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Oxidative damage to cellular components after oxidative stress induction by specific herbicides
Kramná, Barbara ; Wilhelmová, Naďa (advisor) ; Ryšlavá, Helena (referee)
Oxidative stress is caused by overproduction and overaccumulation of ROS (reactive oxygen species). This state is responsible for cellular damage during unfavorable environmental conditions such as drought, low temperatures, salinity. In order to directly study oxidative stress at tobacco plants (Nicotiana tabacum cv. Xanthi) I used specific herbicides, MV (methyl viologen) and 3-AT (3- aminotriazole). There were several markers used for monitoring oxidative damage to cellular components: DNA damage detected by a comet assay, lipid peroxidation, carbonylated proteins and modification of activities of antioxidant enzymes CAT (catalase) and APX (ascorbate peroxidase). Fluorescent microscopy documented changes in a redox state of tobacco cells and a specific signal for peroxisomes was observed after treatment with higher concentrations of MV and 3-AT. Application of both herbicides caused significant DNA damage, while they worked in a different concentrations, MV in µM and 3-AT in mM. Another convincing oxidative stress marker for MV was protein carbonylation. The inhibition of antioxidant enzymes CAT and APX was less significant when compared to the effects of 3-AT. Decreasing membrane stability proved to be an universal oxidative stress marker for both herbicides. On the other hand, lipid...
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The role of mitochondria in adaptation to chronic hypoxia in the spontaneously hypertensive and conplastic rats.
Weissová, Romana ; Kalous, Martin (advisor) ; Rauchová, Hana (referee)
Adaptation to chronic hypoxia provides cardioprotective effects. Molecular mechanism of this phenomenon is not yet completely understood, but it is known that cardiac mitochondria play an essential role in induction of protective effects. The purpose of this diploma thesis is to study effects of continuous normobaric hypoxia (CNH; 10 % O2, 21 days) on spontaneously hypertensive rats (SHR) and conplastic strain that is derived from SHR. These animals have nuclear genome of SHR strain and mitochondrial genome of Brown Norway (BN) strain. Cardiac homogenate was used to measure enzymatic activity of malate dehydrogenase (MDH), citrate synthase (CS), NADH-cytochrome c oxidoreductase, succinate-cytochrome c oxidoreductase and cytochrome oxidase (COX). Using Western blot procedure the protein amount of antioxidant enzymes was measured - manganese superoxide dismutase and copper-zinc superoxide dismutase (MnSOD, Cu/ZnSOD), catalase and chosen subunits of oxidative phosphorylation complexes (Ndufa9, Sdha, Uqcrc2, COX-4, MTCO1, Atp5a1). Under normoxic conditions the conplastic strain has lower amount of complex IV subunit MTCO1 in comparison with SHR. This subunit is encoded by mitochondrial DNA and it is one of the seven protein-coding genes in conplastic strain that differ from SHR. Adaptation to hypoxia causes an...
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Effect of chronic hypoxia on antioxidative capacity of rat myocardium.
Závišková, Kristýna ; Nováková, Olga (advisor) ; Žurmanová, Jitka (referee)
Adaptation to chronic hypoxia activates endogenous signaling cascades, which lead to cardiac protection against acute ischemia/reperfusion (I/R) injury. The molecular mechanism of this phenomenon has not been fully clarified yet. However, it was proved that reactive oxygen species (ROS) take part in cardioprotective signaling pathway inducted by chronic hypoxia. The high level of ROS must be precisely regulated by antioxidative system of a cell. The aim of diploma thesis was to examine the effect of intermittent hypobaric hypoxia (IHH, 7 000 m) on relative amount of antioxidative enzymes (peroxiredoxin 6 - PRX6, thioredoxin 1 and 2 - TRX1 and TRX2, thioredoxin reductase 1 - TRXR1) and also enzymes of iron metabolism (heme oxygenase 1 and 2 - HO1 and HO2, aconitase 1 and 2 - ACO1 and ACO2), which participate in regulation of cell redox state. Moreover, we studied the effect of adaptation to IHH and an antioxidant tempol on relative amount of calcium-independent phospholipase A2 (iPLA2). iPLA2 can remove peroxidized fatty acids from membrane phospholipids. On the other hand, iPLA2 can damage cell in I/R conditions. All enzymes were studied in homogenates from normoxic and IHH adapted rat left ventricular myocardium by Western blot. Adaptation to IHH caused a decrease of PRX6 and on the opposite an increase of...
