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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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Genomic analysis of Paenibacillus larvae and its relation to virulence
Vlková, Kateřina ; Hrabák, Jaroslav (advisor) ; Fišer, Radovan (referee)
Paenibacillus larvae is a Gram-positive sporulating bacterium that causes American foulbrood (AFB). It is one of the most dangerous bacterial pathogens of the honeybee (Apis mellifera). P. larvae spores are highly infectious to bee larvae and resist physicochemical influences. P. larvae is subtyped using repPCR with ERIC primers (Enterobacterial Repetitive Integrance Consensus) into five genotypes (ERIC I-V), which possess different colony morphology, metabolism and especially virulence. There is a significant genetic variability among isolates of P. larvae, which may contribute to differences in virulence. P. larvae isolates used in this work were obtained from clinical cases of American foulbrood as well as from a debris collected from bee hives with no American foulbrood symptoms from all over the Czech Republic in cooperation with the Beekeeping Research Institute, s.r.o., Dol. The isolates were obtained from larvae and hive debris. Both virulet and avirulet strains were sequenced using the SMRT (single molecule real time) method on the Sequel platform (PacBio). This method is suitable for Whole Genome Sequencing (WGS), because it allows sequencing of long reads with high accuracy, eliminating the effect of a large number of repetitive sequences during the genome assembly. Furthermore,...
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Mechanisms of antibiotic action and their relationship to the shape of the inhibition zones in E-test
Brzobohatá, Hana ; Fišer, Radovan (advisor) ; Vrana, Branislav (referee)
The aim of this work is antibiotic susceptibility testing, especially the method of Etest. Etest is a widespread technique due to its simplicity and accuracy in both research and clinical conditions. Understanding the method and its influencing factors is necessary for accurate reading of the MIC (minimum inhibitory concentration) and understanding of bacteria- antibiotic interactions. The purpose of the study was to demonstrate connections between antibiotic action mechanism and zone of inhibition shape. Also, to point out the obstacles that might complicate results interpretation. The pictures of Etest results from research papers were compared to the models of antibiotic diffusion. The zone of inhibition shape is affected by the diffusion rate, molecular characteristics of antibiotic, by the bacterial strain performances and by the specific bacteria-antibiotic interactions.
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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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