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Utilization of pulse labelling techniques for studying the dynamics of proteins and protein complexes
Polák, Marek ; Novák, Petr (advisor) ; Obšil, Tomáš (referee) ; Vrbacký, Marek (referee)
(In English) Mass spectrometry (MS) techniques are routinely used to probe the structure and dynamics of proteins and protein complexes. Although MS techniques lack the high resolution of data provided by X-ray crystallography, NMR, or cryo-EM, they excel in providing insights into analyte dynamics, structure, and interactions with other components, such as ligands. This doctoral thesis presents a contribution to the field of structural biology employing and extending covalent labelling approaches, namely Fast Photochemical oxidation of Proteins (FPOP) and oxidation by singlet oxygen (1 O2). These approaches were followed to study the structure, dynamics, and interaction of proteins, nucleic acids, and protein-DNA complexes in solution. Initially, FPOP was used to investigate the interaction interface of FOXO4 and DAF16-DNA response element and to show the possibilities of analyzing such a complex using both 'bottom- up' and 'top-down' approaches. Furthermore, an isotope depletion strategy combined with multiCASI-ECD proved effective in delivering structural information with the highest possible resolution for mapping protein-DNA interfaces. This research showcases how information derived from structural proteomic methods can guide the construction of in-silico models for protein-DNA complexes with...
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Analyses of lignans in selected variants of linseed (Linum usitatissimum L.)
DOŠEK, Miroslav
Oilseed flax is one of the traditional agricultural crops. Flax seeds contain, among other beneficial components, lignans, which are polyphenolic substances that provide the plant's defense against biotic and abiotic stress. The main naturally occurring lignan in flax is secoisolariciresinol diglucoside (SDG). Flaxseed represents the richest natural source of lignans, which have been observed to have many positive effects on human health, including antioxidant and other biological activities. One method that can be used to determine SDG is high-performance liquid chromatography with mass spectrometry (HPLC-MS), which was used in this study for the quantitative analysis of SDG. Total polyphenol content (TPC) and antioxidant activity expressed as the ability to quench the DPPH radical were also determined. Correlation analysis was performed to determine the relationships between the studied parameters. Both variety and year had a significant influence on all three studied parameters. The highest average content of polyphenols and SDG was found in the Agram variety, while the highest antioxidant activity was determined in the Floral variety. A significant positive correlation was observed only between the total polyphenol content and the SDG content. The results of this study demonstrate the considerable influence of variety and growing conditions on the content of lignans and the antioxidant potential of flaxseed, which predetermines its health benefits.
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Development of mass spectrometric methods for structural analysis of fatty acids and estolides
Cudlman, Lukáš
(EN) This doctoral thesis summarizes the use of modern mass spectrometric methods for the structural identification of fatty acids and triacylglycerol estolides (TG-EST) and their subsequent characterization in the lipidome of vernix caseosa, i.e., the biofilm covering the skin of the human fetus during the last trimester of in-utero development. Vernix caseosa has mainly antimicrobial properties and the ability to heal wounds and burns. The first part of the doctoral thesis focuses on the diversity of fatty acids in the lipidome of vernix caseosa. These acids form the structural subunits of complex lipids, such as the abundant triacylglycerols and wax esters. By studying fatty acids and their structural features, this thesis has provided valuable insights into the structural variability of the vernix caseosa lipidome. Without a full understanding of the structure of these key subunits, it would be impossible to complete the analysis of this lipidome in the future. In this thesis, fatty acids using high-performance liquid chromatography (HPLC) and modern mass spectrometry methods, namely ultraviolet photodissociation (UVPD) and ozone-induced dissociation (OzID), were characterized. The coupling of HPLC and mass spectrometry (HPLC-MS) provided unambiguous information on the positions and geometry of...
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Monitoring process contaminants in coffee roasting using LC-MS
Ilyushchenkova, Alexandra ; Juglová, Zuzana (referee) ; Diviš, Pavel (advisor)
The bachelor thesis focuses on monitoring changes in the concentration of process contaminants during coffee roasting using liquid chromatography with mass detection. Green coffee was roasted at 220 °C for 14 minutes with two-minute intervals. Samples for analysis were taken from 10. by 24. minutes of roasting. Practical monitoring of the acrylamide’s formation during coffee roasting was not carried out, since it was not possible to detect acrylamide in coffee using a mass detector, even after its derivatization with thiosalicylic acid. The relevant data were taken from other scientific articles, which reported a linear increase in concentration with the roasting time up to the maximum value and a subsequent exponential decrease at the end of roasting. By monitoring 5-hydroxymethylfurfural, it was found that during roasting, this substance is gradually formed in coffee due to the decomposition of carbohydrates, however, with increasing roasting time, the degradation of this substance occurs quite quickly. The concentration of 5-hydroxymethylfurfural was also determined in coffee samples purchased in the trade network. Concentrations of 5-hydroxymethylfurfural in these samples ranged from 0,3 to 0,38 mg/kg, which are corresponded to those reported in other expert studies.
