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The role of CSL proteins in oxidative stress response of Schizosaccharomyces pombe
Tvarůžková, Jarmila ; Převorovský, Martin (advisor) ; Rallis, Charalampos (referee)
Oxidative stress represents a complex and intensely studied phenomenon tightly linked to a range of human diseases, and to aging in many organisms. A plethora of key cellular regulators, including the Notch signaling pathway, have been recently described to respond to the cellular redox status. We have characterized the role of CSL (CBF1/Su(H)/LAG-1) proteins, the effectors of Notch signaling pathway in metazoa, in oxidative stress response in fission yeast. Schizosaccharomyces pombe contains two CSL paralogs, Cbf11 and Cbf12, that have antagonistic functions in the regulation of cell cycle and cellular adhesion. Both proteins are able to bind the canonical CSL motif and activate transcription and, thus, function as genuine CSL transcription factors. We have determined that the strain lacking cbf11 is resistant to hydrogen peroxide but not to menadione, a source of superoxide anion radical. Using double knock-outs to assess genetic interactions we have revealed that the resistance of cbf11 knock-out is dependent on the antioxidants catalase and sulfiredoxin. Genes encoding these antioxidants are under transcriptional control of the Sty1 MAP kinase pathway and the Pap1 transcription factor which are also required for the resistance of Δcbf11 cells. Cbf12 is believed to play only a minor role in...
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The effect of oxidative stress on the structure and function of sperm cell cytoplasmic membrane during the fertilization process.
Tojnerová, Kateřina ; Krylov, Vladimír (advisor) ; Šanovec, Ondřej (referee)
Oxidative stress is caused by an imbalance between antioxidants and oxidants, and thus a consequent excess of reactive oxygen species (ROS). It can also be caused by insufficient antioxidant protection, which normally reduces ROS levels and protects the cell from damage. Oxidative stress causes changes in the structure and function of the sperm cytoplasmic membrane during the fertilization process. Here it has a positive effect on physiological functions, where low levels of ROS are necessary for normal sperm development such as sperm capacitation or sperm hyperactivation. However, at higher levels ROS exerts a pathophysiological effect, reducing membrane fluidity due to membrane lipid peroxidation, DNA fragmentation, loss of motility and subsequent inability of the sperm to fertilize can also occur. Keywords: oxidative stress, cytoplasmic membrane, sperm cell, fertilization
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The influence of electrochemically generated hydrogen peroxide on the stress response of microorganisms for the purpose of increased PHA production
Najbrtová, Johana ; Nováčková, Ivana (referee) ; Ehlich, Jiří (advisor)
The subject of the presented thesis is the effect of electrochemically formed hydrogen peroxide on the production of polyhydroxyalkanoate. In this work, hydrogen peroxide was generated directly in the medium with bacteria by two-electron reduction of oxygen. This thesis is divided into a theoretical part, which is devoted to approaching the topic of polyhydroxyalkanoate, oxidative stress, electrochemical production of H2O2 and the connection of electrochemistry with biotechnology. The experimental part is focused on the application of these findings. Hydrogen peroxide was generated by applying an electrical potential to an electrode system composed of a stainless-steel working electrode, a titanium counter electrode, and a pseudo-reference Ag/AgCl electrode embedded in a polypropylene holder. After initial electrochemical characterization of the prepared system, a potential of 0,8 V and -1 V was selected for application in cultivation experiments. Here, the effect of H2O2 on the stress response of the bacterium Cupriavidus necator H16 from the Czech collection of microorganisms was investigated. From the results of the cultivation experiments, it was evident that the electrochemically produced H2O2 does not have a significant effect on the development of the bacterial culture. However, the electrode system itself positively affects bacterial growth. The influence of H2O2 was not noticeable due to the low production in medium. This was caused by a low concentration of oxygen in the medium. During this work, a new electrode system with higher H2O2 production was designed, which could use atmospheric oxygen for reduction to H2O2. However, even with this system, a desired stress response was not achieved, because increased production of H2O2 was prevented by the formation of biofilm on the electrodes. Furthermore, it was found that the materials used for the electrode system support the formation of biofilm, mainly titanium, stainless-steel mesh and polypropylene.
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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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