Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Investigation of the Pathology of Brain Derived Endothelial Cells in In-Vitro Hypoxia Models(01. Izmir Institute of Technology, 2021) Erdemli, Kısmet Tuğçe; Tosun, ÇiğdemThe blood brain barrier (BBB) is a vital structure that protects brain homeostasis. Endothelial cells (EC) have a significant role in regulating the BBB structure and function. Several studies have revealed the association of SUR1-TRPM4 channels that regulate this secondary damage of CNS injuries. After the activation of the channel, Na+ influx causes depolarization, cell swelling (edema) and ultimately oncotic cell death. Hypoxia inducing factor (HIF) transcription factor that has been reported to activate more than 100 genes to adapt to a hypoxic condition. Once Hif1-⍺ is translocated into the nucleus, it can dimerize with HIF1-ß to produce HIF that is critical in hypoxic conditions and regulate cell cycle arrest or cell death pathways. Hypoxia can occur in an O2 dependent and independent manner. In this study, CoCl2 and hypoxia chamber which was cost-effective and reliable were optimized. Cellular death was calculated with Trypan blue staining in this novel hypoxia chamber model and compared with CoCl2 models. In addition, morphological changes were observed in microscopic analysis. Hif1-⍺, caspase-3 and NF-κB translocation to the nucleus localization were quantified. Cell viability was different between the CoCl2 model and novel hypoxia chamber model at 24 hours. The cellular death increased with CoCl2 exposure, where no change was noted in the hypoxia chamber model. Time dependent Hif1-⍺ upregulation was also demonstrated that peaked at 12-hours. Finally, NF-κB translocation into the nucleus was significantly increased at 24 hours of hypoxia exposure. The results reveal that the inflatable hypoxia chamber model could be reliably used to mimic hypoxia in an in-vitro setting. Hif1-⍺ activated in a time dependent manner, along with NF-κB. The upregulation of these transcription factors can ultimately affect the cellular death mechanisms differentlyMaster Thesis The Effect of Glibenclamide in Lipopolysaccharide Stimulated Brain Microvascular Endothelial Cells(Izmir Institute of Technology, 2019) Cihankaya, Hilal; Tosun, ÇiğdemEndothelial cells are essential components of the blood brain barrier (BBB) that regulate the exchange of solutes between the vasculature and the brain parenchyma. The integrity of the BBB is disrupted after central nervous system (CNS) injuries, and it has been associated with the Sur1-Trpm4 channels. Once these channels are opened, Na+ flows into the cells causing edema and cell death. To mimic CNS injuries in vitro, lipopolysaccharide (LPS) was used as an endotoxin to initiate proinflammatory mediators to increase endothelial permeability, and glibenclamide was used as an antagonist of Sur1-Trpm4 channels to reduce the inflammatory response and to maintain the structural integrity of BBB proteins. To demonstrate the role of glibenclamide following LPS stimulation, we determined the cytotoxicity of LPS in bEnd.3 cells by cleaved caspase-3 expression and propidium iodide staining. We also investigated the protective effect of glibenclamide on NF-B translocation, and BBB proteins; collagen IV (COL IV) and zonula occludens 1 (ZO-1) in LPS stimulated bEnd.3 cells. Our results revealed that 1g/ml LPS exposure was sufficient for NF-B nuclear translocation, along with a statistically significant decrease in the expressions of COL IV and ZO-1 proteins, and a significant increase in cell death. We also demonstrated that glibenclamide was able to restore the expressions of COL IV and ZO-1, significantly reduce NF-B translocation, and cell death. Taken together, LPS induction in bEnd.3 cells can be used to investigate endothelial cell dysfunction due to inflammation in stroke and trauma models and glibenclamide can be used as a protective drug to inhibit LPS stimulated inflammatory response, cell death and disruptions in the structures of key BBB proteins.Master Thesis Effects of Polyether Antibiotics on Autophagy(Izmir Institute of Technology, 2017) Khan, Nasar; Tosun, Çiğdem; Bedir, Erdal; Tosun, Çiğdem; Bedir, ErdalTreatment of cancer is one of the crucial enigma for scientific world and that’s why much effort needs to be put in place for the resolution of this challenge in alternative ways. Autophagy is believed to have an important role in tumor development and progression. The natural polyether antibiotics might be important chemotherapeutic agents to cure cancer by modulating autophagy. The primary goal of this study was to investigate the cytotoxic effects and autophagic mechanism of actions of three polyether antibiotics, one of which was a new secondary metabolite isolated from the marine Streptomyces cacaoi. The effects of these polyether antibiotics were investigated along with previously known autophagy modulators from the same group (Monensin). To achieve this goal, cytotoxicities of these polyether type compounds on three different type of cancer cell lines along with two healthy cell lines were investigated followed by a search to reveal the effects of these compounds on autophagy in cancer cell lines. Methodology of this study consists of mammalian cell culturing, cytotoxicity screening, staining and quantification of acidic compartments inside the cells and studying different autophagy markers along with other associated proteins under various conditions by using Western blotting. This study revealed that the tested polyether antibiotics were autophagy inhibitors as well as inducers of apoptosis in cervical, colorectal and prostate cancer cells. The obtained results will be of significance for the field of anticancer drugdevelopment; however, before one places these secondary metabolites as potential drug candidates, further studies including in vivo experiments are warranted.