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Metabolic control of the cell cycle in bacteria
Valtová, Aneta ; Lichá, Irena (advisor) ; Fišer, Radovan (referee)
Metabolic control of cell cycle has been studied for a long time in bacteria, but it is not still fully elucitated. The mechanisms described for several decades have been described in more detail and find new connections between basic metabolism and the cell division process itself. Cell cycle regulation, depending on metabolism and nutritional conditions, takes place over all steps of the cycle. The most mechanisms are studied at the level of bacterial division formation. Nutritional deprivation induces stress responses that use low-molecular substances which are involved in signaling pathways and which regulate the cell cycle. One of the most studying is the molecule of guanosine (penta)tetraphosphate (p)ppGpp, which affects cell cycle at the level of genes expression, at the level of proteins involved in the process of creating divisome, even at the level of replication. Recent research revealed that some enzymes with their already known enzymatic function in major metabolic pathways (glycolysis and TCA), also have a function as sensors that transmit a signal about the nutritional change directly to the division apparatus of the cell. These enzymes regulate the formation of the Z ring through the protein FtsZ or its auxiliary proteins most often and have been found in Gram-positive (Bacillus...
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Non-covalent interactions of tryptophan in protein structure
Sokol, Albert ; Fišer, Radovan (advisor) ; Jurkiewicz, Piotr (referee)
A thorough knowledge of non-covalent amino acid interactions within a protein structure is essential for a complete understanding of its conformation, stability and function. Among all the amino acids that usually make up a protein, tryptophan is distinguished both by its rarity and size of its side chain formed by an indole group. It is able to provide various types of indispensable interactions within the protein and between different polypeptide chains, but also between the protein and a biological membrane. In addition, it is the most commonly used natural fluorophore. Databases of solved protein structures are commonly used to study amino acid interactions and allow more or less complex analyzes of the issue. Thus many non-covalent interactions that may occur between tryptophan and other amino acids have been found. However, most of these analyzes focus on specific interactions and do not follow up the tryptophan's environment as a whole, where all amino acids interact. Some newly developed methods have been used in this Thesis, specifically the occurrence profiles of the individual amino acids around the indole group of tryptophan and the results were compared with an available literature. The amino acid that has the greatest preference for tryptophan turned out to be tryptophan again, and...
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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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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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