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Use of selected fluorescence techniques to characterization of microbial cells
Vaněk, Martin ; Pekař, Miloslav (referee) ; Márová, Ivana (advisor)
Carotenoids are one of the most abundant natural pigment. Furthermore, they have great potential for wide industrial utilization. Nowadays, prefered (and cheaper) synthetic method counters progressively improved biotechnological production. Far more cheaper production should be reached via implementing methods of on-line optimization, such as flow cytometry. In this thesis the dependence of yeasts autotofluorescence intensity on carotenoids concentration in dry mass was studied. The dependence was established by means of linear regression. Moreover, the dependence between light scatter and the amount of dry mass was established by means of model utilizing forward scatter, side scatter and number of cells in unit volume. Yeasts' cells was also studied under the lenses of laser scanning confocal microscope. The goal was to localize carotenoids inside cells. Experiments undertaken suggest that they are localized inside the lipidic granules, but more experiments are required for a proof.
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The Effect of Synovial Fluid Constituents on Friction and Lubrication of Articular Cartilage
Čípek, Pavel ; Dr. Prashant K. Sharma (referee) ; Muňoz, Anna Neus Igual (referee) ; Vrbka, Martin (advisor)
Práce se zabývá problematikou biotribologie modelu synoviálního kloubu. Cílem práce je popsat vliv složek synoviální kapaliny na formování mazacího filmu v modelu synoviálního kloubu. Práce je zaměřena na experimentální analýzu součinitele smykového tření a jeho propojení s pozorováním adsorbovaného mazacího filmu v kontaktu pomocí fluorescenční mikroskopie. Součástí práce je návrh tribometru, který umožňuje vizualizaci v průběhu experimentů. Tribometr je vyroben, kalibrován a verifikován pomocí komerčního tribometru. Dále je součástí práce návrh metodiky experimentů včetně návrhu postupu pro zpracování a hodnocení výsledků. Pro zpracování záznamu z vizualizace kontaktu byl navržen speciální software, který pracuje na principu segmentace obrazu. Poslední část práce je zaměřena na aplikaci vyvinuté metodologie spolu s nově navrženým tribometrem, což umožňuje posoudit roli složek modelové kapaliny u formování mazacího filmu v modelu synoviálního kloubu. Experimentální část je založena na měření tření za současné vizualizace kontaktu. Tato práce prezentuje originální výsledky rozšiřující oblast biotribologie synoviálního kloubu, které mohou pomoci dalšímu rozvoji účinné léčby pacientů s nemocnými klouby.
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Lubricant film formation in compliant contact
Jaroš, Tomáš ; Čermák, Jan (referee) ; Nečas, David (advisor)
This diploma thesis deals with the study of film formation in compliant contact. Primary attention is dedicated to the influence of the entrainment speed and slide-to-roll ratio (SRR) on the central film thickness. Based on a research study of the available experimental methods, the fluorescence microscopy was used to achieve the aim of the work. Experiments were realized in ball-on-disk configuration. Firstly, a validation of the method was performed where the solid contact of the ceramic ball and the glass disk was analyzed. Since the measured values were in a good agreement with the theoretical prediction, a ball of NBR rubber material, which is commonly used for production of lip seals and o-rings, was subsequently used. The experimental measurements were performed under pure rolling and rolling/sliding conditions when the entrainment speeds varied in the range from 10 to 400 mm / s. A constant load of 0.2 N was applied. The measured data were then compared to the theoretical predictions for compliant contacts. The results showed that the thickness of the lubricating film gradually increases with increasing entrainment speed, which corresponds to theoretical assumption. Against expectations, evident influence of SRR on the film formation has been observed. In the last part of the thesis the measured data are confronted with previously published studies. Finally, some limitations of this study are discussed and recommendations for further improvement of the used experimental method are suggested.