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Factors affecting uptake and distribution of uranium in plants.
Krejčová, Daniela ; Lhotáková, Zuzana (advisor) ; Tylová, Edita (referee)
Uranium is a radionuclide, which naturally occurs in Earth's soil in rather an insignificant amount. It is not very dangerous in such small concentration; however, this concentration is rising due to anthropogenic activity, therefore an estimation of its increase is at hand. It is necessary to research possibilities of not only effective, but also ecological extermination of this contamination. Phytoremediation could be an appropriate solution, but this method is still in its beginning stages when it comes to uranium contaminations. That's why we need to study influences of uranium on plants and find out to what extent are they suitable for phytoremediation of areas contaminated by uranium. Phytoremediation methods are based on the abilities of plants to receive and accumulate certain pollutants. The factors that influence the amount of uranium received by a plant can be divided into four groups: plant species, uranium's oxidation levels, pH of of the medium or the substrate and ligand, which is attached to the uranium (Mitchell, 2013). Transportation of uranium to the aboveground part of the plant is also a factor here. Plants need to deal with the accepted uranium; aside from the radiological risks resulting from the characteristics of radioisotope, uranium is also a heavy metal. Chemical...
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Antioxidant enzymes response to abiotic stress. Impact of decreased cytokinin level.
Lubovská, Zuzana ; Wilhelmová, Naďa (advisor) ; Prášil, Ilja (referee) ; Kočová, Marie (referee)
Charles University in Prague, Faculty of Science Department of Experimetnal Plant Biology Ph.D. study program: Plant physiology and anatomy Abstract Antioxidant enzymes response to abiotic stress. Impact of decreased cytokinin level. Zuzana Lubovská Supervisor: RNDr. Naďa Wilhelmová, CSc. Supervisor-consultant: RNDr. Helena Štorchová, CSc. Praha, 2015 The response of the antioxidant enzyme system to drought, heat and a combination of these stresses was followed in tobacco plants overexpressing cytokinin oxidase/dehydrogenase1 (CKX1) under the root-specific WRKY6 promoter (W6:CKX1 plants) and under the constitutive 35S promoter (35S:CKX1 plants) and in the corresponding wild type (WT). CKX1 over-producing lines developed a bigger root system, which contributed to their stress tolerance. The high stress tolerance of 35S:CKX1 plants was also associated with a dwarf shoot phenotype and changed leaf morphology. The genes for chloroplastic antioxidant enzymes, stroma ascorbate peroxidase (sAPX), thylakoid ascorbate peroxidase (tAPX) and chloroplastic superoxide dismutase (FeSOD), which are responsible for scavenging of reactive oxygen species produced via electron transfer during photosynthesis, were all strongly transcribed in control conditions. All the tested stresses down-regulated expression of these genes...
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Vliv akumulace manganu na vybrané rostliny
Švec, Pavel
In this work we investigated the effect of high doses on the agriculture important crop plants (Brassica napus, Hordeum vulgare, Zea mays), herb Matricaria chamomilla and freshwater microalgae Scenedesmus quadricauda. In particularly, we studied the growth rate, activity of antioxidant enzymes, and the amount of some secondary metabolites. We have found that high doses of manganese negatively influenced the growth, which resulted in a reduction of biomass which we observed in all studied groups, except algae. Manganese (Mn) is accumulated in roots and part of such absorbed Mn is transported to the shoot system. In the plants, the distribution of Mn is more balanced compared to copper and cadmium. We observed changes in the content of aliphatic organic acids, and the total amount of thiols. Higher activity was determined in several antioxidant enzymes according to the kind of plant. Abundance of manganese has been considered more toxic for the Brassica rapa than for the ontogenetic older Matricaria chamomilla, where we indicated a higher tolerance to the high concentration of Mn. We also found that the nitrogen oxide showed the positive effect on the toxicity of Mn and improved the plant viability. This improvement was reflected by decrease in uptake of Mn ions, decrease of ROS production and lipid peroxidation and also better growth parameters in the tested plants. The bioavailability of the Mn was influenced by pH however the uptake of Mn was not affected. We also found out that competition between Mn2+ and Ca2+ occur in the both land plants and also freshwater microalgae Scenedesmus quadricauda.
