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Application of Separation Techniques Connected with Mass Spectrometry for the Analysis of Environmentally Important Compounds
Mácová, Daniela ; Čelechovská, Olga (referee) ; Márová, Ivana (referee) ; Demnerová, Kateřina (referee) ; Čáslavský, Josef (advisor)
The identification of the hydrolysis and photodegradation products of flexible polyurethane foams (PUFs) with addition of biooriginated and biodegradable additive was the first topic of this dissertation work. Separation of polyurethane foam hydrolysis degradation products, designed for ecotoxicological tests, was managed by high-performance liquid chromatography coupled with mass spectrometry (HPLC/MS). The degradations product structure was elucidated by tandem mass spectrometry (MSn). PUF photodegradation products were obtained by exposure of materials by irradiation at 254 nm. Semi-volatile degradation products were isolated from the exposed polyurethane by n-hexane extraction; volatile compounds were collected by solid phase microextraction (SPME). Gas chromatography with mass spectrometry (GC/MS) and complete orthogonal tandem gas chromatography with mass spectrometry (GCxGC/TOF MS) was used for separation and identification of photodegradation products. The influence of the bio-filler on the character of degradation products and the possible effect of PUF degradation products on the environment was discussed at the end of this section. The determination of isoprostanes – markers of oxidative stress in tissues of beadlet anemone (Actinia equina) was the subject of the second topic. F2-isoprostanes were synthesized from the arachidonic acid. With thereby prepared isoprostanes the method of determination by liquid chromatography with tandem mass spectrometry (HPLC/MS/MS) was developed and optimized. The isoprostane isolation process from the Actinia equina tissues was optimized with solid phase extraction (SPE). The resulting methodology was used to quantify isoprostanes in tissues of anemones, which were exposed to both moderate and high temperature changes. The temperature changes were used to initiate the oxidative stress in organisms. In addition, concentration levels of unknown compounds were also monitored. These unknown compounds were extracted from tissues together with F2-isoprostanes and their identity is discussed in this dissertation work too. The possibility of using isoprostane levels in the Anthozoa tissues for the oxidative stress monitoring is discussed in the conclusion of this work.
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Application of Mass Spectrometry for the Determination of Oxidative Stress Markers and Mycotoxins
Čumová, Martina ; Večeřa, Zbyněk (referee) ; Hajšlová, Jana (referee) ; Vávrová, Milada (referee) ; Čáslavský, Josef (advisor)
The first topic presented in the dissertation thesis is determination of isoprostanes as markers of oxidative stress and other compounds affected by presence of oxidative stress. Isoprostanes iPF2-III, iPF2-VI, iPF2-VI, astaxanthin and polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA) were monitored in Atlantic salmon eggs (Salmo salar). Methods for the determination of these compounds have been developed and optimized using chromatographic separation coupled to conventional or mass spectrometric detection. Freshly laid eggs, eyed embryos and non-viable eggs were used to test a general hypothesis that egg viability can be affected by susceptibility to oxidative stress, either through the specific fatty acid concentration and/or the antioxidant capacity of the eggs. Levels of isoprostanes and arachidonic acid (AA) were significantly higher in non-viable eggs than in control (eyed embryos) as well as relative abundance of PUFA. While no difference of isoprostanes was found between freshly laid and control those from the Atlantic stock except iPF2-VI which was observed under the LOQ in the control. Higher levels of PUFA and AA in comparison with the control were observed in the freshly laid eggs. However, the only statistically significant difference was observed in the amount of astaxanthin. Different levels of PUFA and astaxanthin may be related to their biochemical consumption during the development of eggs. This work evaluated potential effect on the viability of eggs Salmo salar due to the presence of oxidative stress. The monitoring of mycotoxins in food and feed was the subject of the second topic. Mycotoxins are secondary metabolites produced by fungi. They are ubiquitous undesirable natural contaminants that are toxic for humans and animals. Today are known more than 500 mycotoxins. However, only few of them are regulated by the European Union. The European Food Safety Authority (EFSA) was asked by the European Commission to provide a scientific opinion on other mycotoxins for which statutory limits could be developed. In this study is proposed simultaneous screening allowing fast, reliable and sensitive approach, identification and quantification of 17 mycotoxins in food and feed sample. The method includes both mycotoxins regulated by the EU and selected mycotoxins required by the EFSA (aflatoxins, deoxynivalenol, nivalenol, zearalenone, fumonisin, ochratoxin A, T-2 toxin, HT-2 toxin, enniatins and beauvericin). Analytes are isolated by the modified QuEChERS method. For separation and target mycotoxins detection, ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC –MS/MS) was employed. The method also allows determination of ergot alkaloids (ergocornine, ergosine, ergocryptine, ergocristine and their respective epimers). The developed method was used either for monitoring mycotoxins and ergot alkaloids in feed and raw materials and barley and malt prepared from it.
