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The effect of resveratrol and gambogic acid on the DNA damage caused by daunorubicin in neonatal rat cardiomyocytes.
Mašín, Martin ; Jirkovská, Anna (advisor) ; Pávek, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Martin Mašín Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of diploma thesis: The effect of resveratrol and gambogic acid on the DNA damage caused by daunorubicin in neonatal rat cardiomyocytes. DNA Topoisomerases comprise a family of enzymes that are able to alter DNA topology by transient single- or double-strand breaks (DSB) during fundamental processes such as replication and transcription. Inhibition of topoisomerase II (TOP II) is the main mechanism of action of some antitumour drugs, such as anthracyclines (ANT; e.g., daunorubicin). They stabilize the DNA-TOP II complex, leading to the formation of DSBs and later to apoptosis. Other inhibitors, that interact with the enzyme without the DSB formation, can modulate the effect of ANT. In this thesis, we studied the DNA damage caused by daunorubicin (DAU) and its main metabolite daunorubicinol (DAUnol) and the effect of two naturally-derived compounds and TOP II catalytic inhibitors resveratrol (RES) and gambogic acid (GA) in neonatal rat cardiomyocytes. The DNA damage was determined as the extent of histone H2AX phosphorylation (γ-H2AX) and by Comet Assay. It can be concluded that both DAU and DAUnol (1,2 μM) exhibit DNA damage that is...
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The effect of resveratrol and gambogic acid on the DNA damage caused by daunorubicin in neonatal rat cardiomyocytes.
Mašín, Martin ; Jirkovská, Anna (advisor) ; Pávek, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Martin Mašín Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of diploma thesis: The effect of resveratrol and gambogic acid on the DNA damage caused by daunorubicin in neonatal rat cardiomyocytes. DNA Topoisomerases comprise a family of enzymes that are able to alter DNA topology by transient single- or double-strand breaks (DSB) during fundamental processes such as replication and transcription. Inhibition of topoisomerase II (TOP II) is the main mechanism of action of some antitumour drugs, such as anthracyclines (ANT; e.g., daunorubicin). They stabilize the DNA-TOP II complex, leading to the formation of DSBs and later to apoptosis. Other inhibitors, that interact with the enzyme without the DSB formation, can modulate the effect of ANT. In this thesis, we studied the DNA damage caused by daunorubicin (DAU) and its main metabolite daunorubicinol (DAUnol) and the effect of two naturally-derived compounds and TOP II catalytic inhibitors resveratrol (RES) and gambogic acid (GA) in neonatal rat cardiomyocytes. The DNA damage was determined as the extent of histone H2AX phosphorylation (γ-H2AX) and by Comet Assay. It can be concluded that both DAU and DAUnol (1,2 μM) exhibit DNA damage that is...
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The influence of anesthesia on the degree of DNA oxidative damage
Zubáňová, Veronika ; Kuchařová, Monika (advisor) ; Nováková, Veronika (referee)
Background: Oxidative damage is one of the most frequent types of cell components damage leading to oxidation of lipids, proteins and the molecule of DNA. As a consequence, there is a higher occurrence of several pathologies such as atherosclerosis, neurodegenerative diseases, cancer; or diabetes. In our study, influence of whole body anesthesia during minor surgery on the level of DNA damage was examined using comet assay technique. Methods: The basic principle of this method is fixing the cells (lymphocytes) in agarose, their lysis for the removal of membranes, incubation with the specific enzymes and electrophoresis of the released cell nuclei. During the electrophoresis, free low-molecular weight and negatively charged fragments of DNA move towards anode which causes the formation of the typical comet cell shape. Finally, the gels are stained by ethidium bromide (DNA intercalating dye) and visualized. Results: We have observed single strand breakages (SSBs) and, with the use of modified assay using specific enzymes for detection of specific lesions, also oxidized purines and pyrimidines. The extent of DNA damage as determined by the intensity of the tail of the comet was quantified using LUCIA Comet Assay (Laboratory Imaging, Czech Republic) software for image analysis. The results were used...
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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 influence of spinal anesthesia on the degree of DNA damage
Koščáková, Mária ; Kuchařová, Monika (advisor) ; Vokřál, Ivan (referee)
Background: The human organism is exposed daily to many endogenous and exogenous substances that are the source of oxidative damage. Cell structures, including DNA (deoxyribonucleic acid) in the nucleus are damaged due to high concentrations of these substances and accumulation of oxidative stress in cells. The predominance of these damaging processes may later be responsible for human diseases such as cancer, neurodegenerative diseases or heart failure. In our study, we observed oxidative damage at the DNA level due to spinal anesthesia. Methods: Sample processing was performed by comet analysis. The principle consists in fixation of cells (lymphocytes) in the agarose gel, lysis of cell structures for nucleotide release, incubation with specific enzymes and exposure to electrophoresis. Damaged, negatively charged parts of the DNA in the electric field are directed to the positive charged anode, creating a typical comet shape. For visualization, the gels were stained with ethidium bromide (DNA intercalating dye). Results: We have quantified single-strand breaks, oxidized purines and pyrimidines (use of enzymes to detect specific damages). The results are reported in percentage of DNA in the comet's tail. The principle is to compare the intensity of the comet's tail with the total comet intensity....
