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Heme sensor proteins as potential biomarkers of cellular and oxidative stress processes induced by ionizing radiation
Vávra, Jakub ; Martínková, Markéta (advisor) ; Souček, Pavel (referee) ; Tichý, Aleš (referee)
[IN CZECH] Ionizing radiation is a potential inducer of the oxidative stress processes in cells. As a result, reactive radicals are formed in the intracellular space modifying the essential biomolecules. Ionizing radiation has either direct (radiation sickness) or indirect (malignant processes) effect on the organism. Therefore, a fast determination of the dose is required when suspected irradiation of the organism occurs. However, a method routinely applicable, fast enough and at the same time suitable for dose estimation based on biomarkers has not been developed so far. The aim of this thesis is to describe new properties of the selected heme sensor proteins and discuss their potential importance for the cellular adaptation to oxidative stress conditions. Specifically, the thesis is focused on two eukaryotic proteins, heme regulated inhibitor (HRI) and transcription factor p53. The study of functional regulation as well as the conformational changes of these proteins induced by heme is greatly emphasized. Besides, the optimization of the key experimental methods was conducted. Specifically, Phos-tag electrophoresis was applied for the kinetics study of HRI wild type and its Gly202Ser mutant form, which is a characteristics of lung cancer development. Unsurprisingly, for both HRI forms studied,...
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Role of oxidative stress in male infertility.
Dolečková, Barbora ; Tlapáková, Tereza (advisor) ; Šanovec, Ondřej (referee)
Oxidative stress is a phenomenon caused by an excess of reactive oxygen species (ROS), or by insufficient activity of antioxidants, that reduces these ROS levels and thereby protect the organism from oxidative damage. ROS have two types of origin: endogenous, which includes leukocytes and immature sperm, and exogenous, which includes factors such as air pollution caused by heavy metals, smoking tobacco products, obesity and others. Low levels of ROS have a positive effect on the physiological functions of the organism, including the process of spermatogenesis, where ROS participates in the course of hyperactivation and capacitation. However, increased levels of ROS trigger a number of cellular pathologies, whether the loss of fluidity of biological membranes due to lipid peroxidation, deformation of enzymatic proteins or DNA fragmentation, which negatively affects individuals' infertility. Due to the significant positive correlation of ROS scavenging by antioxidants with improving sperm parameters of an infertile individual, antioxidant therapy has recently begun to be used as a possible successful component of male idiopathic infertility treatment.
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Microplastics and their effects on the metabolism of animal cells
Fiedlerová, Gabriela ; Cajthaml, Tomáš (advisor) ; Černý, Jan (referee)
Microplastics are tiny particles smaller than 5 mm in size found in various environments, including seas, rivers and soil. These particles can be intentionally produced as part of cosmetic products or formed by the breakdown of larger plastic objects. Microplastics represent a serious threat to the environment and animals and could eventually reach humans through the food chain. This thesis deals with a critical and synthetic overview of the literature related to the influence of microplastics on the metabolism of animal cells. The thesis also considers the experimental conditions under which the data were obtained. In particular, factors such as the tested microplastics' shape, size and ageing are critically evaluated as these aspects are often neglected in the literature. Current knowledge shows that microplastics accumulate in the bodies of animals and cause physical and metabolic damage, inflammation, disruption of energy metabolism, protein metabolism and amino acid and lipid metabolism. The most serious effects of microplastics can be considered to be the formation of oxygen radicals, neurotoxicity, disruption of gametogenesis and offspring development.
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Electrochemical evolution of hydrogen peroxide for biotechnological applications
Runštuková, Nikola ; Kratochvíl, Matouš (referee) ; Ehlich, Jiří (advisor)
This master‘s thesis deals with the connection of two distinct fields, electrochemistry and biotechnology, where electrochemically generated peroxide was subsequently used in bacterial cultivation. The peroxide was synthesized in an electrolytic cell by reducing oxygen at the cathode to which suitable electrical potential was applied. The aim of this thesis was to investigate the dependence of the amount of hydrogen peroxide produced on a few selected parameters, such as cathode potential, electrode material, the geometry of the measuring cell, or the stirring speed, and then to apply these findings in the continuous stressing of the bacterium Cupriavidus necator H16, in order to increase the production of polyhydroxyalkanoates. The selected parameters were first tested in small-scale measuring cells and then experiments were carried out on a larger scale, in Erlenmeyer flasks. During the experiments, the amount of electric current produced was measured and also the peroxide concentration was examined spectrophotometrically along with the oxygen concentration measured by using an optical sensor. Based on the obtained results, bacteria were cultivated in flasks with a built-in system of steel electrodes, due to which oxidative stress in the form of in situ generated hydrogen peroxide was continuously applied. The bacterial cultures were evaluated by measuring the change in optical density, gravimetric analysis and GC-FID analysis. It was found that even small amounts of hydrogen peroxide (~25 M) can induce a stress response in the organism in the form of increased polyhydroxybutyrate (PHB) synthesis. A percentage increase in the PHB content of the biomass was achieved, however, the overall polymer yield was lower due to lower biomass growth.
