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Mitochondrial subpopulations in rat myocardium - effect of chronic hypoxia
Kovalčíková, Jana ; Nováková, Olga (advisor) ; Žurmanová, Jitka (referee)
Adaptation to chronic hypoxia induces endogenous cardioprotection and increases the heart resistance to ischemia/reperfusion injury. The heart mitochondria, which produce reactive oxygen species (ROS) in addition to ATP, play an important role in these processes. During ischemia/reperfusion, ROS are produced in excessive amounts and damage the cells. However, in lower concentrations, ROS are involved in the signalling pathway of cardioprotection induced by adaptation to chronic hypoxia. In the heart, two mitochondrial subpopulations have been observed, subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria (IMFM), which differ in cell localization as well as in morphological and biochemical properties. The aim of this work was to introduce the method of SSM and IMFM isolation in our laboratory and to analyse their antioxidative capacity after adaptation to chronic hypoxia. Adult male Wistar rats were kept either under normoxic conditions or exposed to intermittent high-altitude hypoxia (IHA; 7000 m, 5 days a week/8 hours a day, totally 25 exposures). Mitochondrial subpopulations were isolated from heart left ventricle and their functionality was verified by measuring oxygen consumption and enzyme activities. The IMFM had higher oxygen consumption in comparison with SSM and activities...
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Biotransformation aspects on novel carbocyclic nucleoside analogs.
Rozumová, Nela ; Mertlíková Kaiserová, Helena (advisor) ; Rumlová, Michaela (referee)
Carbocyclic nucleoside analogs with norbornane moiety that have been synthesized at IOCB AS CR, represent new potential chemotherapeutic agents with significant activity against Coxsackieviruses. The main objective of this work was to study the metabolism and mechanism of action of the original analog carbocyclic nucleoside MS 254, which is characterized by its antiviral and cytostatic effects. The attention was partially paid also to the two structurally related substances (MS 255, MS 320). In this work, we determined cytotoxicity of these compounds in cell culture and the effect of MS 254 on the amount of total and oxidized glutathione, activity of glutathione-S-transferase (GST), glutathione reductase (GR) and the effect on cellular oxidative stress. The kinetics of the conjugation of MS 254 by human GST was also studied. It was found that of the three substances tested MS 255 was the most cytotoxic and MS 254 was the least cytotoxic compound. It was further found that MS 254 does not cause significant oxidative stress and that it increases the activity of GST and GR in a dose-dependent manner. Michaelis-Menten constant of the conjugation of MS 254 with the glutathione (main metabolic pathway) was determined in the milimolar range, indicating a relatively low affinity of MS 254 for GST.
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Proteins demaged by oxidative stress, their role in physiology and ageing of yest cells
Mikešová, Jana ; Palková, Zdena (advisor) ; Borčin, Kateřina (referee)
Reactive oxygen species (ROS) are regularly produced in cells as a by-product of aerobic metabolism. Hence, organisms developed various defence mechanisms, which are able to avoid molecular damages caused by ROS under physiological conditions. In stress conditions, however, such defence mechanisms are not sufficient to avoid molecular damages. Accumulation of oxidized proteins is supposed to be a reason for ageing and many diseases including Friedreich's ataxia, Amylotrophic lateral sclerosis, Alzheimer's disease and many others. During oxidative stress, reactive oxygen species are reflected in oxidation of cystein residues in transcription factors, regulation proteins and active canters centers of enzymes. Oxidative modifications however could lead also to changes in transcription factor activity and activation of specific pathways, including changes in gene expression, cell cycle and proteolysis. This work shows defence mechanisms, ROS and proteins altered by reactive oxygen species that may function as important signalling molecules, which are essential for many cellular processes.
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Oxidative stress as the possible outcome of opioid treatment
Böhmová, Barbora ; Novotný, Jiří (advisor) ; Žurmanová, Jitka (referee)
Free radicals and reactive oxygen species are continuously formed in all aerobic organisms. They may be implicated in ethiology of some diseases such as cancer, atherosclerosis, rheumatoid arthritis, liver, renal, cardiovascular, and neurodegenerative diseases and premature aging. Free radicals and reactive oxygen species can also cause oxidative damage to important cellular components - proteins, lipids, and DNA. Cells are equipped with antioxidant systems to neutralize these radicals. The antioxidant system includes some vitamins (ascorbic acid, α-tocopherol, β-carotene) and enzymes, e.g., superoxide dismutase converting superoxide radical to hydrogen peroxide and molecular oxygen or catalase destroying H2O2. Another important H2O2-removing enzyme in human cells is glutathione peroxidase. Reduced activity of antioxidants leads to excessive production of free radicals resulting in oxidative stress. Opioids belong among pro-oxidants (chemicals inducting oxidative stress). Opioids can decrease activity of antioxidant enzymes and generate reactive oxygen species. The aim of this bachelor thesis is to gather information regarding the opioid ability to elicit oxidative stress. It briefly summarizes our current knowledge about opioids, oxidative stress, free radicals and antioxidants. Key words Opioids,...
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Effect of iron overload on the induction of apoptosis in mammalian cells
Kabíčková, Tereza ; Balušíková, Kamila (advisor) ; Klíma, Martin (referee)
Iron cations are an important metal ions required to number of essential cell functions. On the other hand, ferrous iron can be very toxic as well. When surplus iron is present in cells, it can catalyze the formation of reactive oxygen species (especially hydroxyl radicals) by Fenton reaction. Iron homeostasis is predominantly regulated by very strict mechanisms on the level of iron uptake into the body. Moreover, iron absorption, transport and storage within the body can be also regulated using complex mechanisms which differ on the level of individual cells and on the level of whole organism. Deregulation of iron homeostasis causing an iron overload and generation of reactive oxygen radicals can evoke serious cell damage leading up to apoptotic cell death. Excess iron storage and subsequent development of oxidative stress can affect lot of different tissues in the body. The organ damages such as fibrosis, cirrhosis, hepatocellular carcinoma, heart failure, loss of β cells and glucose intolerance or diabetes mellitus in patients with iron overload are very often seen. Nevertheless, the apoptosis induced by iron overload has not been well elucidated yet. There are no complex informations about the precise mechanism by which oxidative stress affects different cell types or whether there are other...