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The influence of critical condition of patients on DNA damage
Verešpejová, Natália ; Kuchařová, Monika (advisor) ; Bárta, Pavel (referee)
The first cases of patients with pneumonia which grew into an acute respiratory distress syndrome and caused breathing problems began to appear in December 2019. Coronavirus disease 2019 (COVID-19) is the cause of a global pandemic and it is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A complex interplay of factors is responsible for the progression of the disease. Some studies suggest that it promotes oxidative stress and thus may lead to oxidative damage to cells and DNA. The purpose of this study was to observe the relationship between oxidative DNA damage and a critical condition caused by COVID-19 using a comet assay technique. The basic principle of the used method consists in fixation of lymphocytes in an agarose gel, removal of the membrane and cytoplasm of cells, incubation with specific enzymes and electrophoresis. In the process of electrophoresis, negatively charged DNA fragments migrates towards the anode and the cell thus acquires the typical shape of a comet. Comets are visualized using the DNA intercalation dye ethidium bromide. We quantified single - strand breaks and oxidized pyrimidines and purines by using specific enzymes (modification of the method for detecting specific lesions). Results are reported as % tail DNA, thus the percentage of DNA in the...
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Plant virus-based biotechnology
Vaculík, Petr
The latest model of tertiary structure of capsid protein of potato virus X (PVX CP) was used as a template to design new insertion sites suitable for the preparation of PVX-based antigen presentation system. Based on this model, seven insertion sites (A-G) located in putative surface loops were tested. As an antigen inserted into these sites was used 17 amino acids long epitope derived from human papillomavirus type 16 E7 oncoprotein (E7 epitope) fused with either 6xHis tag or StrepII tag in both possible orientations (6xHis-E7 and E7-6xHis, StrepII-E7 and E7-StrepII). Prior to plant expression, modified PVX CPs were expressed in Escherichia coli MC1061. The results showed that only PVX CP carrying StrepII-E7 or E7-StrepII in the insertion site A formed virus particles. The results from transient expression experiments with modified PVX CPs in Nicotiana benthamiana showed that only the insertion site A (located between 24th and 25th amino acid in the PVX CP) could tolerate all tested inserts. Importantly, viral particles were detected only in the presence of StrepII tag and their stability was affected by the insert orientation (StrepII-E7 vs. E7-StrepII) as only the viral particles presenting E7-StrepII could be purified. Besides the preparation of PVX-based antigen presentation system, an...
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The influence of spinal anesthesia on the degree of DNA damage
Koščáková, Mária ; Kuchařová, Monika (advisor) ; Vokřál, Ivan (referee)
Background: The human organism is exposed daily to many endogenous and exogenous substances that are the source of oxidative damage. Cell structures, including DNA (deoxyribonucleic acid) in the nucleus are damaged due to high concentrations of these substances and accumulation of oxidative stress in cells. The predominance of these damaging processes may later be responsible for human diseases such as cancer, neurodegenerative diseases or heart failure. In our study, we observed oxidative damage at the DNA level due to spinal anesthesia. Methods: Sample processing was performed by comet analysis. The principle consists in fixation of cells (lymphocytes) in the agarose gel, lysis of cell structures for nucleotide release, incubation with specific enzymes and exposure to electrophoresis. Damaged, negatively charged parts of the DNA in the electric field are directed to the positive charged anode, creating a typical comet shape. For visualization, the gels were stained with ethidium bromide (DNA intercalating dye). Results: We have quantified single-strand breaks, oxidized purines and pyrimidines (use of enzymes to detect specific damages). The results are reported in percentage of DNA in the comet's tail. The principle is to compare the intensity of the comet's tail with the total comet intensity....
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The influence of anesthesia on the degree of DNA oxidative damage
Zubáňová, Veronika ; Kuchařová, Monika (advisor) ; Nováková, Veronika (referee)
Background: Oxidative damage is one of the most frequent types of cell components damage leading to oxidation of lipids, proteins and the molecule of DNA. As a consequence, there is a higher occurrence of several pathologies such as atherosclerosis, neurodegenerative diseases, cancer; or diabetes. In our study, influence of whole body anesthesia during minor surgery on the level of DNA damage was examined using comet assay technique. Methods: The basic principle of this method is fixing the cells (lymphocytes) in agarose, their lysis for the removal of membranes, incubation with the specific enzymes and electrophoresis of the released cell nuclei. During the electrophoresis, free low-molecular weight and negatively charged fragments of DNA move towards anode which causes the formation of the typical comet cell shape. Finally, the gels are stained by ethidium bromide (DNA intercalating dye) and visualized. Results: We have observed single strand breakages (SSBs) and, with the use of modified assay using specific enzymes for detection of specific lesions, also oxidized purines and pyrimidines. The extent of DNA damage as determined by the intensity of the tail of the comet was quantified using LUCIA Comet Assay (Laboratory Imaging, Czech Republic) software for image analysis. The results were used...
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