Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Deciphering Functions of Aberrant Hemichannels Formed by Connexin 26- I30n and D50y Mutations(Izmir Institute of Technology, 2015) Aypek, Hande; Meşe Özçivici, GülistanCells need to communicate with each other for maintenance cellular and tissue homeostasis. Gap junctions are channel-forming structures that are formed by docking of two hemichannels on the plasma membrane of adjacent cells. Connexins are subunits of gap junctions. Connexin 26 (Cx26) is one of the connexin isoform and mutations on the Cx26 gene (GJB2) cause non-syndromic and syndromic deafness. Keratitis-ichthyosis-deafness (KID) syndrome is one of the syndromic deafness disorders caused by Cx26 mutations. Among these mutations, Cx26-I30N and D50Y missense mutations were shown to form aberrant hemichannels but their effect on protein biosynthesis and functions have not studied. In this study, we aimed to decipher in vitro functions of aberrant hemichannels formed by Cx26-I30N and D50Y mutations. First of all, the effect of Cx26-I30N and D50Y mutations on localization, mRNA expression and protein synthesis properties were investigated in HeLa, N2A and HaCaT cells. Results suggested that Cx26-I30N and D50Y mutants were not able to form gap junction plaques on the plasma membrane and were localized in the Golgi apparatus. In addition, mutations resulted in a reduction in mRNA expression and protein synthesis. After, functional analysis was performed in Cx26-I30N and D50Y transfected N2A and HaCaT cells. Internal Ca2+ content measurement, measurement of released ATP, measurement of cell size and apoptosis assays were performed. Ca2+ measurement results showed that both Cx26-I30N and D50Y mutations deregulate Ca2+ balance in both N2A and HaCaT cells. Result of ATP release assay indicated that ATP amount in the extracellular environment decreased in N2A cells having Cx26-I30N and D50Y clones. Finally, apoptosis assay showed that number of necrotic cells increased when N2A cells were transfected with Cx26-I30N and D50Y constructs. Therefore, it was shown that aberrant hemichannels formed by Cx26-I30N and D50Y mutations may induce necrotic cell death by disrupting Ca2+ balance and ATP amount in cells.Master Thesis Design and Fabrication of Microfluidic Device That Allows Investigation of Distance Dependent Interactions of Two Different Cell Types(Izmir Institute of Technology, 2014) Sağlam, Murat; Pesen Okvur, Devrim; Özyüzer, LütfiThe main studies of in this thesis, the mold and a microfluidic device are achieved by using SU-8 photoresist and PDMS polymer. Firstly, molds are obtained which are thickness ranging from 30 to 400 μm by using SU-8 photoresist with UV lithography technique and this molding will use for shaping polydimethylsiloxane (PDMS) polymer. Finally, PDMS molds combined with the glass surface to create a three dimensional reservoirs. Microfluidic device that allows investigation of distance dependent interactions, two factors are positioned at certain distances from each other and the microfluidic device is allowed to investigation of distance dependent interaction of two factors. There is an alternating width channel between two channels which have each of two factors. These three channels are separated from each other by colonnades, not by walls, therefore physical, chemical and biological interactions are possible between the factors. Necessary physical, chemical, and biological conditioning can be provided by the reservoirs which are neighbor of channels including factors. Microfluidic chip has a lot of advantages that are small liquid volumes (pL-μL), precise spatial & temporal control, successfully mimic the physiological context, highthroughput analysis, low fabrication costs; portable and safer therefore it facilitates us to refine our methods of analysis and development in cell biology investigations and determining the content of chemical samples.Master Thesis Synthesis, Characterization of Cdsxse1-X Quantum Dots and Evaluation of Their Real-Time Motions in Live Cells(Izmir Institute of Technology, 2011) Ünal, Gülçin; Özçelik, SerdarThe use of quantum dots as fluorescent labels in bioimaging is the most intensively studied subject. The aim of this study is to elucidate locations of quantum dots and track their motions in real time through confocal microscopy and to evaluate influence of surface chemistry on diffusions of quantum dots in live cells. In this study, trioctylphosphine oxide (TOPO) capped CdSxSe1-x quantum dots were synthesized and then TOPO molecules were exchanged with 3-mercaptopropionic acid and N-acetyl-Lcysteine to make quantum dots water dispersible for cellular imaging. Human lung adenocarcinoma epithelial cells (A549) and human bronchial epithelial cells (BEAS-2B) were incubated 1 hour with CdSxSe1-x quantum dots with a concentration range of 1-10 g/mL. Localizations and real time motions of quantum dots were tracked by a spinning disc confocal microscope. The center of fluorescent spots of quantum dots was determined by 2D Gaussian fitting with a sub-pixel resolution (<100nm/pixel). The mean square displacements, diffusion coefficients and trajectories in which quantum dots made motions were analyzed by the software ImageJ with a plug in Spot Tracker. Confocal images showed that both MPA and NAC cappped quantum dots were observed in the cytoplasm of cells. Trajectories of quantum dots in cellular environment demonstrated that the quantum dots performed various types of motions in live cells. Unimodal, trimodal and multimodal distribution histograms of the diffusion coefficeints were obtained for different capping agents (MPA and NAC) and cell types (A549 and BEAS-2B). We conclude that the surface chemistry regulates the motion of the quantum dots in the cellular environment.