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Hyaluronane interactions with hydrophobic solutes
Slezáková, Dagmar ; Knotková,, Kateřina (referee) ; Pekař, Miloslav (advisor)
The diploma thesis is based on the study of hydrophobic interactions of the native hyaluronan with selected solutes. On the basis of a literature search were chosen fluorescent probes and fluorescing biologically active substances, which are useful for investigation of colloids as 6-(p-toluidino)-2-naphthalenesulfonic acid (polarity probe), lipophilic vitamin (±)-alpha-tocopherol, pyrene (polarity probe) and finally hydrophilic vitamin riboflavin. In the experimental part of this thesis was studied the influence of solvents with different polarities, or more precisely dielectric constant, on the emission spectra, as well. There were investigated interactions of native hyaluronan with TNS and then interactions, which were influenced by the ionic strength. Such influenced interactions were not observed, that was probably due to the strong solvation´s wrapping of the hyaluronan. Interactions were observed after the process of lyophilisation followed-up by the rehydratation of the samples. For the next study of interactions the riboflavin was chosen and was investigated the REES effect in the native hyaluronan in different concentrations of its different molecular weights. In this case were not observed any shifts in the emission maximum with the excitation wavelenght shift and that is why the interactions of hyaluronan with riboflavin were not demonstrated in the field of chosen concentrations. By using another probe alpha-tocopherol was investigated the associative behaviour of hyaluronan and moreover was observed anisotropy of alpha-tocopherol in different concentrations of different molecular weights of native hyaluronan. The anisotropy reached high values in contrast to the reference solute that was the mixture of glycerol and ethanol. The anisotropy depended more on the molecular weight than on the concentration of hyaluronan. Interactions of hyaluronan were also studied by using the polarity probe pyrene in different concentrations of different molecular weights of the hyaluronan. The pyrene 1:3 ratio did not show the concentration dependence within the chosen concentrations except for the molecular weight 253.9 kg mol–1. Both probes alpha-tocopherol and pyrene were performed by the process of lyophilisation followed-up by the rehydratation, which improved interactions of these probes with hyaluronan.
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Biological role of PHA cycle in bacterium Rhodospirillum rubrum
Šarköziová, Patrícia ; Obruča, Stanislav (referee) ; Slaninová, Eva (advisor)
The diploma thesis deals with the study of polyhydroxyalkanoates (PHAs) production and their protective effects during exposure of the bacterial strain Rhodospirillum rubrum to various stress factors. The theoretical part of this work is focused on the characterization of PHAs and used bacterial strain. In the first part of the experimental work carbon substrates for biomass growth and PHA production in different time intervals (24, 48, 72 and 96 hours) were investigated. Acetate, fructose, hexanoate and malate served as a carbon source. The second part of this work was focused on the selection of a suitable fluorescent probe for the bacterium Rhodospirillum rubrum (propidium iodide, SYTOXTM, fluorescein diacetate), which was used to determine the viability of bacterial cells using flow cytometry. This method was used to determine the viability of bacterial cells after their exposure to various stress factors (thermal stress, freezing and thawing, ethanol stress, osmotic stress, peroxide stress, pH stress and UV stress). Finally, the growth curve of bacteria Rhodospirillum rubrum was determined by measurement of the optical density.
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Fluorescence probes for immobilized photocatalyst activity monitoring
Blašková, Martina ; Dvoranová, Dana (referee) ; Dzik, Petr (advisor)
This diploma thesis deals with the use of fluorescent probes for evaluation of photocatalytic activity of immobilized photocatalyst. To the evaluation of photocatalytic activity of TiO2 were used three different fluorescent probes – terephthalic acid, coumarin and benzoic acid, wherein was monitored the increasing intensity of fluorescence of their oxidation products – hydroxyterephthalic acid, 7-hydroxycoumarin and salicylic acid for the photochemical degradation of various fluorescent probes. To the evaluation of photocatalytic activity was used solid phase (photocatalyst) – liquid phase (probe) system and was used three sources of radiation. Fluorescence of oxidation products was monitored by the fiber spectrometer and a conventional cuvette fluorometer.