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Study of the use of crops for phytoremediation of contaminated soil by pharmaceuticals
Hamplová, Marie ; Zlámalová Gargošová, Helena (referee) ; Mravcová, Ludmila (advisor)
Pharmaceuticals leaking into the environment from wastewater and livestock farms raise concerns about the impact on the ecosystem and human health. Residues of pharmaceuticals present in soil are subject to a number of biotic and abiotic processes, including uptake, translocation and accumulation by plants. The ability of plants to accumulate or degrade pharmaceuticals could have potential applications in the process of 'phytoremediation', where plants and their associated micro-organisms are used to stabilise, degrade or remove contaminants from the environment. In the framework of this thesis, experiments on phytoremediation of drug-contaminated soil by the cover crop oilseed radish (Raphanus sativus L. var. Oleiformis) under controlled conditions were conducted. Soil samples were extracted by ultrasonic extraction, plant samples by QuEChERS method and the final analysis of drugs was performed by UPLC-MS/MS. Oilseed radish produced a large amount of aboveground biomass compared to the other two crops and the presence of drugs had no negative effect on its growth. However, the determined levels of each drug in the soil and in the crop showed insufficient effectiveness of phytoremediation. The accumulation of drugs by the crop was low except for residues of the antidepressant venlafaxine. Therefore, according to these results, oilseed radish is not a suitable crop for phytoremediation of drug-contaminated soil. Experiments were also carried out with the root vegetable radish (Raphanus sativus L. var. Sativus) and carrot (Daucus carota L.) to assess the risk to human health associated with the accumulation of drugs in vegetable roots. The risk has not been demonstrated in terms of the amount of established drugs in the roots. Only the metabolite clofibric acid accumulated to a higher extent and the antibiotic trimethoprim was the most translocated to the aerial parts of the crop. The rate of uptake and translocation of drugs by the plant depended on many factors, as their content varied considerably between drug groups and between parts and types of crops.
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Studz of direct and indirect plasma application on onion seeding bulbs
Krejsová, Lenka ; Enev, Vojtěch (referee) ; Krčma, František (advisor)
Nowadays, agriculture is based on conventional methods that involve the application of pesticides, agricultural chemicals and fertilizers. These substances have a negative impact on nature and human health, so more environmentally friendly methods are being sought. This thesis deals with the analysis of fragrances in onions treated with plasma before planting. The treatment was carried out to see if it would improve onion growth while maintaining or increasing the fragrance content. The theoretical part deals with the soil and it is characterization methods, plasma, quantitative and qualitative methods for the determination of volatile substances. It contains the principles of UV-VIS spectrometry, atomic absorption spectrometry (AAS), mass spectrometry (MS) as well as tandem connection of gas chromatography with mass spectrometry (GC–MS). The experimental part was carried out over a period of three years at 22 sites. The preparation and analysis of soil samples are described, which was also evaluated. Furthermore, the procedures for treating the bulbs with corona discharge and plasma-activated water before planting were described. In total, four treatment variants were carried out and bulbs from the fifth variant were not additionally treated. Each treatment variant was planted in four replications of ten bulbs each. Twenty selected grown onions from one site were individually converted to liquid samples and the saturated vapor was analysed by proton transfer reaction time of flight mass spectrometry (PTR–TOF–MS). Thanks to this analysis, the concentrations of volatile substances were determined. Some samples were analyzed by gas chromatography with mass spectrometer as detector to identify the compounds. Subsequently, the data were processed and evaluated. From the results obtained, it is evident that plasma treatment can be useful for the treatment of onions in agriculture, because it confirmed harvest increase without decrease of fragrances concentrations. Thus, it is necessary to carry out experiments on a larger scale in the future.
