Viral vectors derived from mouse polyomavirus are a convenient tool for studying the targeted delivery of therapeutical agents into the cells and cellular organelles. Vectors derived from mouse polyomavirus face difficulties similar to other nanoparticles, as they often end up trapped inside an endosome where they are subsequently degraded. This diploma explored the potential of vector modifications, which have the potential to make the transport to the nucleus or cytosol more effective. This work had particularly focused on increasing the transduction efficiency by modifying particle's internally localized VP3 capsid protein with covalently bound membrane-penetrating peptides. Primary covalent genetic modification to the VP3 protein was the polyhistidine peptide KH27K. Its potential of improving the transduction effectivity was compared with two other peptide modifications - LAH4 and R8. The results of the transduction test showed that covalently bound R8 peptide had many-fold improved the transport to the nucleus when compared to the unmodified particles. The modification with LAH4 peptide had been regarded more effective only when was associated with the particles non-covalently. In such scenario the transduction efficiency rose 40-times when compared with unmodified particles. Polyhistidine...

This work was focused on the preparation of model stabilized phospholipid membranes formed on porous polycarbonate carrier. 1,2-dipalmitoyl-sn-glycero-3-phosphocholin was used for their formation in hydrophilic pores of polycarbonate carrier. For characterization of the formation of phospholipid layers, their changes and a study of transport processes, electrochemical impedance spectroscopy and voltammetry were used. Transport of cadmium and copper ions was studied in the presence and in the absence of ionophore calcimycin which was incorporated into the formed of phospholipid membrane. Because these ions are often bound in complexes with various substances, such as low molecular weight organic acids (LMWOAs), this work was also focused on the transport of copper and cadmium ions across the model phospholipid membranes in the presence of malic acid, citric acid and oxalic acid at different pH. Besides the use of ionophore, some pilot experiments were performed to realize the transfer of copper ions using two peptides, nisin and transportan 10. Formation of phospholipid membranes and the transport processes were characterized by two proposed electric equivalent circuits which correspond to the covered and to the uncovered polycarbobate carrier. Keywords: Phospholipids, Membranes, Ionophore, Peptid....

1 Abstract Adenylate cyclase toxin (CyaA) is one of the major virulence factors of bacterium Bordetella pertussis, which is a causative agent of whooping cough. CyaA belongs to the family of RTX toxin-hemolysins. The toxin targets primarily cells expressing integrin receptor CD11b/CD18 but it can also penetrate cells lacking this receptor. CyaA acts on host cells by two independent activities. One is formation of small cation-selective channels, which can lead to colloid osmotic lysis of target cells. The second is disruption of cell signaling through the translocation of the adenylate cyclase (AC) domain to host cell cytosol, which leads to the conversion of ATP into cyclic AMP. It was recently shown that cholesterol affects endocytosis of CyaA. CyaA translocates it's AC domain after relocation of CyaA molecule to the cholesterol-rich lipid raft (Bumba et al. 2010). In this work I examined the effect of cholesterol on channel- forming activity and selectivity of ion channels created by CyaA. For measurements I used artificial membranes enriched with cholesterol. CyaA channels are voltage-dependent. The positive membrane potential on the side of toxin is rquired for incorporation of CyaA molecule into cell membrane. I tried to find out whether the value of voltage has effect on channels opening time....

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...

The adenylate cyclase toxin-hemolysin (CyaA) plays a key role in virulence of the whooping cough agent Bordetella pertussis. It translocates an AC enzyme into cytosol of CD11b+ phagocytes and subverts their bactericidal functions by unregulated conversion of ATP to cAMP. In parallel, CyaA permeabilizes cellular membrane by forming cation-selective pores. The goal of my diploma thesis was an analysis of the mechanism of interaction of the segment linking the invasive adenylate cyclase domain and the RTX hemolysin moiety of CyaA with target membrane. Our data show that the segment linking the AC to the hydrophobic domain of CyaA is directly involved in the interaction of the toxin with the membrane and controls the formation of small cationt-selective pores. Our results generate new knowledge that will be of relevance to the entire field of toxin biology and will enable the design of improved CyaA- based vaccines. Keywords: Bordetella pertussis, adenylate cyclase toxin, membrane translocation, pore- forming activity, black lipid bilayers, liposomes

This diploma thesis presents reader with current knowledge in the field of gray wastewater reuse. The theoretical part describes the current state of water in the world, thus characterizes wastewater and describes its treatment. Further, this section describes the possibility of obtaining thermal energy from wastewater. At the end of the theoretical part, the reader is presented with some existing buildings in the Czech Republic that use the wastewater re-use technology system. In the following practical part, all the knowledge from the theoretical part are applied for a concrete project of construction of a graywater re-use technology system in the area of Vlněna in Brno city.

