NAD+ ) attached to 5' end of some small regulatory RNAs (sRNA). Later it has been shown, that RNA polymerase can attach NAD+ at 5' of RNA ab initio, meaning that RNA polymerase can utilize NAD+ as a substrate for transcription initiation. To some extent substrate for transcription initiation is chosen based on promoter sequence. Crucial requirement is presence of adenine at +1 position of DNA coding strand. This thesis focuses on promoter sequence requirements for transcription initiation with NAD+ . As a template for transcription four promoters with different modifications and their chimeras are used: RNA1, Pveg, lac UV5 and rrnB P1. Also, I tried to compare RNA polymerase from E. coli and B. subtilis in terms of transcription initiation substrate usage. Lastly, I describe here isolation of NudC, enzyme that cleaves NAD+ to nicotinamide mononucleotide (NMN) and adenosine monophosphate (AMP). NudC will be used for upcoming...
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Biomolecular corona of Si and Au nanoparticles and its impact on interaction with cells
Javorová, Pavlína ; Hubálek Kalbáčová, Marie (advisor) ; Fišer, Radovan (referee)
Biological response to presence of gold and silica nanoparticles is extensively researched area of science. However there is only limited knowledge and understanding of the effects of small and ultrasmall nanoparticles. Regarding the unique physical and chemical properties that originate from the small size have these nanoparticles ability to interact very specifically on molecular level with organisms. Once the particle enters the complex physiological environment of the body molecules (predominantly of protein character) adsorb on the surface and form a polymeric case called biomolecular corona. There is a presumption that the first contact of the nanoparticle with the cell is mediated through the molecules of this corona and are important in subsequent steps of interactions of nanoparticle-biocorona complex. Therefore the genesis and structure of biocorona is as essential as the structre of the nanoparticle itself. Nanoparticles enter and are internalized within the cell and cellular compartments through the same mechanisms like naturally occurring molecules and substances. There are slightly different patterns of behavior of small and ultrasmall nanoparticles that are not fully researched and understood. Response of mamallian cells to the presence of the ultrasmall nanoparticles is very...
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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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