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Influence of Storage Conditions on Metabolic Profile of Apples
Duroňová, Kateřina ; Vávrová, Milada (referee) ; Kráčmar, Stanislav (referee) ; Márová, Ivana (advisor)
The goal of presented dissertation has been complex analysis of changes in content of fatty acids, enzymatic and low-molecular antioxidants in apples and related evaluation of perception of originators of storage diseases for apples stored in various conditions. Main part of the work has been dedicated to study of the impact of storage of apples in modified atmosphere with reduced amount of oxygen and in reference “normal” atmosphere for six months. Next part of the work has been dedicated to study how apple storage in common, consumer affordable, conditions (storage in a cellar, in a refrigerator, and in room temperature) affects content of low-molecular antioxidants. For testing has been selected apple kinds Jonagored, Golden Delicious, Idared, Šampion, and Granny Smith. Within the scope of this work has been optimized the method for determination of fatty acids in plant material with higher content of wax. The measured values imply the apples are valuable source of many important nutrition substances like vitamins, provitamins and antioxidants. During the storage process these substances exhibit considerable protective function. Long-term storage, mainly in the atmosphere with reduced amount of oxygen (FAN), enables preservation of majority of these important nutrition substances depending on the kind of apple and conditions of storage. Freezing process is conservative to apples (mainly in the presence of protective substances), while in the process of drying the values of all monitored antioxidants decrease depending on temperature and conditions of drying. Upon the choice of the storage method one must consider nutrition, sensoric characteristic and consumer demands.
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Changes of enzyme activities in fruits during long-term storage
Ferdová, Jitka ; Melounová,, Jitka (referee) ; Márová, Ivana (advisor)
This study is focused on study of changes of enzyme and low-molecular weight antioxidants in different fruits during long-term storage. In theoretical part individual low-molecular weight antioxidants and enzymes are described. The main causes of fruit decay and some possibilities of fruit preservation and storage are summarized. As biological material some common fruits were chosen - green and red apples, peaches, plums and white grapes. The fruits were stored in laboratory, cellar, in refrigerator and in freezer. In freezing experiments some ways of fruit preparation and processing were tested and their influence on fruit antioxidant status was compared. Shortened storage experiment was applied on blueberries, cranberries, raspberries and strawberries too. In fruits some group parameters – total antioxidant status, dry mass content, ascorbate level, total flavonoids and total phenolics were analyzed spectrophotometrically. Individual flavonoids and phenolics were determined by RP-HPLC/UV-VIS and on-line LC/PDA/ESI-MS. Antioxidant enzyme activities (superoxide dismutase SOD, catalase CAT, polyphenol oxidase PPO and lipoxygenase LOX) were measured by spectrophotometry. The surface microscopy and cultivation of moulds from fruit surface were performed too. Influence of storage conditions on biological activities is dependent on fruit sort. Freezing is the most suitable procedure for long-term storage without significant changes of active substance content. Long-term storage in controlled temperature conditions and/or atmosphere is usable for fruits with longer storage period. In these fruits stabile levels of antioxidant enzymes are stored for relatively long time. Some of enzymes act synergistically. Enzyme activities differed according to storage phase; at the beginning mainly high SOD and LOX activities were observed. CAT and PPO are probably activated as defence systems in rippened and/or damaged fruits. Levels of total as well as individual low molecular weight antioxidants varied during storage in all sorts, generally, increased course with longer storage period can be observed.
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Influence of storage conditions on content of biologically active substances in apple fruits.
Ferdová, Jitka ; Čarnecká, Martina (referee) ; Márová, Ivana (advisor)
This study deals with antioxidants in diet and their effects on human organism. Further, it summarizes the agents affect the quality of apples in the course of long-term storage and it outlines the possibility of defence against them. In the experimental part methods of determination of antioxidant enzymes superoxid dismutase (SOD), catalase and polyphenol oxidace (PPO) in apples were introduced. The enzymes were measured in apples tissues in liquid nitrogen after 158 days in normal or modified atmosphere. In Apple the quantitative and qualitative analysis of proteins was realized. Further, some low molecular antioxidants (total phenolics, total flavonoids and vitamin C) as well as total antioxidant status were measured in frozen raw juice. This values were compared with values from apples analyzed immediately after the harvesting. Artificial inoculation with fungi was made in the last part and the fruitfulness was observed.
