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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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Molecular physiology of low-voltage-activated T-type channels in neuropathic pain
Pelant, Tomáš ; Weiss, Norbert (advisor) ; Fišer, Radovan (referee)
Low-voltage activated T-type channels contribute significantly to signal transmission in ascending pain pathway. Their electrophysiological and biochemical properties allow them to modulate neuronal excitability and neurotransmitter release. Alterations of electric currents associated with a number of neuronal disorders, including neuropathic pain and epilepsy, have been linked to this subtype of calcium channel, suggesting its prominent role in modulation of neuronal response to various noxious stimuli. Multiple diseases, such as diabetes, cancer or chronic nerve injury, are accompanied by painful neuropathic conditions. Specific inhibitors of T-type channels have been demonstrated to alleviate symptoms of neuropathic pain in mouse models, showing their potential for development of novel type of drugs possibly more effective than traditional analgesics, which exhibit minor effect in neuropathic pain treatment.
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Bacterial toxins translocating across the membrane of eucaryotic cells
Poledňák, Jan ; Fišer, Radovan (advisor) ; Žáčková Suchanová, Jiřina (referee)
The bacterial protein toxins endowed with the ability to translocate accross the plasmatic membrane are often crucial virulence factors of pathogenic bacteria invading eucaryotic organisms. These toxins translocate either their own protein domains carrying toxic activity or can form pores transfering other substances like small ions, DNA, RNA or proteins. By observing the translocation of these molecules together with others artificially prepared agens on synthetic membranes 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 investigation tools for characterization of new translocated molecules. One of such example is the transfer of nucleotides or whole nucleic acid molecules accross the membrane pore of α- hemolysine of S. aureus. This applications is in recent days commercially used for DNA sequencing.
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Artificial phospholipid membranes - method of prepatation, properties and their usage
Hryzáková, Klára ; Fišer, Radovan (advisor) ; Sýkora, Michal (referee)
Heterogenita biologických membrán vedla ke vzniku širokého spektra zjednodušených modelových systémů, jejichž uspořádání, velikost a tvar se dají přizpůsobovat různým aktuálním požadavkům. Existují dva rozdílné přístupy k vytváření umělých fosfolipidových membrán. První z nich je založen na vzniku membrán ve vodném prostředí. Do této skupiny patří černé lipidické membrány, dvojvrstvy na pevném podkladu, dvojvrstvy vzniklé z monovrstev na rozhraní vody a vzduchu a liposomy. Ve druhém případě vznikají dvojvrstevné membrány v množství organické fáze metodou dvojvrstev na kapičkovém rozhraní. Každý typ umělých membrán má své experimentální výhody a nevýhody, což se používá ke studiu různých problémů sahajících od chování jednotlivých fosfolipidů a proteinů až po fúze membrán. Umělé fosfolipidové membrány jsou vhodným nástrojem pro elektrickou charakterizaci dvojvrstev a nebo membránových proteinů. Tato práce je ucelený přehled nejpoužívanějších metod vhodných pro vznik umělých fosfolipidových membrán. Klíčová slova: membrána, lipid, fosfolipidová dvojvrstva, liposom, černá lipidová membrána, dvojvrstvy na pevném podkladu, dvojvrstva na kapičkovém rozhraní Abstract The heterogeneity of biological membranes has led to development of a wide spectrum of simplified model systems whose composition, size and...
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Functional analysis of Spr1057 protein in Streptococcus pneumoniae
Stehlíková, Zuzana ; Branny, Pavel (advisor) ; Fišer, Radovan (referee)
Functional analysis of Spr1057 protein Streptococcus pneumoniae The genome of important human pathogen Streptococcus pneumoniae encodes a single gene of an eukaryotic type serine/threonine protein kinase StkP. Analysis of the global transcriptome of a mutant strain with inactivated stkP gene identified spr1057 gene whose expression was significantly repressed in ∆stkP strain. This gene is coding for Spr1057 protein which is a member of haloacid dehalogenase family. The analysis of the substrate specifity of the Spr1057 protein confirmed nucleotidase activity of this protein in vitro. To study the function of this protein in vivo we prepared several mutant S. pneumoniae strains. Growth characterictics of mutant strains were observed in the presence of modified nucleotides, 5-fluoro-2'-deoxyuridine (5-FdU) and 5-bromo-2'-deoxyuridine (5-BrdU). In addition, we monitored the rate of incorporation of 5-BrdU into the chromosomal DNA of the mutant strains in comparison with the wild type S. pneumoniae strain. The growth of the Δspr1057 strain was significantly inhibited in the presence of the modified nucleotides and increased incorporation of 5-BrdU in DNA was showed. Neither growth inhibition nor incorporation of 5-BrdU in DNA was observed for the wild type strain. The expression of an ectopic copy of spr1057...
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The substrate specificity of adenylation domains of synthetases in secondary methabolism.
Vobruba, Šimon ; Janata, Jiří (advisor) ; Fišer, Radovan (referee)
The crucial part of the biosynthesis of lincosamide antibiotics lincomycin and celesticetin is the condensation of amino sugar and amino acid moieties. This reaction is catalysed by the oligomeric enzyme lincosamide synthetase (LS). One of the most important components of LS is adenylation domain recognizing and activating amino acid precursor. The substrate specificity of adenylation domain is determined by "nonribosomal code", 10 amino acids residues which side chains are in close contact with the activated substrate. The homologous adenylation domains LmbC from biosynthesis of lincomycin and CcbC from biosynthesis of celesticetin exhibit strong substrate specificity for their natural substrates (2S,4R)-4-propyl-L-proline (PPL) and L-proline, respectively. At first the effect of selected amino acid residues of LmbC nonribosomal code on the substrate specificity of the whole domain was tested. The amino acids residues, most important for preference of PPL substrate over L proline, were determined: G308, A207 and L246. Then the effect of double mutations in nonribosomal codes of both LmbC and CcbC on their substrate specificity was evaluated. The double mutants LmbC G308V + A207F and CcbC V306G + F205A were prepared and tested biochemically. The results brought new evidence of validity of homologous models...
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