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Application of Selected Methods for Oxidative Stress Analysis
Lízalová, Martina ; Tremlová, Bohuslava (referee) ; Beklová, Miroslava (referee) ; Vávrová, Milada (referee) ; Lojek, Antonín (advisor)
Chronic pancreatitis (CP) is a heterogeneous disease defined as chronic inflammatory changes of the pancreatic tissue caused by variety of aetiologies. Oxidative stress accompanying the inflammatory processes has been suggested as an important factor contributing to CP development. The aim of this study was to determine levels of lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), together with nitrites, the total antioxidant capacity, cytokines, biochemical and haematological parameters in the plasma of patients with CP and control subjects. Levels of MDA and 4-HNE were analyzed using high-performance liquid chromatography. The total antioxidant capacity of plasma against peroxyl radicals was evaluated using chemiluminescence determination. Nitrites were determined using Griess reaction. Cytokines - TNF-alfa; TNF RI; PDGF-AB; TGF-beta, together with myeloperoxidase and hyaluronan were determined using ELISA Kits. Biochemical and haematological parameters were measured by standard methods.
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Myocardial phospholipases A2 and oxidative stress
Závišková, Kristýna ; Míčová, Petra (advisor) ; Chytilová, Anna (referee)
Heart and vascular diseases, in which oxidative stress plays an important role, are the most frequent cause of death in developed countries. Therefore today's researches focus on clarifying signaling pathway and their components that operates in these processes. One of the possible key factors can be phospholipases A2. These enzymes hydrolyze unsaturated fatty acids from C2 position on phospholipids, which easily subject to the oxidative modification due to their double bonds. There are five groups: the secreted, the cytosolic, the calcium independent, the platelet-activating factor acetylhydrolases and the lysosomal phospholipases A2. They differ in the requirement for calcium ions for its activity, molecular weight, position of disulfide bonds and amino acids sequences in the active site. Their main importance is to keep the stability and integrity of cell membranes and produce lipid signaling molecules (free fatty acids, lysophospholipids, eicosanoids). The phospholipases A2 play a dual role during oxidative stress in myocardium. First, they have a positive effect by protecting membranes against oxidative stress due to their higher affinity for removal of peroxidized fatty acids from membrane phospholipids. On the other hand, the phospholipases A2 can also cause destructive effects during...
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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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Oxidative damage studied in rat hepatocyte model
Hauptmanová, Gabriela ; Hodek, Petr (advisor) ; Černá, Věra (referee)
Levels of oxidation damage caused by reactive oxygen and nitrogen species is very often measured in vitro in cells. For example, measurements are performed using fluorescence probes. These substances react with certain kinds of reactive species by emitting fluorescent radiation. It is also possible to observe cellular damage of DNA, lipids or proteins. Many techniques were developed for this purpose but most of them don't provide unambiguous objective results. Measures are usually contaminated by large number of interferences. These techniques are often selective just for certain types of molecules, therefore their results do not correspond to generalized level of oxidation damage. Topic of my bachelor thesis is optimization of selected techniques used in oxidation damage levels measurements. Specifically, determination of malondialdehyde and measurement of reactive oxygen and nitrogen species levels using DCFDA and DHR fluorescence probes. Rats primary hepatocytes were used as an experimental system for measuring. These techniques were successfully optimized and can be used for further study of antioxidation characteristics of natural flavonoid compounds in rats as model organisms. Key words fluorescence, probe, lipid peroxidation, lipopolysaccharide, ethanol
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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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Myocardial phospholipases A2 and oxidative stress
Závišková, Kristýna ; Míčová, Petra (advisor) ; Chytilová, Anna (referee)
Heart and vascular diseases, in which oxidative stress plays an important role, are the most frequent cause of death in developed countries. Therefore today's researches focus on clarifying signaling pathway and their components that operates in these processes. One of the possible key factors can be phospholipases A2. These enzymes hydrolyze unsaturated fatty acids from C2 position on phospholipids, which easily subject to the oxidative modification due to their double bonds. There are five groups: the secreted, the cytosolic, the calcium independent, the platelet-activating factor acetylhydrolases and the lysosomal phospholipases A2. They differ in the requirement for calcium ions for its activity, molecular weight, position of disulfide bonds and amino acids sequences in the active site. Their main importance is to keep the stability and integrity of cell membranes and produce lipid signaling molecules (free fatty acids, lysophospholipids, eicosanoids). The phospholipases A2 play a dual role during oxidative stress in myocardium. First, they have a positive effect by protecting membranes against oxidative stress due to their higher affinity for removal of peroxidized fatty acids from membrane phospholipids. On the other hand, the phospholipases A2 can also cause destructive effects during...
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Application of Selected Methods for Oxidative Stress Analysis
Lízalová, Martina ; Tremlová, Bohuslava (referee) ; Beklová, Miroslava (referee) ; Vávrová, Milada (referee) ; Lojek, Antonín (advisor)
Chronic pancreatitis (CP) is a heterogeneous disease defined as chronic inflammatory changes of the pancreatic tissue caused by variety of aetiologies. Oxidative stress accompanying the inflammatory processes has been suggested as an important factor contributing to CP development. The aim of this study was to determine levels of lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), together with nitrites, the total antioxidant capacity, cytokines, biochemical and haematological parameters in the plasma of patients with CP and control subjects. Levels of MDA and 4-HNE were analyzed using high-performance liquid chromatography. The total antioxidant capacity of plasma against peroxyl radicals was evaluated using chemiluminescence determination. Nitrites were determined using Griess reaction. Cytokines - TNF-alfa; TNF RI; PDGF-AB; TGF-beta, together with myeloperoxidase and hyaluronan were determined using ELISA Kits. Biochemical and haematological parameters were measured by standard methods.
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