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Mitochondrial production of reactive oxygen species and its role in physiological regulations
Holzerová, Eliška ; Mráček, Tomáš (advisor) ; Kalous, Martin (referee)
The production of mitochondrial reactive oxygen species and the resulting oxidative stress is an important phenomenon driving long-lasting research and intense discussions. Knowledge of exact mechanisms of reactive oxygen species production and pathways leading to their formation could help us to directly affect their production, a task with potential terapeutic implications. The molecular nature of the production of reactive oxygen species by some enzymes has already been well documented, but others still remain controversial and current theories are obviously far from the truth. Much more interesting is the question of physiological importace of this production. The reactive oxygen species were considered harmful factors clearly distorting the integrity of the organism for a long time. However, recent research suggest that their existence can also be beneficial and effective. Evidently they can serve as a signaling molecules in several metabolic and regulatory pathways occurring in the organism. This bachelor thesis offers insight into the current state of knowledge. It focuses on the most detailed description of the reactive oxygen species production by mitochondrial respiratory chain enzymes. Furthermore, it deals with some signaling cascades, where involvement of mitochondrially generated...
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The influence of oxidative stress on mammalian sperm quality and fertilization ability.
Dudková, Barbora ; Komrsková, Kateřina (advisor) ; Svoboda, Petr (referee)
This bachelor's thesis deals with the oxidative stress and its influence on structures and physiologic functions of mammalian sperm. Oxidative stress is a consequence of imbalance between pro-oxidants and anti-oxidants in the body. It is associated with impaired quality of sperm and it is considered to be one of the major reasons of male infertility, including humans. This work is presented as a research review targeting how and where reactive oxygen species are generated in the male reproductive tract. It summarizes specific structures and physiologic functions that are influenced by reactive oxygen species either positively or negatively and it also covers a protective role of antioxidants in seminal fluid.
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Diversity of methods used for characterization of molluscan hemocytes
Jindrová, Zuzana ; Skála, Vladimír (referee) ; Horák, Petr (advisor)
Hemocytes are the main immune cells of invertebrates; therefore they can be found in molluscs, too. They differ both in morphology and function. The two generally accep- ted morphological types, granulocytes and hyalinocytes, vary in the level of phagocy- tosis and encapsulation, production of reactive oxygen species and nitrogen oxide, and presence of some enzymes. There is an array of methods by means of which hemocy- tes can be characterized. Microscopy serves particularly for study of morphology. An- tigens localized on the surface can be determined by monoclonal antibodies or lectin probes. Hemocytes can be divided on the basis of cell size and granularity using gra- dient centrifugation or flow cytometry. Production of nitrogen oxide and reactive oxy- gen species is monitored by adding appropriate substrate which changes its proper- ties after reaction with the radical. It may become fluorescent, change absorbance of the solution or form a visible precipitate. Another possibility is the use of chemilu- miniscence. The objective of hemocyte research is to explain mollusc-pathogen inter- action. 1
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Molecular characterization of NADPH oxidase in the gut of the tick \kur{Ixodes ricinus}
KUČERA, Matěj
This thesis focuses on characterization of one member of NADPH oxidases - dual oxidase (DUOX) which has been described as the main factor of epithelial immunity in the gut of model organism Drosophila melanogaster. We have identified an orthologous gene coding for DUOX in the tick I. ricinus and described its tissue expression profile. The DUOX is mainly expressed in the gut of unfed ticks and seems to be downregulated upon artificial microbial infection. A fragment of tick DUOX was prepared as recombinant protein and used for preparation of specific antibodies to be used for further characterization of the enzyme. Our main aim is to highlight the importance of tick DUOX producing the reactive oxygen species and their role in the defence against pathogenic organisms within the tick gut.
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Study of senzitizers phototoxicity on molecular level and possibilities their use for photodynamic tumors therapy
ZEMAN, Jan
Photodynamic therapy is a modern and promising method of treating some types of tumors. It can supplement and in some cases even replace the current methods. It is based on the use of a photosensitive substance, a senzitizer, which is located in tumor cells and subsequently exposed to the light of an approporiate wave length, which brings about photodynamic reaction. Photodynamic reaction arises due to the combination of four agents: a senzitizer, molecular oxygen, light of optimum wave length, and a biological substrate (proteins, nucleic acid or other cell components). After the application of the senzitizer, whose prior uptake takes place in the pathologically altered tissues, visible light of a wave length corresponding with the absorption maximum of the photosenzitizer is applied, which brings about tumor cells destruction at active participation of the reactive oxygen forms as superoxide anion, hydrogen peroxide and hydroxyl radicals. My thesis was focused on testing photodynamic properties of ZnTPPS4 senzitizer on tumor cell lines. The production of oxygen reactive forms was tested by Synergy HT spectrofluorimeter and Olympus IX 81 fluorescence microscope with image analysis. Production of reactive oxygen forms was assessed with the use of molecular symbol CM-H2 DCFDA. LED emitters adapted for working with cell cultures were used to irradiate the cell structures. The results proved that ZnTPPS4 is photodynamically effective and the production of reactive forms of oxygen depends on senzitizer ZnTPPS4 concentration as well as on the dose of radiation.
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