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Lipid Membranes at the Nanoscale: Single-Molecule Fluorescence Approach
Koukalová, Alena ; Černý, Jan (advisor) ; Malínský, Jan (referee) ; Benda, Aleš (referee)
The complexity of cell membranes is far from being only a simple assembly of lipids and proteins separating cells from the surrounding environment. Each of the thousands of different membrane components performs its specific role in cellular functions, since a multitude of biological processes is mediated by membranes. The understanding of the molecular basis of these processes is one of the important aims of current biological research. Our research employing single- molecule fluorescence methods (e.g. FCS, FCCS, FLIM-FRET) has made a contribution to the knowledge of membrane lateral organization or mechanism of membrane fusion. Furthermore, we revealed the mechanism of membrane activity of a small natural compound. As native cell membranes are very complex structures, we performed the experiments on simplified model lipid membranes that allow studying lipid-lipid or lipid-protein interactions at the molecular level in a controlled way. The first part of this thesis deals with the mode of action of a membrane active secondary metabolite didehydroroflamycoin (DDHR). We demonstrated that DDHR is a pore-forming agent and that this activity is influenced by the presence of cholesterol. Direct visualization of intrinsic fluorescence of DDHR revealed its preferential partitioning into membrane areas...
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New Possibilities in Fluorescence Correlation Spectroscopy
Benda, Aleš ; Hof, Martin (advisor) ; Hozák, Pavel (referee) ; Valenta, Jan (referee) ; Vácha, František (referee)
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Biological imaging by super-resolution microscopy
Adamová, Zuzana ; Komrsková, Kateřina (advisor) ; Benda, Aleš (referee)
zuzana_adamova_abstract_eng.txt[11.05.2017 20:26:29] Fluorescence microscopy is one of the most widely used imaging techniques in biological research. Despite its numerous advantages, it can be used only for studies of structures larger than 200 nanometres, due to diffraction limit caused by a wave nature of light. The value of 200 nanometres is the best reachable value of optical resolution, in other words, the smallest distance of two objects, which can be separately recognized by conventional optical systems. Up to the end of the 20th century it was therefore impossible to observe finer details of cells. However, recently several breakthrough imaging techniques, named super-resolution microscopy techniques, managed to bypass the diffraction limit and enabled biologists to study much more delicate structures, such as small organelles, virions, protein complexes or even particular proteins, while still using a visual light. This thesis introduces some selected super-resolution methods, explains briefly their principles and presents some of their applications in biology.
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Membrane interactions studied by advanced fluorescent techniques: From ions to macromolecules
Pokorná, Šárka ; Hudeček, Jiří (advisor) ; Konopásek, Ivo (referee) ; Benda, Aleš (referee)
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...
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Dynamic saturation optical microscopy using photoswitchable proteins
Kolářová, Marie ; Benda, Aleš (advisor) ; Obšil, Tomáš (referee)
Fluorescence microscopy is an essential technique for live cell imaging. One of its drawbacks is a rather low diffraction limited spatial resolution, which is described by Abbe diffraction law. Therefore, in the last decade a lot of new methods improving spatial resolution were developed. One of them is dynamic saturation optical microscopy (DSOM) that is based on spatial monitoring of reversible transition kinetics between bright and dark states of fluorophores. The dark state is possible to obtain for example by using reversibly photoswitchable fluorescent proteins such as Dronpa and its variants. These proteins undergo reversible transition from fluorescent to nonfluorescent state after irradiation by blue and ultraviolet light. In my work I focus on employing the kinetics of controllable photoswitching of Dronpa in improving the overall image quality, including the spatial resolution. The experiments were performed on yeasts expressing selected proteins labelled with Dronpa. Firstly, photoswitching behaviour of Dronpa was confirmed. Secondly, experimental conditions were optimized by studying dependence of switching rate on laser intensities and on excitation wavelength and by studying protein photostability. Experiments were performed on different timescales and for various proteins. Using the optimal...
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Piston for 110kW 4-stroke SI-engine
Benda, Aleš ; Dundálek, Radim (referee) ; Svída, David (advisor)
In this bachelor, graduation theses we will follow up the process of combustion engine basic proposal, piston size proposal, strength control of a dangerous cross-sections. Its part is also a summary of combustion engines development and detailed description of piston group components.
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