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Analysis of bacrerial cells employing flow cytometry and flurescence microscopy
Müllerová, Lucie ; Mravec, Filip (referee) ; Obruča, Stanislav (advisor)
This thesis focuses on fluorescent analysis of viability and PHA content in bacterial cultures, the main methods of investigation were flow cytometry and fluorescent microscopy. In order to determine viability of C. necator H16, several viability probes were tested, nevertheless, only BacLightTM kit and propidium iodide can be used to estimate portion of viable and live bacterial cell in samples. Further, Acridine orange was used to monitor physiological state of bacterial culture and two hydrophobic probes, Nile Red and BODIPY 493/503, were used to investigate PHA content in bacterial cells. Application of BODIPY 493/503 seems to be promising since this probe does not require permeabilization of bacteria cells and it can be used along with propidium iodide. Furthermore, several fluorophores were tested in the microscopic part. In was found that concentrations used in cytometric analyses were too high for microscopic use. Emission from the SYTO9 fluorophore is seen mainly in the green channel but because of the high concentration some emission was visible in the red channel. Cells stained with BODIPY 493/503 had very high fluorescence intensities when the stain concentration was 10 . At the same time, negative amplitudes of fluorescence were measured in both strains of C. necator, but in case of C. necator H16 that amplitude was much more pronounced. In this strain surprising high concentration of BODIPY stain was observed on the surface of PHB granules. Anisotropy of the fluorophore was nearing 0 which is very surprising.
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Cytotoxicity of selected naphthoquinones on prostatic cell cultures
Mondeková, Věra ; Gumulec, Jaromír (referee) ; Provazník, Ivo (advisor)
This master´s thesis discusses cytotoxicity of selected naphthoquinones on prostatic cell cultures. The introductory part is dedicated to general characteristic of naphthoquinones with focus on their cytotoxicity, testing of cytotoxicity and mechanisms of cytotoxicity. This part is followed by chapters about cytotoxicity, characteristics and biological activities of selected naphthoquinones; plumbagin and naphthazarin. The last part of this thesis’ theoretical section speaks about fluorescence microscopy and its use in research of naphthoquinones cytotoxicity. The practical part is dedicated to evaluation of cytotoxical tests’ results and to analysation of pictures of cells obtained by fluorescence microscope. At the end of thesis, all finding are summarized and put in the context.
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Intrinsic fluorescence of bacteria Cupriavidus necator
Marková, Kateřina ; Obruča, Stanislav (referee) ; Mravec, Filip (advisor)
This thesis focuses on autofluorescence of flavins in gram-negative bacteria Cupriavidus necator H16 and its mutant strain PHB-4. The main methods used were fluorescence microscopy and flow cytometry. To confirm the presence of flavins, excitation and emission spectra of the bacterial suspension were measured, which were compared with flavin standards. In the part of testing cells without stress response, the autofluorescence of bacteria in PBS buffer and cell suspensions stained with fluorescence probe BODIPY 493/503 was measured. The ratio of short fluorescence lifetime to long autofluorescence lifetime, and its dependence on fluorescence probe was compared with previous conditions. Autofluorescence of the supernatant was measured; it was found that the relative amplitude of long lifetime was multiple times higher than in the cell. In the part devoted to the stress response, this thesis was focused on the amount of dissolved oxygen in the production medium and the effect on bacterial autofluorescence. Then differently concentrated hydrogen peroxide was used, the best results were obtained from the concentration of 100 mM in media. For comparison a combination of hydrogen peroxide with ferro-ammonium sulphate was used, but there was no big difference. Sodium azide and antimycin A were selected as substances that directly influence on bacterial respiratory chain. Both compounds affected change in the ratio of the relative amplitudes, but the distribution of these lifetimes and the autofluorescence change over time was affected only by sodium azide.