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Changes of the intracellular redox state during virus infections
Kompas, Maroš ; Mělková, Zora (advisor) ; Trejbalová, Kateřina (referee)
Viruses are infectious agens, which cause disruption of a host cellular redox homeostasis. This effect is mediated by cellular defense machinery or via viral gene products. In order to restore cellular redox enviroment, activation of cellular adaptive response takes place. That is mediated by transcription factor Nrf2, which leads to upregulation of gene expression of antioxidant enzymes. Under suboptimal redox condition, or by detecting foreign nucleic acid, redox sensitive transcription factor Nf-κB is also being activated, what leads to expresion of proteins mediating cellular imunne responses. It is important to remember that these proteins might show malignant effects to surrounding tissues during long term inflammations. With respect to that, viruses have evolved mechanisms, through which they are able to overcome or hijack these pathways, in order to propagate the infection. Key words: intracellular redox state, ROS, RNS, oxidative stress, antioxidant enzymes, regulation of gene expression, virus infections
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Vliv herbicidu s-metolachloru na raná vývojová stádia raka mramorovaného
MATERNA, Jan
S-metolachlor is a substance belonging to the group of herbicides, widely used in agriculture to protect crops. This substance with the chemical composition C15H22CINO2 and is used as the pre-emergent herbicides to control unwanted weeds. The aim of this work was to evaluate the influence of s-metolachlor on mortality, behavior, growth, ontogenetic development, oxidative stress level and activity of antioxidant enzymes of early developmental stages of marbled crayfish (Procambarus fallax f. virginalis). The effect of s-metolachlor was evaluated on the basis of embryo-larval toxicity test. Early developmental stages of crayfish were exposed to three concentrations of s-metolachlor, 1.1 micrograms/l, 11 micrograms/l and 110 micrograms/l for 45 days. All tested concentrations significant showed higher mortality, delay of ontogenetic development, lower growth and changes in behavior (total movement distance and movement speed). Moreover, significant decrease in catalase, glutathione-s-transferase, superoxide dismutase concentration, and reduced glutathione levels at the two higher test concentrations (11 micrograms/l and 110 micrograms/l) of s-metolachlor were observed. No significant changes in oxidative damage (TBARS levels) were observed in all the tested groups. Based on the results of this study, early developmental stages of marbled crayfish can be recommended as suitable organisms for toxicity test and their use for biomonitoring of the aquatic environment by herbicides.
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Teplotní závislost motility spermatu u různých druhů ryb
DADRAS ASYABAR, Hadiseh
The reproduction of fish either in nature or controlled condition in aquaculture is a biological event that can be strongly related to the reproductive success. In addition, study of sperm physiology and control of sperm quality can be taken into account as an issue to obtain successful production in aquaculture systems. It is often admitted that sperm quality as one of the limiting factors of the fertilization success is affected by different factors, so some researches have paid attention to sperm quality. Motility is regarded as one of the most reliable parameters for predicting sperm quality, and to evaluate the sperm fertilizing potential, observation of decrease in sperm motility performance under in vivo or in vitro circumstances has been commonly used. These studies were conducted based on the hypothesis that aims to assess sperm motility characteristics and some physiological changes of spermatozoa (such as antioxidant enzymes activity and lipid composition) in out range of spawning temperature of each species. The investigation of the fish spermatozoa motility function and enzymatic response in relation to different in vitro temperatures was carried out using activation of spermatozoa from taxonomically distant fish species (common carp, rainbow trout and sterlet) at 4, 14, and 24° C. Special attention was taken to the sperm motility parameters (motility rate, duration and velocity), activity of catalase (CAT) and superoxide dismutase (SOD), and thiobarbituric acid-reactive substance (TBARS) content as marker of lipid peroxidation. As results of this study, duration of motility in three aforementioned species were shown to be longest at 4° C compared to 14 and 24° C. In rainbow trout and carp, velocity was significantly increased at 14 °C after 10 and 30 s of spermatozoa activation respectively. While, at 60 s post-activation, the velocity of spermatozoa in sterlet was highest at 24° C. Activity of CAT was in the highest level at 4° C in rainbow trout and sterlet, while no difference was observed in carp spermatozoa. As second aim of the present study, lipid composition was analyzed in motile or immotile carp spermatozoa at different in vitro temperatures (4, 14 and 24° C). The lower proportions of some fatty acids, 18:3 (n-3) and 22:6 (n-3) in motile spermatozoa were detected at 24° C. With respect to importance of phospholipids as source of energy in fish spermatozoa movement, the total phospholipid content was lesser in motile than in immotile spermatozoa at 24° C. At 24° C also, phosphatidylcholine and phosphatidylserine proportions were lesser in motile than immotile spermatozoa. On the basis of these data, lipid composition of carp motile spermatozoa is affected by temperature, with decrease in proportion of some lipid compositions at higher temperature, elevation of sperm curvilinear velocity and decreased duration of the period over which motility is sustained. The third study was designed to investigate role of different factors that might control the activation of spermatozoa motility, using physiological functions of osmolality, calcium (Ca2+) ion and particularly temperature in burbot. Spermatozoa motility was tested at temperatures of 4 and 30° C in following media: seminal fluid (SF), isotonic media (plus Ca2+ and without it) and hypotonic media (plus Ca2+ and without it). With regard to activation of burbot spermatozoa motility in seminal fluid at high temperature (30° C), it seems that no risk of semen handling will occur during hatchery routine practice. The results suggest that initiation of spermatozoa motility in burbot is mediated by the simultaneous involvement of Ca2+ and temperature. Thus, it might be concluded that burbot spermatozoa is more sensitive to high temperature for initiation of motility relative to other freshwater fish species.