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Úloha regulačních proteinů pro pohyblivost rybích spermií
DZYUBA, Viktoriya
The investigation of the energetic aspects of spermatozoon motility implementation (Chapter 2) was carried out using demembranated spermatozoa of taxonomically distant fish species (common carp and sterlet). Special attention was given to the functioning of ATP regeneration systems: adenylate kinase (AK), and creatine kinase (CK). It was shown for the first time that the phosphocreatine/CK system is present in sterlet spermatozoa and plays an essential role in ATP regeneration. Spermatozoa of carp and sterlet were shown to have similar systems for ATP regeneration, while the efficacy of the studied systems differs in these species. The low baseline activity of CK in carp and AK in sterlet suggest these to be the source of the most pronounced effects of their inhibition on energy supply for flagella movement in the respective species. The presence of a maturational process during the post-testicular transit of sperm in sturgeon was recently ascertained in our laboratory (Chapter 3). This discovery prompted investigation of the factors that regulate this process including the involvement of proteolysis regulators and prooxidant-antioxidant system. As a result of this study (Chapter 3.3), we found that there was no significant difference between proteolytic profiles of seminal fluids (SF) of testicular sperm (TS) and Wolffian duct sperm (WS). It suggests that the majority of proteases present in SF of mature sperm originate in the testis. Measure of amidase and anti-proteolytic activities in the SF of sterlet sperm showed significant decrease in activities as the sperm passed through the kidneys and Wolffian ducts. Considering our observation that trypsin inhibition during in vitro TS maturation blocked the maturation process (Chapter 3.1), and based on zymography, amidase and anti-proteolytic activity determination, we think that the decrease in anti-proteolytic activity of spermatozoa surroundings during their post-testicular transit could be needed to prepare them for maturation. The present study showed that maturation of sturgeon spermatozoa and different times of storage in Wolffian ducts (in vivo storage), are accompanied by significant alterations in motility parameters as well as in SF redox balance (Chapter 4.1). A high level of TBA-reactive substances (TBARS) and a high activity of antioxidant enzymes were found in immature TS compared to those in WS. The high activity of the enzymatic antioxidant system (AOS) allows TS to cope with the deleterious effects of excessive reactive oxygen species production and to retain the ability to become motile after post-testicular transit, or after in vitro maturation. The increase in TBARS content during in vivo storage was not accompanied by a corresponding increase in activity of AOS. We suggest that extended time in the Wolffian ducts resulted in sperm oxidative damage resulting from inadequate AOS efficacy and, finally, in decreases in motility parameters. Short-term hypothermic in vitro storage of sterlet sperm resulted in a significant decrease in motility and velocity without changes of AOS activity (Chapter 4.2). It means that AOS of sterlet sperm is effective enough to prevent the development of oxidative stress during short-term storage. Short period of tench sperm exposure to hypotonic conditions was shown to induce oxidative stress and, as a result, sperm quality decline (Chapter 4.3). The combined results of the study concerning the regulation of sperm prooxidant-antioxidant status (Chapter 4) during spermatozoa maturation, motility activation and sperm in vivo and in vitro storage may confirm a dual role of reactive oxygen species (regulatory or damaging depending from the levels of their formation and elimination) in fish sperm physiology.