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Aflatoxins in food and their influence on DNA and cell lines
Šislerová, Lucie ; Pernicová, Iva (referee) ; Brázda, Václav (advisor)
Aflatoxins present a great danger due to their high toxicity and carcinogenicity, which is not easily avoided in everyday life. Intoxication with aflatoxins causes a wide range of diseases ranging from mild diseases to organs necrosis or death. Aflatoxins mostly affect the liver, where it degrades and the formation of subsequent metabolites, which are the most toxic to the body. For this reason, their precise determination and understanding of the principle of their effect is very important. In this work, methods for monitoring and closer determination of aflatoxin effects on human cells were calibrated. The methods that were used are: MTT viability assays, fluorescence microscopy and flow cytometry. Next, the amount of aflatoxins present in different foods with different storage conditions was measured. For this analysis were used ELISA assays RIDASCREEN Aflatoxin Total and RIDA Aflatoxin column. Calibrated methods were compared with the methods already used to determine the effect of aflatoxins and the results of the ELISA tests were compared with the limits of aflatoxin levels permitted by the Czech legislation. None of the controlled foods contained above-the-limit concentration of aflatoxins, which in the Czech Republic is set at 4-10 µg/l (varies for different types of food). Foods that were poorly stored but not visibly affected by fungi showed the highest levels of aflatoxins. The LD50 value for aflatoxin B1 was determined to 12,25 µM. The type of cell death caused by aflatoxins was determined by flow cytometry and these data were further confirmed by fluorescence microscopy images.
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Analysis of lubricated compliant contact
Dočkal, Kryštof ; Dzimko, Marián (referee) ; Nečas, David (advisor)
This diploma thesis deals with the proposal of methodology for film thickness eva- luation within compliant contacts. With respect to characteristics of such contact pairs, like variable film thickness, high surface roughness, or poor conductivity and reflectivity, the usage of conventional experimental methods is particularly complicated. In present study, an optical method based on the principle of fluo- rescent microscopy was employed in present thesis. An evaluation algorithm in- volving background normalization and calibration of fluorescent intensity to film thickness was created in a form of experimental software. The proposed algori- thm was validated by using elastohydrodynamic contact formed between ceramic ball and glass disc. The measured film thickness was compared with theoretical prediction, while very good agreement of obtained data was observed. Further, a series of experiments with compliant samples was conducted, while the central film thickness was evaluated as a function of mean speed, applied lubricant, ap- plied load and slide-to-roll ratio. The last part of the thesis is focused on results analysis and discussion considering the previously published literature.
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Multiparametric fluorescence spectroscopy
Lacko, Kata ; Venerová, Tereza (referee) ; Mravec, Filip (advisor)
This diploma thesis deals with the possibilities of multiparametric fluorescence spectroscopy, since the main objective of this experiment was to evaluate the possibilities of multiparametric measurements in the fluorescence spectroscopy laboratory. A suitable fluorescence probe was proposed for this type of experiment that shows high sensitivity for pH changes in the environment, SNARF-4F AM, based on a literature research. The fluorophore was dissolved in solutions of different pH and this system was examined using a time-resolved spectrofluorimeter. The method named TRES (time-resolved emission spectra) was used to obtain the emission spectra of the probe and to find the emission maximum. Fluorescence intensity decay measurements as a function of wavelengths allowed to create deconvolution of the emission spectra, which provided information about the fluorescent lifetime and the relative representation of the states of probes in the solution. Later, the probe was dissolved in solutions of different density and pH - this system served for anisotropic measurements, during which the individual correlation-rotational times of the fluorophore were obtained. The obtained results were then used as the basis for multiparametric analysis, which was performed by using a fluorescence correlation microscope and a spectrograph. This combination allows to measure the necessary fluorescence parameters in one step. A standard operating procedure was created for the spectrograph’s control. On the basis of the obtained information the suitability, accuracy and sensitivity of the multiparametric analysis were qualified.
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Phytotoxicity of selected naphthoquinones on a selected plant model
Rucký, Jakub ; Masařík,, Michal (referee) ; Provazník, Ivo (advisor)
The introductory part of this thesis is focused on the theoretical analysis of solved problems as examining the toxicity of naphthoquinones plumbagine and juglone, especially with regard to their allelopatic action. The next section is focuses on the plant stress, caused by the action of stress factors leading to their death. There is an experimental protocol and the possibilities of determination the effect of naphthoquinones on the plant model. Experimental section discusses changing growth parameters of the plant samples in different concentrations of the naphthoquinone. There is examined cell viability and changes in the synthesis of the secondary metabolites. Data obtained by using spectrophotometric and microscopic analysis are evaluated with STATISTICA software and statistical significance are plotted.
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