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Oksidatif Stres Karşısında Hava Yolu İnflamatuvar ve Yapısal Hücrelerinde Antioksidan Cevabın Araştırılması

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HAYRİYE AKEL BİLGİÇ DOKTORA TEZ_ 29_01_2020 (3)yeni.pdf (12.01Mb)
Date
2020
Author
Akel Bilgiç, Hayriye
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Abstract
Free radicals are very short-lived chemical species that contain one or more unshared electrons in their outer orbits. Free radicals, which have a highly reactive structure, act on important components of cells such as lipids, protein, deoxyribonucleic acid (DNA) and carbohydrates and cause their structure to deteriorate. There are many defense mechanism in order to prevent the formation of reactive oxygen species (ROS) and to avoid their damage. These mechanisms are called as “antioxidant defense systems” or simply “antioxidants”. The shift of the balance between antioxidant and oxidant in favor of oxidant is called oxidative stress. Oxidative stress contributes various pathological conditions and diseases including cancer, neurological disorders, hypertension, diabetes, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, Chronic Obstructive Pulmonary Disease (COPD) and asthma. In this study, the response of antioxidant defense mechanism to increase oxidative stress in inflammatory cells (eosinophil, monocyte) and airway structural cells (epithelium, fibroblast, endothelium) which was involved in the pathogenesis of asthma was investigated at RNA and protein levels. To stimulate oxidative stress, cigarette smoke condensate (CSC) and tert-butyl hydroperoxide (TBHP), which are frequently used in the literature, are used. Viability / cytotoxicity tests and total oxidant measurements were performed to determine the dose and incubation time in the cells after oxidant stimulation, where death and cytotoxicity were not observed and there was a significant increase in free radicals. Appropriate dose and incubation time for each cells were determined after MTT test, LDH test, EtBr / Kalsein AM live / dead cell staining, Caspase activity measurement and total oxidant measurement. In these conditions, which are different for each cell, the cells were stimulated and the total antioxidant capacity of the cells and SOD, GPx and Catalase enzyme activities in the primary antioxidant category were measured and thus the antioxidant response of the cell was investigated at the protein level. In the last stage of the thesis, qPZR array method was used to determine the gene expression that changes when cells are stimulated under specified conditions. With this method, 84 gene expressions in the anthoxidant and oxidant pathways were determined. In line with the hypothesis of the thesis, it was assumed that asthma is a disease consisting of different phenotypes and the oxidant and antioxidant responses to oxidative damage in different cells involved in the pathogenesis will be different. As a result of the experiments, it was determined that each cell responded differently to oxidant stimulus. According to these results, the antioxidant response of eosinophil cells at the protein level is strong, monocyte cells are resistant to oxidative stress and endothelial cells, which are structural cells, are sensitive to oxidant stimulation. It is thought that the findings obtained from the thesis study will contribute to the literature in determining the cellular source of increased oxidant capacity in asthma by determining the oxidant and antioxidant response specific to different cell types and in understanding the antioxidant-oxidant balance. The antioxidant response of the cells involved in the pathogenesis of asthma against oxidative stress was evaluated collectively for the first time in this study.
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http://hdl.handle.net/11655/22713
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