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Determination of vibality of rhizobacterial cultures
Svobodová, Lucie ; Slaninová, Eva (referee) ; Obruča, Stanislav (advisor)
This thesis focuses on the study of viability of plant growth promoting rhizobacteria (PGPR). Viability was determined in three strains of Azotobacter vinelandii, namely CCM 289, DSM 87 and DSM 720, using flow cytometry with fluorescent probe PI, SYTOXTM Blue and DAPI after 120 and 72 hours of cultivation. Optimization of the appropriate fluorescent probe for the strain was performed, with the PI probe for strain CCM 289 being the most suitable. PI and SYTOXTM Blue probes can be used for strains DSM 87 and DSM 720. For the following experiments, strain DSM 87 was selected and subjected to the influence of different crosslinking reagents. Using a flow cytometer and staining with a fluorescent PI probe, the viability was verified after application of calcium chloride, barium, copper, ferric, aluminium and calcium sulphate solutions of 2, 0.2 and 0.02 wt. % to the culture. Calcium chloride, barium and calcium sulfate solutions had no significant effect on cell viability. On the other hand, when ferric chloride was used, a trend was observed where dead cells decreased with decreasing concentration of the solutions. This effect was also achieved with aluminium and copper chloride, but the use of the most concentrated solution resulted in the inactivation of a greater number of cells than in the previous case, whereas aluminium chloride resulted in the loss of viability of most of the cells present. Viability was also verified for cells released from the prepared gels. For the experiments, solutions of the aforementioned crosslinking agents were chosen at a concentration of 2 wt.%, and the culture was subjected to gelation under the experimentally determined conditions. A portion of the gels was subsequently left in phosphate buffer to allow for the re-release of cells. To facilitate this release, the enzyme alginase was added to break down the alginate. It was found that a concentration of 2 wt % of the selected crosslinking agents did not affect cell viability, i.e., the cells released from the gel appeared to be viable.
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Anisotropy techniques in study of cytoplasm
Sýkorová, Kateřina ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
The main goal of this thesis was to compare experiments using time-resolved anisotropy and steady-state anisotropy for measuring in bacteria strain Cupriavidus necator. Fluorescent probe for anisotropy imaging was chosen BCECF_AM, which is derivate of fluorescein. Using experiment in system glycerol/water with fluorescein, anisotropy has been verified and calculated molecular hydrodynamic volume of a single fluorescein molecule, which approximately corresponded with real value. By using fluorescence imaging anisotropy microscopy, images and values of average anisotropy in cells were taken. Images of living cells (bacteria) of CN H16 and mutant CN PHB-4 showed differences, mainly in the uniformity of the inside environment.
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Support of physical interaction between hyaluronan and selected hydrophobic solutes
Michalicová, Petra ; Kislinger, Jiří (referee) ; Pekař, Miloslav (advisor)
The method of fluorescence spectroscopy was applied for the studying of the way of the hyaluronan-fluorescence probe system`s preparation on mutual interaction in water. Several experiments with fluorescence probes pyrene, prodan, perylene and diphenylhexatriene (DPH) had been submitted. The first part of the experiments deals with the verification of the results of T. Brown`s work and it was focused on the study of simple interaction between hyaluronan and hydrophobic compounds. In the second part of the work hyaluronan was dried at higher temperature followed by adding of the fluorescence probe. The aim of this method was the distraction of hyaluronan`s moisture packaging and the opening up the hydrophobic parts of the chain for the fluorophore. Although wide concentration ranges of fluorescence probes had been tested in the first experiments, the interactions hadn`t been observed. The similar results were obtained in the second part of the experiments.
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Phospholipids as the basis of biodegradable delivery systems
Burdíková, Jana ; Čeppan, Michal (referee) ; Mravec, Filip (advisor)
This thesis is focused on investigation of phospholipid-hyaluronan system. First, appropriate method for preparation of bulk solution of phospholipid/lipid and suitable fluorescence probe were chosen. Sonification was selected as a method for preparation of bulk solution and pyrene was chosen as a fluorescence probe. From the group of phospholipids lecithin was selected. Next to phospholipid, lipid with no phosphate group (DPTAP) was utilized for comparison, alternatively a mixture of lipid (DPTAP) and phospholipid (DPPC). Instead of hyaluronan another polyelectrolytes (sodium polystyrene sulfonate, sodium alginate) were used too. Measurements were performed in water environment and in phosphate buffer saline (PBS). All investigation was accomplished by fluorescence spectroscopy and dynamic light scattering.
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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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Fluorescence study of hydrophilic domains of associating colloids
Londinová, Monika ; Knotková,, Kateřina (referee) ; Pekař, Miloslav (advisor)
The properties of the hyaluronan were investigated by using different fluorescence probes, because hyaluronan is a hopeful carrier of an active matter in medicine and cosmetics. Selected fluorescence probes were: cationic acridine orange, Nile Blue A, methylene blue, amphiphilic 4-Di-2-ASP and anionic fluorescein. Except from fluorescence and absorption spectra of the probes were observed electrostatic and hydrophobic interactions as well. The probes in solvents with different polarity (MeOH, EtOH, DMSO) showed the bathochromic shift in the emission maximum and quenching of the fluorescence with the increasing polarity of the solvents. The influence of the ionic strength on fluorescence properties of the probe acridine orange and 4-Di-2-ASP was investigated in aqueous solutions of chlorides. The formation of acridine orange dimer is inhibited with increasing ionic strength. CaCl2 increased the ionic strength the most, then prevented repulsion of carboxylate groups, so it means the expansion of hyaluronan cluster into the solution. However, the emission of the probe 4-Di-2-ASP was quenched with the addition of CaCl2 the most. The first additions of COO– groups cause the formation of dimers of AO shown as decreasing in extinction coefficient and fluorescence intensity. Next addition of the hyaluronan caused a depolymerization of formed dimers and the increase of the emission intensity. The repolymerization caused the decrease and then again the increase. In case of 4-Di-2-ASP was the pattern of the fluorescence (the intensity and the position of the emission) firstly the same, but at the concentration of 1 g dm-3 the emission intensity increased. The probes MB and F were used for spectroscopic studies of the interaction between methylene blue-fluorescein complex and anionic and cationic surfactants. The absorbance of separate MB and F changed only with the addition of surfactants with the opposite electric charge. Absorbance of the mixture MB-F changed with the addition of the CTAC surfactant, while the addition of SDS into the mixture caused only the change of MB absorption spectra.
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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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Study of interaction between hyaluronan and phospholipid in a presence of biocompatible non-ionic surfactant
Burdíková, Jana ; Klučáková, Martina (referee) ; Mravec, Filip (advisor)
This work is based on investigation of aggregation behavior of sugar surfactants and sn-glycero-dipalmitoylphosphatidylcholin (DPPC) in aqueous environment and on study of the interactions in nonionic surfactant-DPPC and DPPC-nonionic surfactant-hyaluronan systems. Sugar surfactants were used from non-ionic surfactants. The behavior of each systems was studied by fluorescence spectroscopy. Values of critical micellar concentrations (CMC) of sugar surfactants, begining of aggregation of DPPC, DPPC aggregation effect of sugar surfactants and the effect of hyaluronan on the system non-ionic surfactant-DPPC has been investigated. Also CMC of the CPC (cetylpyrimidium chloride), which was used for fluorescence quenching in the determination of aggregate numbers of sugar surfactants, has been determined. Fluorescent probes pyrene, perylene, nile red, acridine orange and hydrophobic dye sudan red were used for measurements.
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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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