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Role reaktivních forem kyslíku a proteinové fosforylace na funkci spermií ryb
GAZO, Ievgeniia
Spermatozoa of externally fertilizing fish species after releasing into aqueous environment are particularly vulnerable to damage mainly due to alterations in composition of media surrounding sperm. Among factors affecting spermatozoa movement in external medium are water pollutants, temperature, pH and osmotic conditions. The goal of this thesis was to investigate the possible effects of oxidative stress on sperm performance and intracellular signaling, particularly the effect of pollutants occurring in water environment. In addition the molecular mechanisms of stress response and motility activation for spermatozoa of several freshwater fish species were analyzed. Our results show that xenobiotics, such as vinclozolin, induce a dose-dependent reduction in sterlet (Acipenser ruthenus) spermatozoa motility and velocity at environmentally relevant concentrations. Increased levels of lipid oxidation (LO) and protein carbonylation (CP), as well as changes in antioxidant activity of superoxide dismutase (SOD) indicate the development of an oxidative stress in spermatozoa exposed to xenobiotic. Moreover, increased DNA fragmentation as well as a reduction of the level of ATP was observed in spermatozoa incubated with xenobiotic in vitro. These results demonstrated that sterlet spermatozoa are highly susceptible to the presence of pollutants, which induce excessive ROS production even at low concentrations. Further studies were performed in order to evaluate the role of ROS production in fish sperm and protective properties of seminal plasma. The ROS were generated in carp (Cyprinus carpio L.) spermatozoa by in vitro incubation with xanthine - xanthine oxidase system (X-XO). A time- and dose-dependent reduction in spermatozoa motility and velocity was observed as well as increased LO, CP and DNA fragmentation. Moreover, it was shown that O-linked N-acetylglucosamine transferase and septin-8-A changed their phosphorylation state on tyrosine residue, and acid phosphatase activity decreased in response to oxidative stress. On the other hand, catalase (CAT), superoxide dismutase (SOD), and glutathione (GTH) in combination with seminal plasma can reduce oxidative stress in carp spermatozoa and improve sperm quality. Our next study was applied to determine how the protein phosphorylation pattern changed after motility activation in carp and sterlet spermatozoa, where phosphorylated proteins are located in spermatozoon and to identify proteins involved in sperm motility. It was shown that the pattern of protein phosphorylation and their localization differs significantly between two species. Phosphorylation on serine and tyrosine residues, as well as protein kinase A (PKA) and protein kinase C (PKC) substrates play an important role in spermatozoa motility activation and regulation in both species. Numerous signaling proteins involved in carp and sterlet spermatozoa movement were identified in this study, giving a better understanding of molecular mechanisms underlying sperm motility. As a conclusion, the results of this study provide new data on the effect of xenobiotics and oxidative stress on fish spermatozoa motility, DNA integrity, lipid and protein oxidation, antioxidant defense system and intracellular signaling. These data proved the toxicity of water pollutants and ROS for fish spermatozoa and proposed the use of CAT, SOD, or GTH in combination with seminal plasma to reduce oxidative stress in these cells. Moreover, we identified many spermatozoa proteins involved in stress response and motility. In practice, the data presented in this thesis could be useful for elaboration of suitable medium for cryopreservation and artificial propagation of freshwater fish species.
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Vliv vybraných kardiovaskulárních léčiv nalézaných ve vodním prostředí na ryby
STEINBACH, Christoph Antonius
Cardiovascular pharmaceuticals are among the most prescribed drugs. As a result of the high consumption, these pharmaceuticals have been frequently detected in waste and surface waters. Verapamil, diltiazem and atenolol are very important representatives of cardiovascular pharmaceuticals; therefore, the present research focused on their acute and sub-chronic effects, bioconcentration, half-life time and metabolism in fish. Moreover, unified protocol for the quantitative assessment of histopathological alterations on the heart ventricle and coronary blood vessels employing heart index calculation was developed with the aim to better assess histopathological changes in fish heart which is one of the targets of cardiovascular pharmaceuticals and other chemicals. The effects caused by high concentrations of the studied substances, verapamil, diltiazem and atenolol, in fish can be considered similar to the therapeutic effects and side effects that are found in humans. The acute exposure to verapamil at the human therapeutic plasma level reduced the heart rate in common carp embryos and larvae. In addition, the acute and chronic exposure to this substance caused peripheral edema and gastrointestinal haemorrhage in carp. Similarly, the histological changes in heart and the blood vessels of the liver in diltiazem exposed rainbow trout suggested vasodilatation similar to the pharmacological effect of diltiazem in the human body. In rainbow trout sub-chronically exposed to atenolol at a human therapeutic blood plasma concentration, histopathological changes in the cardiovascular system were found. The bioconcentration of verapamil, diltiazem and atenolol in fish can be classified as low. Bioconcentration factor (BCF) of verapamil in whole body homogenates of common carp ranged between 6.6 and 16.6. The BCF of diltiazem was also relatively low (0.5-194) in analysed tissues of trout, following the order kidney liver muscle blood plasma. BCF of atenolol in rainbow trout tissues was the lowest among the tested substances (BCF = 0.002-0.27), following the order of liver > kidney > muscle. In the blood plasma, the concentration of atenolol was below the limit of quantification. Verapamil showed a longer half-life time (10.6 days) in fish compared to the human body, indicating the slow rate of biotransformation and/or elimination of verapamil in fish. Estimated half-life times of diltiazem in liver (1.5 h) and kidney (6.2 h) were in the same order of magnitudes as those determined for the human blood plasma. The half-life time of atenolol in trout was not studied, because of its very low bioconcentration. In diltiazem exposed rainbow trout, 8 groups of metabolites of diltiazem with 17 different isoforms were identified using liquid chromatography/high resolution mass spectrometry method. Diltiazem was found to undergo a biotransformation involving desmethylation, desacethylation and hydroxylation in fish. These results showed that diltiazem was metabolised in fish in a similar way like in the human body by desmethylation and desacethylation. On the other hand, hydroxylation, which was involved to a minor extent, seemed to be species specific. Verapamil had no effect on early life stages of common carp at the environmentally relevant concentration after one month lasting exposure. On the other hand, atenolol and diltiazem in environmentally realistic concentrations caused after 42-day exposure some physiological changes in rainbow trout. Namely, atenolol affected haematological and biochemical parameters of the blood in exposed rainbow trout and diltiazem caused changes in the activity of antioxidant enzymes in trout liver and gills. These data indicated that atenolol and diltiazem, when present in the aquatic environment, could be a source of sub-lethal detrimental effects in fish.