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Fluorescence spectroscopy in the research of aminoclays
Jančík Procházková, Anna ; Pekař, Miloslav (referee) ; Mravec, Filip (advisor)
Aminoclay belongs to a large group of organic modified materials based on clays. These materials could find many potential practical applications and, in particular, may be used as a matrix with the ability of the organisation and stabilisation of incorporated substances. The utilisation of aminoclay in the field of medical applications seems highly prospective, but there is the need to visualise the clay system and the processes within it. This essay deals with the issue of aminoclay visualisation by means of fluorescent spectroscopy. Through fluorescent spectroscopy, aminoclay was discovered to be autofluorescent. The autofluorescence of aminoclay is not usable in practical applications because of the low fluorescence intensity. This is the reason for the research into other possibilities for modifications of the internal structure of aminoclay by europium ions. These ions in chelated form are able to be fluorescent and modifications of the external structure are possible by electrostatic implementation of fluorescent dyes. Characterisation of the prepared complexes was provided by spectrofluorimetry and fluorescent microscopy for the purpose of evaluation of the practical application of aminoclay.
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Biological role of PHA cycle in bacterium Rhodospirillum rubrum
Šarköziová, Patrícia ; Obruča, Stanislav (referee) ; Slaninová, Eva (advisor)
The diploma thesis deals with the study of polyhydroxyalkanoates (PHAs) production and their protective effects during exposure of the bacterial strain Rhodospirillum rubrum to various stress factors. The theoretical part of this work is focused on the characterization of PHAs and used bacterial strain. In the first part of the experimental work carbon substrates for biomass growth and PHA production in different time intervals (24, 48, 72 and 96 hours) were investigated. Acetate, fructose, hexanoate and malate served as a carbon source. The second part of this work was focused on the selection of a suitable fluorescent probe for the bacterium Rhodospirillum rubrum (propidium iodide, SYTOXTM, fluorescein diacetate), which was used to determine the viability of bacterial cells using flow cytometry. This method was used to determine the viability of bacterial cells after their exposure to various stress factors (thermal stress, freezing and thawing, ethanol stress, osmotic stress, peroxide stress, pH stress and UV stress). Finally, the growth curve of bacteria Rhodospirillum rubrum was determined by measurement of the optical density.
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Molecules in Cell Membranes
Timr, Štěpán ; Jungwirth, Pavel (advisor) ; Böckman, Rainer (referee) ; Ettrich, Rüdiger (referee)
Biological membranes are actively involved in a multitude of processes in living cells; therefore, a detailed characterization of their structure, dynamics, and function is essential for an understanding of living organisms at the molecular level. In this work, we made use of the high spatial and temporal resolution offered by computer simulations to investigate the behavior of several molecular species which associate with cellular membranes. Using a combination of classical molecular dynamics simulations and ab initio electronic structure calculations, we were able to characterize nonlinear optical properties of membrane- embedded fluorescent probes and thus contribute to establishing two-photon polarization microscopy as a tool of structural biology. Moreover, our molecular dynamics simulations provided an atomistic picture of the reversible membrane binding of recoverin, a neuronal calcium-sensing protein involved in vision adaptation, and they also yielded an important insight into the mechanism of its calcium-induced myristoyl switch. In addition, we examined the biological role of cholesterol oxidation and compared two methods of representing transmembrane voltage in molecular dynamics simulations.
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The comparison of the performace of selected carbocyanine dyes in fluorescent probing of yeast cell membrane potential.