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Evaluation of glutathione content in plants as a marker of heavy metals environmental contamination
Borková, Marie ; Stoupalová, Michaela (referee) ; Opatřilová,, Radka (advisor)
Dependence of glutathione concentration on the amount of thallium in the plant was studied. Observed plant was maize (Zea mays) which was divided to two parts – root and overground. Two culture procedures were elaborated where seeds and young seedlings were cultivated in a solution of thallium of concentration 0, 1, 3, 5, 8, a 10 µmol/l. Extraction agents used during extraction were phosphate buffer and solution of ascorbic acid. Determination of glutathione was realized by capillary electrophoresis (CE) and high performance liquid chromatography (HPLC). Diode array detector (DAD) was used in both methods. Quantification of the thallium amount in the plant was done by method of inductively coupled plasma-atomic emission spectrometry (ICP-AES).
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Redox signaling to chromatin
Plšková, Zuzana
Chromatin is a highly dynamic structure, which is constantly subjected to regulation in response to environmental conditions. This response is mediated by chromatin modifiers, such as histone acetyltransferases, which deposit histone post-translational modifications, resulting in changes in gene expression. One of such modifiers in plants is GENERAL CONTROL NON-REPRESSIBLE 5 (GCN5). Despite its importance in plant growth and development, it remains poorly understood how GCN5 is regulated. Emergine evidence points towards redox regulation of chromatin remodeling that can be executed via redox modifications of chromatin modifiers. To investigate possible redox regulation of GCN5, we used redox insensitive lines, carrying GCN5 with mutated cysteine to serine. Even though gcn5 mutants displayed enhanced susceptibility to paraquat-induced oxidative stress, the mutated lines phenocopied the wild type. We further probed the interactome of GCN5 to identify putative functional partners, whose association with GCN5 could be altered under oxidative stress conditions, or affected by its redox status. Mutating of a single cysteine residue in GCN5 did not result in significant changes of its interactome, suggesting that additional single and higher order mutants need to be explored.
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The effect of yttrium and samarium oxide nanoparticles on plant development
Brandová, Zuzana ; Soudek, Petr (advisor) ; Petrová, Šárka (referee)
This thesis is based on hydroponic experiment conducted on Hordeum vulgare with nanoparticles of rare earth elements in concentration 2·10-4 mol/L and REEs chlorides in concentration 3·10-4 mol/L. The comparison of the the effect on their acumulation of another nanoparticles in the sloution and normal medium is included as well. In this case the hydroxyapatite NPs were chosen in the same concentration as yttrium and samarium NPs. The role of type and form of the elements on their acumulation and impact is also studied. Negative effect of REEs was proven by enzymatic analysis that detects reducting enzymes and determination of plant pigment levels by high performance liquid chromatography. Because both types of substances can reduce reactive oxygen radicals. Acumulation of REEs was determined by ICP spektrometry. Key words: nanoparicles, reare earth elements, reductive enzymes, plant pigments, acumulation, oxidative stress
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The impact of mutant huntingtin on oxidative stress in primary fibroblasts isolated from a new Huntington's disease knock in porcine model
Sekáč, Dávid ; Ellederová, Zdeňka (advisor) ; Hanzlíková, Hana (referee)
Huntington's chorea is a dominantly inherited disease caused by trinucleotide (Cytosine-Adenine -Guanine) expansion in a gene coding huntingtin protein. Carriers of these mutation show symptoms associated with motor impairment, a cognitive and psychiatric disturbance, which is called Huntington's disease (HD). The major sign of HD is striatal atrophy in the middle age of life. Since it is known that huntingtin protein participates in a lot of cellular processes, such as transcriptional regulation and metabolism, these processes change by its mutation. One of the features observed in HD pathogenesis is the presence of oxidative stress. The aim of the work was to monitor the molecular changes preceding the HD manifestation in the knock-in minipig model. As a material for monitoring molecular changes leading to this condition, primary fibroblasts were used. Whereas, the oxidative stress arises from an imbalance between oxidants and antioxidants, level of reactive species and lipid peroxidation together with expression of antioxidant response associated genes was measured. At the same time, expression of metabolic and DNA repair related genes was monitored. Although the differences in oxidative stress level or the expression of antioxidative response genes were not detected, the changes in the...