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Effect of oxidative stress on the differentiation of mesenchymal stem cells.
Bura, Radek ; Tlapáková, Tereza (advisor) ; Pacherník, Jiří (referee)
Mesenchymal stem cells are capable of forming different types of tissue such as muscle, bone, fat or cartilage tissue, thanks to the ability to divide and transform into another cell type. Mesenchymal stem cells obtained from various tissues are used for cell therapy and regenerative medicine. Knowledge of the influence of various factors on the differentiation of these multipotent cells is important. Currently, little is known about the effect of reactive oxygen species (ROS) on the differentiation potential of mesenchymal stem cells. The aim of this bachelor's thesis is to summarize the knowledge of the effect of oxidative stress on the differentiation of mesenchymal stem cells and the affected signaling pathways.
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Sublethal effects of pesticide mixtures on crayfish
HOVORKOVÁ, Anna
Prometryn, a triazine herbicide, was banned in Europe in 2004. Metazachlor, a chloracetamide used to treat mainly oilseed crops, is one of the herbicides still in use. Individual pesticides or their metabolites can cause adverse effects in the aquatic environment, and the resulting mixtures of pesticides that can form in the environment are another issue in terms of adverse effects in the environment. The aim of this study was to assess the effect of mixtures of pesticides mixed from selected concentrations (0.01 mg.l-1, 0.1 mg.l-1, 1 mg.l-1). Pesticides were also tested individually at selected concentrations. The biochemical profile of hemolymph and antioxidant biomarkers and oxidative stress biomarker in hepatopancreatic, muscle and gill tissues of marbled crayfish were determined. Toxicity was assessed using a subchronic toxicity test performed over a period of 8 days. Metazachlor concentrations (0.1 and 1 mg.l-1) caused decreasing ALT, Cl-, Mg2+ and Ca2+ levels and increasing NH3 levels in hemolymph. The effect of all tested mixtures was observed on the increase of GLO, ALT and NH3 levels, here a synergistic effect of the mixtures was observed. The activity of antioxidant biomarkers was affected only in CAT for prometryn (0.01 mg.l-1) and metazachlor (0.01 and 1 mg.l-1). LPO levels were affected by metazachlor (0.01 mg.l-1). There was no effect of pesticide mixtures on LPO levels and antioxidant enzyme activities.
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Akutní a chronické působení neonikotinoidových látek na vodní organismy
STROUHOVÁ, Alžběta
Neonicotinoids, synthetic substances derived from the natural alkaloid nicotine, are one of the newest class of pesticides. Neonicotinoids are selective for invertebrate nicotinic acetylcholine receptors (nAChRs) and are widely used to treat crops around the word. The aim of this study was to evaluate acute and chronic toxicity of neonicotinoid substance acetamiprid and neonicotinoid product Mospilan 20 SP, which contains 20 % of acetamiprid, to aquatic organisms. Chronic effects to the biochemical profile of haemolymph, oxidative stress biomarker and antioxidant biomarkers have been evaluated too. Marbled crayfish (Procambarus virginalis) was used as a model organism to provide the toxicity. The acute toxicity (96hLC50) of the pure neonicotinoid substance acetamiprid to marbled crayfish is 0.8 mg.l-1, of Mospilan it is 2.71 mg.l-1. The theoretical acute toxicity of Mospilan is 4.0 mg.l-1. It was calculated based on the acute toxicity of pure acetamiprid as the active substance of Mospilan. Additives in the preparation probably contributes its toxicity more than the active substance itself. Acetamiprid and Mospilan impact on changes in the levels of biomarkers of the biochemical profile of haemolymph, biomarker of oxidative stress and antioxidant biomarkers. Changes in the levels of biomarkers are already manifested at concentrations corresponding to 0,1 % and 1 % of 96hLC50. These changes include increases in GLU, AST, ALT and LDH levels, decreases in CAT and GST activity and changes in LPO level.