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Development of mass spectrometric methods for structural analysis of fatty acids and estolides
Cudlman, Lukáš ; Cvačka, Josef (advisor) ; Kozlík, Petr (referee) ; Lísa, Miroslav (referee)
(EN) This doctoral thesis summarizes the use of modern mass spectrometric methods for the structural identification of fatty acids and triacylglycerol estolides (TG-EST) and their subsequent characterization in the lipidome of vernix caseosa, i.e., the biofilm covering the skin of the human fetus during the last trimester of in-utero development. Vernix caseosa has mainly antimicrobial properties and the ability to heal wounds and burns. The first part of the doctoral thesis focuses on the diversity of fatty acids in the lipidome of vernix caseosa. These acids form the structural subunits of complex lipids, such as the abundant triacylglycerols and wax esters. By studying fatty acids and their structural features, this thesis has provided valuable insights into the structural variability of the vernix caseosa lipidome. Without a full understanding of the structure of these key subunits, it would be impossible to complete the analysis of this lipidome in the future. In this thesis, fatty acids using high-performance liquid chromatography (HPLC) and modern mass spectrometry methods, namely ultraviolet photodissociation (UVPD) and ozone-induced dissociation (OzID), were characterized. The coupling of HPLC and mass spectrometry (HPLC-MS) provided unambiguous information on the positions and geometry of...
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Study of low-temperature plasma products using mass spectrometry and their relation to thin film chemistry
Maršálek, Blahoslav ; Bránecký, Martin (referee) ; Čech, Vladimír (advisor)
The aim of this thesis was to analyse and interpret the spectra of tetravinylsilane as a function of plasma discharge power in order to find a relationship between plasma products, layer deposition and thin film chemistry. Another objective was to carry out a literature search in the field of plasma-enhanced chemical vapour deposition (PECVD) and mass spectrometry. Low temperature organosilicate-based plasma technology enables the synthesis of specific materials with controlled chemical and physical properties. The targeted synthesis of surfaces with controlled properties is determined by the atomic and molecular processes in the plasma, which are responsible for building the chemical structure and the resulting material in the form of a thin film. In this work, mass spectrometry has been used to detect and quantify the particles produced in the PECVD process, which is one of the methods that allow the characterization and identification of plasma products. Analysis of the mass spectra revealed that the molecules responsible for the growth of the layer contain carbon and silicon. The deposition rate determined by in situ spectroscopic ellipsometry correlates quantitatively with the flux of carbon and silicon particles that are chemisorbed on the film surface. The ratio of carbon and silicon deposited on the surface also correlates strongly with the C/Si flux ratio of the power driven plasmas. The contribution of silicon-containing particles as building blocks to the film growth decreases with increasing power and accounts for 20% (2 W), 5% (10 W) and only 1% (75 W) of the total chemisorbed fraction. This ratio between bound silicon containing particles and carbon particles affects the elemental composition and chemical structure of the deposited layers. The relationships between plasmachemical processes and particle adhesion on the surface are quite complex. The adhesion of silicon particles first increases sharply to a maximum at 25 W and then gradually decreases, which is characteristic of the so-called precursor-deficient PECVD. Similarly, the concentration of vinyl groups incorporated into the deposited layer and the fraction of sp2 hybridization of carbon correlate with the particle fluxes of the corresponding plasma. This work has demonstrated that mass spectroscopy is a suitable method for the study of plasmachemical deposition from the gas phase (PECVD). PECVD technology is promising for the deposition of silicon-containing layers, which is technologically applicable in many directions of materials research.
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Determination of linear synthetic aromatic compounds in abiotic and biotic matrices
Tulková, Tereza ; Čáslavský, Josef (referee) ; Vávrová, Milada (advisor)
Synthetic musk compounds are artificial organic compounds with a smell similar to the odor of a natural musk. The most recently discovered and also the least known group of artificial musks is linear musk compounds. These substances are now often used in cosmetics, personal care products, detergents and various cleaning products. They have leaked into all the major environment components, especially into the hydrosphere. There has been great attention drawn to synthetic musk compounds in the last few years because of their environmental impact. The aim of this thesis is to identify twelve linear musk compounds (linalool, 2-cyclohexylethanol, fresco menthe, citronellol, HSA, isobornyl acetate, arocet, aroflorone, allyl cyclohexyl propionate, lilial, isoamyl salicylate and hexylcinnamic aldehyde) in waste water samples. The samples were taken at three different waste water treatment plants (WWTP) in South Moravian region (WWTP Brno-Modřice, WWTP Luhačovice and WWTP Hodonín). Each of these treatment plants has a different equivalent number of inhabitants. At first, a working method was optimized, then applied on real waste water samples. Solid-phase microextraction (SPME) technique was used for the extraction of analytes. Gas chromatography–mass spectrometry (GC/MS) was then used for identification and quantificaton of the studied compounds.
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