Advanced fluorescence techniques were used to explore tree distinct topics concerning biological membrane and their interactions. Following thesis is according to the topic divided into three parts: 1) Ionic effects were studied employing time dependent fluorescence shift experiments and molecular dynamic simulations. Combination of these two approaches are suitable to reveal characteristic like mobility and hydration of particular bilayer segment, lipid packing or ion binding sites. Halide anions were reported to adsorb to the cationic lipid bilayer specifically, altering membrane mobility and organization. Changes in observed parameters follows Hofmeister order. Their effect is mediated either by direct ionic interaction (soft, polarizable ions) as well as via alteration of water structure (hard, non-polarizable ions) in proximity of ion molecule. Further, divalent calcium was shown to bind strongly to neutral and negatively charged lipid bilayers. Several types of binding sites depending on calcium concentration were identified. 2) Two complementary lipopeptides, CPK and CPE, incorporated into distinct lipid bilayers serve as a minimal model inducing membrane fusion. Effectiveness of fusion event might be influenced by lipopeptide-membrane and lipopeptide-lipopeptide interaction. To reveal...

Viral particles derived from mouse polyomavirus can be potentially used as a delivery system for therapeutic genes and drugs into target cells. This thesis focuses on preparation and characterization of polyomaviral particles that are modified with cell-penetrating peptides in order to increase efficiency of transduction of reporter genes into human cells. Viral particles that are composed of major capsid protein VP1 in combination with minor capsid protein VP2 and minor capsid protein VP3 that is modified with octaarginine, LAH4 peptide or with transduction domain of adenoviral protein VI are analysed in transduction assays. The thesis also provides information about the effect of the modification on encapsidation of heterologous DNA. The results of transduction assays performed with modified particles containing encapsidated luciferase gene revealed that efficiency of transduction did not increase but decreased in comparison with unmodified particles. These findings help to elucidate the role of polyomaviral minor capsid proteins in gene transfer mediated by viral particles and contribute to the design of new strategies for modifications of viral particles derived from mouse polyomavirus for their successful application in nanomedicine. Key words: mouse polyomavirus, pseudovirions, virus-like...

The diploma thesis deals with the topic of lipids and it is focused on the processing in the form of educational materials (educational text and educational animations) for chemistry and biology on secondary school. It is devided into two parts. The theoretical part defines the key concepts serving as theoretical background: interdisciplinarity, visualization, animation. There is also performed the analysis of RVP G and the representation of the topic of the diploma thesis in this curricular document. The final chapter of the theoretical part is dedicated to determing the criteria for the analysis of the educational materials and the characteristics of the animation program Adobe Animate CC. The analysis of available educational materials is performed according to predetermined criteria in the practical part, specifically animations available on the Internet. There are also introduced the educational materials so as the complex file of educational animations created in the program Adobe Animate CC and the educational text that serves as a guiding material to educational animation program. The materials include methodological guide for users. The created materials focus on the topic of the lipids and biological membranes, they emphasize interdisciplinarity of chemistry and biology and also connect...

In this diploma thesis the preparation of ceramic catalytic materials was studied. They were studied by means of catalytic activity on powder samples and in a form of membrane as well. At first were multicomponent perovskite materials with the help of “glycine-nitride synthesis” synthesized. Two types of perovskite systems were prepared. First system was on the basis of nickelates LaNiO3 and the second one on the basis of cobaltites SmCoO3. Both of them were doped with aluminium and calcium. Ceramic powders were catalytic tested during reformed reaction. A part of powders was pressed with hydraulic biaxial press. Then the membranes were calcinated, sintered and polished. The membranes were tested to specify the catalytic activity. At first they were in hydrogen atmosphere reduced and afterwards came through the partial oxidation. The appearance of coke fibers on the surface was discussed.