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Vliv chloridazonu na raná vývojová stádia raka mramorovaného
ŘEHOŘEK, Martin
The main objective of this bachelor thesis is to evaluate the influence of chloridazon on the early growing stages of marbled crayfish (Procambarus virginalis). The influence of chloridazon is assessed using various factors such as biomarkers of oxidative stress, growth, mortality, ontogenetic development, and crayfish behaviour. The acquired results from this research will extend the current knowledge and data on the influence of chloridazon on crayfish and on water ecosystems.
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The effect of morphine withdrawal on G protein-coupled receptors and redox balance in rat brain
Kočárková, Veronika ; Novotný, Jiří (advisor) ; Černá, Barbora (referee)
Although morphine is one of the most effective analgesics available, its long-term use is severely limited by the development of tolerance and dependence. Molecular mechanisms responsible for these effects have been the focus of intensive research for decades aimed at developing safer and more effective pain treatments. The aim of this thesis was to contribute to a better understanding of the molecular actions of morphine, as an understanding of these processes is essential to develop better therapeutic approaches. Using western blot analysis, we investigated the effect of 10-day treatment with increasing doses of morphine and its subsequent withdrawal on key components of opioid signalling in the rat cerebellum and cerebral cortex. Amounts of opioid receptors, various heterotrimeric G protein subunits, GRK3 kinase and β-arrestin 2 were examined. Furthermore, the levels of adenosine A3 receptor and cannabinoid receptor type 1 were detected. Finally, we also investigated the effect of morphine and its withdrawal on oxidative stress markers. Morphine administration resulted in a significant decrease in GRK3 kinase levels in both brain regions, and these changes remained even after withdrawal. However, there was no significant change observed in the content of opioid receptors, different G protein...
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Effect of morphine preconditioning and mKATP channel activity on survival of differentiated SH-SY5Y cell line after oxidative stress
Paluba, Michal ; Hejnová, Lucie (advisor) ; Kolář, David (referee)
Morphine as one of the most effective analgesics has been used in medicine for more than a century. However, morphine is also known for its neuroprotective and cardioprotective effect in ischemic disorders, where it induces increased cellular resistance to oxidative stress. One of the aims of our study was to analyze the effect of chronic morphine treatment on the viability of differentiated cells of the neuroblastoma cell line SH-SY5Y after induction of oxidative stress by tert butyl hydroperoxide (tBHP). However, the detailed mechanism of the protective effect of morphine is still unknown. Current research on this topic, and in particular on morphine-induced preconditioning, has focused much attention on mitochondrial ATP- sensitive potassium channels (mKATPs). There is evidence that morphine, through activation of opioid receptors, mimics the protective effect of ischemic preconditioning precisely through activation of mKATP channels. Modulation of mKATP channel activity is thought to be responsible for the functional state of the mitochondria by altering mitochondrial membrane potential and regulating reactive oxygen species (ROS) production. Thus, the next aim of our work was to reveal the role of mKATP channels in morphine preconditioning and the effect of their activity on cell survival. The...
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Plant responses to combined arsenic and cadmium stress
Burešová, Andrea ; Mašková, Petra (advisor) ; Podlipná, Radka (referee)
Arsenic and cadmium are both non-essential, highly toxic and carcinogenic elements that often occur together in the environment. Given the frequent co-contamination of the environment, it is necessary to investigate both plant strategies for dealing with one metal(loid) and the mechanisms that lead to tolerance or, conversely, sensitivity in the presence of both elements. Simultaneous exposure to multiple toxic elements may lead to extensive plant damage, however it may also result in the increasing engagement and intensity of defence strategies. Contamination of the food chain through crops growing on contaminated soils is a major concern, one that poses a risk to both human and animal lives. The aim of this research field is to reduce accumulation and translocation to aboveground edible parts as much as possible, for example by understanding the mechanisms behind heavy metal accumulation and translocation, or by stabilising toxic elements in the soil, or by cleaning up contaminated soil. One method of clean-up is phytoremediation, which usually utilizes plants with high tolerance to toxic elements, called hyperaccumulators. The knowledge of how defence strategies and mechanisms are affected by the interaction of multiple elements is important for identifying plant species capable of activating...
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