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Změna abundance mRNA, oxidativních produktů a aktivit antioxidačních enzymů spojených se stárnutím rybích oocytů
MOHAGHEGHI SAMARIN, Azadeh
In fish, delayed spawning in nature, delayed egg collection in capture and delayed fertilization after egg stripping lead to excessive oocyte ageing and finally over-ripening. Until now there has been only poor understanding about the processes and underlying mechanism of oocyte ageing in fish as well as other vertebrates. Some studies on other vertebrates have suggested the oxidative stress as initiating factor for the progress of oocyte ageing. The present work investigated possible changes in the mRNA levels of transcripts involved in oxidative damage, mitochondrial function and stress response, with roles in fertilization, embryo development, transcriptional regulation and cell cycling as well as the ones related to the apoptosis during the egg over-ripening. This study investigated the oxidation status of oocytes during post-ovulatory ageing by measuring TBARs as the marker of lipid oxidation and carbonyls which shows the extension of protein oxidation. In addition the role of oxidative stress in the progress of oocyte ageing was assessed by evaluation the activity of antioxidant enzymes, CAT, SOD, GPX and GR. Possible changes in lipid class and fatty acid composition during fish oocyte aging were also examined. Egg viability parameters and larval ploidy levels were examined. Complete loss of egg viability for African catfish (Clarias gariepinus) occurred at 16 and 24 Hours Post Stripping (HPS) when eggs were stored at 25°C and 4°C respectively. Under both storage temperatures, the embryo mortality and larval malformation rates increased significantly over time and were the highest in the most aged oocytes. We determined the time in which the eggs retain their fertilizing ability 10 hours for tench (Tinca tinca) at 20 °C, 18 hours for goldfish (Carassius auratus) at 20 °C and 14 hours Post Ovulation (HPO) and more than 10 Hours Post Stripping (HPS) for common carp (Cyprinus carpio) at 20 °C. Our results demonstrated no significant changes in the mRNA levels of oxidative stress related genes and genes involved in cell cycling during the progress of oocyte ageing in any of species; African catfish, goldfish and common carp. Additionally, with elapsing time following ovulation the amount of TBARs and carbonyls, did not change in any of the oocytes from our experimented species; African catfish, tench, goldfish and common carp. However an increase in the mRNA abundance of apoptotic related genes were observed. Antioxidant enzyme analysis indicated no significant changes in the activity of CAT and SOD during post-ovulatory ageing of common carp, goldfish and tench oocytes. However, a significant decrease in the activity of GPX was observed during post-ovulatory ageing of tench oocytes which could play a major role in the overall drop of egg quality. This observation in GPX activity was not observed in common carp and goldfish oocytes. Besides the direct effects of oxidative stress on oocytes, also changes of fatty acid and lipid composition caused by oxidation could affect the functionality. We observed that post-ovulatory ageing of the oocytes does not change the fatty acids and lipid class composition of the oocytes. Therefore, according to our obtained results, oxidative stress is not the main initiator or promotor of the oocyte ageing process. However, complementary tests and analysis are required to clearly clarify its involvement. Increased mRNA levels of apoptotic related transcripts during oocyte ageing in this study demonstrates that apoptotic pathway might be involved in the molecular changes during the progress of oocyte ageing. Investigation of epigenetic changes associated with fish oocyte ageing seems to be interesting for future research works.
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