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Vliv koncentrace, teploty a doby působení na biochemické parametry raka mramorovaného (Procambarus fallax f. virginalis) exponovaného neonikotinoidovému pesticidu, thiaklopridu
HUMMELOVÁ, Světlana
The aim of this work was to evaluate the effect of concentration, water temperature conditions and duration of action of thiacloprid on biochemical parameters of marbled crayfish (Procambarus virginalis). The time of this experiment was taken for 56 days (28 days of exposure and 28 days of depuration). Crayfish were divided into groups according to selected concentrations: control group without thiacloprid (K), highest detected environmental concentration 4.5 ?g?l-1 (TEN) and concentration 64.6 ?g?l-1 (TLC) (10 % of the 96-hour lethal concentration for marbled crayfish) and different water temperatures (17 and 23 °C). Crayfish, which was exposed to a lower water temperature conditions (17 °C) during the experiment, have been generally shown lower activity in behaviour against crayfish exposed to a higher temperature (23 °C). The effect of concentrations of thiacloprid (4.5 and 64.6 ?g?l-1) caused in the muscle and hepatopancreas LPO and changed GST activity. Statistically significantly (P<0.01) changes were observed in all tissues (muscle, hepatopancreas and gills), caused by effect of concentrations of thiacloprid (4.5 and 64.6 ?g ? l-1) and temperature condition (17 and 23 °C) during the experiment in SOD, CAT and GSH. The effect of thiacloprid concentration, water temperature condition and duration of action caused in haemolymph significantly (P<0.01) increase in GLU, NH3, AST, ALT and CK. Other parameters in haemolymph (LDH, TP, Ca2+, Mg2+) were affected only by the water temperature condition. Crayfish in higher water temperature condition (23 °C) showed higher values of these indicators in opposite to crayfish in colder temperature condition (17 °C). No different changes in haemolymph were observed only in two parameters (ALB and GLOB). The highest statistically significantly (P <0.05) changes of individual parameters inside each one group were observed especially after 14 and 28 days of exposure. Especially in higher concentration of thiacloprid and temperature was found that 28 days of depuration time was not sufficient to balance biochemical parameters to normal control values. At the end, it is apt to mention that water temperature condition had influence on individual parameters themselves, when in several cases different values of monitored biomarkers were measured between temperatures in groups exposed to the same concentrations of thiacloprid, even though their control groups showed no differences between temperatures. The results of this work confirm the synergistic effect of thiacloprid, temperature and duration of action on non-target aquatic organisms and could be used for other studies to evaluate the effect of thiacloprid and other pesticides on organisms.
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Molecular consequences of electron transport chain deficiency in proliferating and quiescent cells
Magalhães Novais, Silvia Carina ; Rohlena, Jakub (advisor) ; Panicucci Zíková, Alena (referee) ; Eelen, Guy (referee)
Oxidative phosphorylation (OXPHOS) system is best known for producing ATP in mitochondria. As a major metabolic hub, OXPHOS also supports other biological functions that are unrelated to ATP synthesis. Are these additional roles of OXPHOS unique to different cell states, such as to proliferation and quiescence, two of the most contrasting states of a mammalian cell? In this thesis we used genetic models of OXPHOS deficiency to show that in proliferating cells and in tumors OXPHOS primarily supports biosynthesis of nucleotides, whereas in quiescent cells OXPHOS provides oxidative stress resistance. Mechanistically, in growing tumors OXPHOS drives dihydroorotate dehydrogenase (DHODH), an enzyme crucial for de novo synthesis of pyrimidines. Complex II of OXPHOS also acts as a sensor of mitochondrial dysfunction and regulates pyrimidine synthesis for energy conservation. In contrast, in quiescent cells OXPHOS maintains autophagy by continuous low level ROS generation and together with the antioxidant defense provides constitutive oxidative stress protection. In summary, beyond ATP production OXPHOS has additional vital roles tailored to the particular requirements of quiescence and proliferation, with relevance for pathophysiology in cancer and vascular biology.
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Oxidative Post-translational Modifications and Their Importance
Šimková, Anna ; Míšek, Jiří (advisor) ; Petrák, Jiří (referee)
Aerobic life brings the inevitable exposure of living systems to reactive oxygen, nitrogen and chlorine species. A wide variety of oxidants can alter the structure of biomolecules such as proteins, lipids and DNA. Oxidative post-translational midifications play an important role in a number of cellular processes, for example, they are involved in redox signaling, gene transcription and metabolism. The increase of oxidants in cells leads to oxidative stress, which is associated with the development of neurodegenerative, cardiovascular and autoimmune diseases in humans. Considering reactive species as specific molecular agents can shed light on the complexity of the connection between redox processes and develop the emerging field of redox medicine.
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