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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 monitoring of intracellular ion concentrations in microbial cells
Vodáková, Adéla ; Plášek, Jaromír (advisor) ; Krůšek, Jan (referee)
The Master Thesis focuses on monitoring of intracellular ion concentrations in bacteria Escherichia coli and yeast Saccharomyces cerevisiae using genetically encoded fluorescent probes with green fluorescent protein (GFP). Aquired knowledge about this protein and its spectral characteristics is summarized in the introduction. For experimental study a pH-sensitive sensor which displays a ratio change of two excitation fluorescence peaks - pHluorin - was chosen. This probe was tested in bacteria and yeast cells. The experiments concentrated on the ability of the cell to maintain a constant cytosolic pH under various conditions like different pH values of the suspension, addition of glucose or KCl to the suspension. Another topic discussed in the thesis is the elimination of the cell autofluorescence from the GFP signal. For this purpose the synchronous fluorescence scan technique was succesfully used. I have found out that by using this method the measurements of cytosolic pH values are even more accurate thanks to the improved signal to noise ratio.
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Bioapplications of novel nanostructured materials
Fučíková, Anna ; Valenta, Jan (advisor) ; Polívka, Tomáš (referee) ; Plášek, Jaromír (referee)
Title: Bioapplications of novel nanostructured materials Author: Anna Fučíková Department / Institute: Department of Chemical Physics and Optics, Faculty of Mathematics and Physics, Charles University in Prague Supervisor of the doctoral thesis: Doc. RNDr. Jan Valenta, Ph.D., Department of Chemical Physics and Optics, Faculty of Mathematics and Physics, Charles University in Prague Abstract: This work is aimed at development of new fluorescent labels based on silicon nanocrystals. Nanodiamonds and commercial CdSe quantum dots have been used as comparative materials. Silicon nanocrystals are relatively small (1-4 nm) compared to other studied nanomaterials. They are prepared by electro-chemical etching and their surface can be activated by various molecules which strongly influences luminescence properties. Luminescence quantum efficiency can be as high as 30 % and perfectly photostable even in biological environment. Si nanocrystals are biodegradable in a living organism within reasonable time scale and non-toxic. We are able to detect luminescence of single nanocrystals, even inside living cells, with use of our micro-spectroscopy apparatus. Nanodiamonds have weak luminescence; they are toxic at higher dosages and very stable in living bodies (without available technique how to remove them). Studied CdSe...
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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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Fluorescence spectroscopy of calcofluor stained yeast cell suspensions.
Dostál, Marek ; Plášek, Jaromír (advisor) ; Heřman, Petr (referee)
Title: Fluorescence spectroscopy of calcofluor stained yeast cell suspensions Author: Marek Dostál Department: Fyzikální ústav UK Supervisor: prof. RNDr. Jaromír Plášek CSc. Abstract: We were finding and we found fast and easy fluorescence spectroscopy method which we can use for cell wall research of different yeast types that were cultivated on various medium. Gauging was under way in solution of different ionic forces. The growth medium has the greatest effect on quality of cell wall. We use calcofluor as fluorescent colour. After exposing calcofluor by UV (λex ≈ 365 nm) calcofluor emites blue fluorescence with maximum intensity around 420 nm in dependence on suspension concentration. We must be careful about inner filter that can influence our result during research. We validated diversity of cell wall structures in various yeast types by ratio spectral method (spectral fingerprint). Keywords: Fluorescence, spectroscopy, cell wall, calcofluor, inner filter.
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Atomic Force Microscopy (AFM) used to reveal differences in the cell wall structure of of yound and old S. cerevisiae yeast cells.
Schwarzová, Tereza ; Plášek, Jaromír (advisor) ; Shukurov, Andrey (referee)
The main goal of this MSc. is to use Atomic Force Microscopy (AFM) in studies of yeast cell wall structure aimed at (i) expected differences between young and aged S. cerevisiae cells, (ii) the effects of glucose concentration in culthuring media on the quality of yeast cell wall. AFM can image biological systems with molecular resolution in real time, and under natural condition. Numerous AFM images of the surface of S. cerevisiae yeast cells (strains AD 1-3,IL,US and JC 482A, AD-JC 488A) have been obtained, which surpassed that one of standard results of still-rare AFM studies of yeast cells. No clear effect of glucose concentration has been found. On the other hand, AFM studies have revealed ageing-related changes in the structure of yeast cells (crumpling, rough surface). Highresolution images of yeast bud scars, including their fine details were also obtained.
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Measurement of the membrane potential using ion-selective electrode sensitive to tetraphenylphosphonium
Lábajová, Anna ; Kofránek, Jiří (advisor) ; Sigler, Karel (referee) ; Plášek, Jaromír (referee)
103 ABSTRACT The computerized device for membrane potential measurement using the tetraphenylphosphonium-selective electrode was constructed. Signal acquisition, processing and data storage were realized by MATLAB/Simulink software. The selective membrane was optimized incorporating TPP+ TPB- precipitate into the membrane. The electrode had a Nernstian response from 3.10-6 M TPP+ . The TPP+ TPB- precipitate was later replaced by sodium tetrakis[3,5-bis(1,1,1,3,3,3-hexafluoro-2- methoxy-2-propyl)phenyl]borate (NaHFPB). The electrode with incorporated NaHFPB had a Nernstian response from 1.10-6 M TPP+ and had better sensitivity than commercially available electrodes. The values of selectivity coefficients for K+ , Na+ , Ca2+ and Mg2+ were calculated. The device was used for mitochondrial membrane potential measurement of isolated mitochondria and for evaluating the respiratory chain function of digitonin- permeabilized cells (hepatocytes, HeLa G, BSC-40 and control transmitochondrial cybrids). This method was used also for monitoring the mitochondrial permeability transition pore (MPTP) function of isolated mitochondria and permeabilized hepatocytes. MPTP opening was induced by high calcium concentration and the action of calcium was enhanced by pro-oxidant tert-butyl hydroperoxide (t-BHP). This...
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