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Bacteriocins produced by gram-negative bacteria with a focus on microcins - their mechanism of action.
Leštach, Oliver ; Dolejšová, Tereza (advisor) ; Grobarčíková, Michaela (referee)
Bacteriocins are ribosomally synthesised antimicrobial peptides and proteins produced by bacteria. One of the groups of bacteriocins are microcins. Microcins are antimicrobial peptides with size under 10 kDa, produced by bacteria of the Enterobacteriaceae family. Microcins form a heterogenous group of bacteriocins, not only in terms of mechanism of action but also other characteristics presented in this work. These characteristics include genetic determinants of microcins, posttranslational modifications, transport and aslo how immunity of the producing cell is ensured. Compared to other bacteriocins, microcins are less researched. However, there is a growing number of articles pointing to their potential use in medicine. This bachelor thesis includes current knowledge about the structure and mechanism of action of most researched microcins (B17, J25, C7 and E492), which are all very different from each other. The knowledge about microcins presented in this work is the basis for further research concerning their practical use. Key words: bacteriocins, microcins, antimicrobial peptides, posttranslational modification, Enterobacteriaceae
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Cellular factors influencing the antibiotic resistance by ABC-F proteins
Kýr, Jan ; Balíková Novotná, Gabriela (advisor) ; Dolejšová, Tereza (referee)
Antibiotic resistance is one of the main problems modern medicine has to face. In order to control it, it is important first to understand the mechanisms by which resistant pathogens bypass antibiotic treatment. One of the important protein families conferring resistance to 50S binding antibiotics is the ARE ABC-F protein family. A member of this protein family is the MsrA protein, which confers resistance to 14- and 15-membered macrolides. Loss-of-function mutations in the non-essential chaperone ClpX were found to a significant ly enhance the action of the MsrA protein. This leads to the significant increase in the resistance conferred by this protein. The exact mechanism by which ClpX affects MsrA function is still unknown. In this the diploma thesis was demonstrated that chaperone protein ClpX affects the resistance to erythromycin conferred by MsrA protein, due to the interaction with an unknown essential protein, which is mediated by a functional N-terminal zinc-binding domain of the chaperone. Furthermore, it was demonstrated in this work that loss of GluTR function influence the MsrA ability to confer resistance. The results of this work will bet he basis for further reasearch, which will lead to a more detailed understanding of the mechanism of resistance conferred by these proteins and...
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Elucidation of the properties and structure of the pore-forming domain of colicin U produced by bacterium Shigella boydii.
Dolejšová, Tereza
Colicin U is a protein produced by bacterium Shigella boydii. It belongs to the group of pore-forming colicins. These colicins interact with receptors in the outer membrane of bacteria closely related to a producing colicinogenic strain. After interaction with the receptor, colicin is translocated across the outer membrane and periplasm to the cytoplasmic membrane where it forms pores. Consequently, the pore formation leads to membrane depolarization and cell death. In this thesis I decided to study the pore-forming properties of colicin U and its membrane topology. It is shown that colicin U pores are formed by only one colicin molecule and they are voltage dependent. Using measurements with nonelectrolytes we estimated a theoretical inner profile of the pore and its inner diameter to be between 0.7 and 1 nm. Above that, a membrane topology of colicin U pore-forming domain (PFD) is studied. BLM measurements with biotinylated colicin U showed that a significant part of colicin's PFD was translocated to the opposite side of the membrane after the pore opening. The segment between substituted amino acids F463 and D486 was evidenced to be on the trans side of the membrane after the pore opening. Additionally, properties of peptide H1, which reflects a significant part of the first α- helix of colicin...
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Elucidation of the properties and structure of the pore-forming domain of colicin U produced by bacterium Shigella boydii.
Dolejšová, Tereza ; Fišer, Radovan (advisor) ; Krůšek, Jan (referee) ; Osička, Radim (referee)
Colicin U is a protein produced by bacterium Shigella boydii. It belongs to the group of pore-forming colicins. These colicins interact with receptors in the outer membrane of bacteria closely related to a producing colicinogenic strain. After interaction with the receptor, colicin is translocated across the outer membrane and periplasm to the cytoplasmic membrane where it forms pores. Consequently, the pore formation leads to membrane depolarization and cell death. In this thesis I decided to study the pore-forming properties of colicin U and its membrane topology. It is shown that colicin U pores are formed by only one colicin molecule and they are voltage dependent. Using measurements with nonelectrolytes we estimated a theoretical inner profile of the pore and its inner diameter to be between 0.7 and 1 nm. Above that, a membrane topology of colicin U pore-forming domain (PFD) is studied. BLM measurements with biotinylated colicin U showed that a significant part of colicin's PFD was translocated to the opposite side of the membrane after the pore opening. The segment between substituted amino acids F463 and D486 was evidenced to be on the trans side of the membrane after the pore opening. Additionally, properties of peptide H1, which reflects a significant part of the first α- helix of colicin...
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Survey of bacterial nucleotide-based second messengers
Beneš, Tomáš ; Branny, Pavel (advisor) ; Dolejšová, Tereza (referee)
Second messengers are small molecules that belong to one of the fundamental types of cell signalling. Their function is to transmit signals from extracellular or intracellular receptors to specific effector proteins. This type of signal transduction is evolutionarily ancient and conserved, occuring in every cellular organism. However, individual taxa differ in the specific compounds they use in signal transduction. In bacteria, different nucleotide derivatives are mostly used. The most important examples are cAMP, (p)ppGpp, c-di-GMP and c-di-AMP. Bacterial second messengers are involved in the regulation of metabolism, biofilm formation, stringent response, osmoregulation, protection against viral infection and many other processes. In addition to describing these signalling pathways, this work also deals with enzymes for synthesis and degradation of these small signalling molecules.
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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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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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Compensation for non-material damage in the event of injury and death in collateral proceedings
Dolejšová, Tereza ; Tejnská, Katarína (advisor) ; Mulák, Jiří (referee)
1 Abstract Compensation for non-pecuniary harm in the event of personal injury and death in collateral proceedings The topic of the diploma thesis is the issue of compensation for non-pecuniary harm in the event of personal injury and death in collateral proceedings. This issue is currently often debated due to, among other reasons, a change in the legal regulation of compensation for pecuniary and non-pecuniary harm effective from January 1, 2014. The specificity of this subject lies in the combination of two legal branches - criminal and civil law, as collateral proceedings are part of criminal proceedings and decide on claims which are of a civil nature. The thesis deals with the criminal law institutes of the injured party and the collateral proceedings themselves. An extensive space is then devoted to the private law regulation of compensation for non-pecuniary harm in the event of personal injury and death, especially in accordance with Sections 2958 and 2959 of the Civil Code with regard to legal practice and court decisions. The aim of this work is to describe and evaluate the legal regulation of compensation for non-pecuniary harm in the event of personal injury and death and the procedural possibilities of the injured party to attain this private law claim in criminal proceedings. With regard to...
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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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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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