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The effect of metribuzine on oxidative stress and antioxidant enzymes of signal crayfish
LIDOVÁ, Jaroslava
The aim of this study was to investigate effects of the triazine herbicide metribuzine on oxidative stress level and antioxidant enzymes activity in gills, muscle and hepatopancreas of signal crayfish (Pacifastacus leniusculus Dana) and also extension of knowledge about effect of metribuzine on the environment. The experiment took up 60 days. Crayfish were exposured to metribuzine concentrations of 0.52 micrograms.l-1 (real environmental concentration) and 3.06 mg.l-1 (10% 96hLC50) for the first 30 days. Then a second phase followed depuration without metribuzine (30 days). Changes in the oxidative stress level (TBARS), superoxiddismutase (SOD) activity and catalase (CAT) activity were observed in all examined tissues. Changes in glutathionreductase (GR) activity were observed only in hepatopancreas. Chronic exposure of metribuzine demonstrated an oxidative damage of cell lipids, proteins and also changes in antioxidant activity in examined crayfish tissues.The results of this study suggest that crayfish are a very suitable organisms for toxicological tests and simultaneously extend knowledge about effect of metribuzine on the environment.
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Vliv triazinových pesticidů na ryby
STARÁ, Alžběta
Fish and crayfish are widely used as biological monitors of environmental levels of anthropogenic pollutants. The present thesis is a contribution to the assessment of the toxicity and effects of long-term effect of triazines on the different developmental stages of common carp (Cyprinus carpio L.) and adult red swamp crayfish (Procambarus clarkii). The carp was selected as a model fish due to its economic importance, e.g. carp farming contributes about 90% to total fish production in the Czech Republic. Crayfish are easily identified species representing given locality, they are widespread, and they provide a sufficient amount of tissue for individual biochemical and chemical analyses. The results of these studies provide further data on chronic exposure to triazines for consideration in risk assessment. We selected three active substances of triazine herbicides which are the most frequently detected in surface waters such as prometryne, simazine and terbutryn. The findings contribute to knowledge of the toxic potential of triazine herbicides to carp and crayfish at environmentally relevant concentrations in Czech rivers. There is a scarcity of information regarding the toxicity of triazines on freshwater organisms. During the tests we monitored several parameters: behaviour, mortality, biometric, haematological, biochemical blood, histopathology, oxidative stress and antioxidants. The data obtained from all tests performed during my thesis are very valuable for assessment and evaluation of long-term effects xenobiotics on aquatic organisms, especially fish and crayfish. In the future, I would extend the focus of my research for study of possible synergic or amplifying effect of mixtures triazines with other xenobiotics which are often found in the aquatic environment. This approach is recently accented as the aquatic environment is polluted by mixtures of different compounds. Therefore, more research is needed in order to clarify the more detailed effects of xenobiotics on non-target aquatic organisms.
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Effect of oxidative stress on antioxidant enzymes in \kur{Drosophila melanogaster}.
SZAKOSOVÁ, Klára
Oxidative stress reflects an imbalance between reactive oxygen species and antioxidant defense, which can result in cellular damage. The reactive radicals can interact with cell components, including proteins, lipids and nucleic acid. Antioxidant enzymes are the main defense against oxidative stress, representing by two the most important enzymes, catalase and superoxide dismutase. In this thesis I studied the effect of oxidative stress on activity of antioxidant enzymes in Drosophila melanogaster and Locusta migratoria, after exposure to the herbicide paraquat.
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