Mudroňová, Kateřina ; Plášek, Jaromír (advisor) ; Krůšek, Jan (referee)
The membrane potential is one of the most important parameters of the living cell. It can be measured using carbocyanine fluorescent probes. In this thesis we examined parameters of several dyes of this family. For further experiments three of them were chosen - diOC3(3), diIC1(3) a diIC2(5) as a supplement to diSC3(3) and diSC3(5), which represent standard probes used at biophysical department of Institut of Physics. We compared the rates of their accumulation in S. cerevisiae cells to determine if they were MDR pumps' substrates. The other goal of this work was to decide whether the results obtained using different probes are equivalent and to determine if the presence of a probe affects the spectral characteristics of another. For this purpose we have chosen diSC3(3) and diSC3(5). With those dyes we examined the influence of the acidification on membrane potencial of the yeast S. cerevisiae. We showed that the information on depolarization obtained using both probes were matching very well.
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The comparison of the performace of carbocyanine dyes disC3(3) a diSC3(5) in fluorescent probing of yeast cell membrane potential.
Matunová, Petra ; Plášek, Jaromír (advisor) ; Krůšek, Jan (referee)
Membrane potential represents a voltage across a membrane and it is an important parameter that helps to describe processes in cells. Carbocyanine fluorescent probes diS-C3(3) and diS-C3(5), for which a common short chemical name 3,3'- dipropylthiadicarbocyanine iodide is used, are suitable for monitoring membrane potential changes of cells in which microelectrodes can not be used because of a small size of the cells. These changes can be measured on the scale of mV. A spectral analysis of cell suspensions containing a fluorescent probe makes it possible to determine the ratio of extracellular and intracellular concentrations of the probe. Using it we can calculate the value of membrane potential changes which can be induced by an outer stimulus. This Bc. thesis presents a comparison of the rate of accumulation of the above mentioned fluorescent probes in yeast cells, as well as experiments aimed for studying an inuence of different substances and their various concentrations on free and bound component of the dye.
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Investigation and inhibition of α-synuclein aggregation
Afitska, Kseniia ; Yushchenko, Dmytro (advisor) ; Žídek, Lukáš (referee) ; Bařinka, Cyril (referee)
α-Synuclein (AS) is a small intrinsically disordered protein expressed in neurons and abundantly present in synapses where it is involved in regulation of synaptic vesicle-mediated protein trafficking. Misfolding of AS into amyloid fibrils is a key process in progression of Parkinson's disease (PD), the second most common neurodegenerative disorder which has no cure to date. Inhibition of AS aggregation and blocking of cell-to-cell spreading of AS fibrils is a promising strategy for PD treatment. However, rational design of inhibitors of this type remains complicated due to the lack of thorough knowledge about the mechanisms of aggregation. Therefore, the aim of this thesis was to gain deeper knowledge about AS aggregation and to apply it for developing inhibitors of AS fibrillization. In my work on the mechanisms of AS aggregation, I first determined that the concentration of AS that enables the fibril growth is an order of magnitude lower than the concentration of AS required for initial fibril formation from monomers. I explored fibril disaggregation at AS concentrations below its Kd value, and characterized AS aggregation at low micromolar concentrations. I then investigated how different modifications of AS C-terminus (namely, extensions of various sizes and charges) affect fibril growth and...
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Molecules in Cell Membranes
Timr, Štěpán ; Jungwirth, Pavel (advisor) ; Böckman, Rainer (referee) ; Ettrich, Rüdiger (referee)
Biological membranes are actively involved in a multitude of processes in living cells; therefore, a detailed characterization of their structure, dynamics, and function is essential for an understanding of living organisms at the molecular level. In this work, we made use of the high spatial and temporal resolution offered by computer simulations to investigate the behavior of several molecular species which associate with cellular membranes. Using a combination of classical molecular dynamics simulations and ab initio electronic structure calculations, we were able to characterize nonlinear optical properties of membrane- embedded fluorescent probes and thus contribute to establishing two-photon polarization microscopy as a tool of structural biology. Moreover, our molecular dynamics simulations provided an atomistic picture of the reversible membrane binding of recoverin, a neuronal calcium-sensing protein involved in vision adaptation, and they also yielded an important insight into the mechanism of its calcium-induced myristoyl switch. In addition, we examined the biological role of cholesterol oxidation and compared two methods of representing transmembrane voltage in molecular dynamics simulations.
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