Master Degree / Yüksek Lisans Tezleri

Permanent URI for this collectionhttps://hdl.handle.net/11147/3008

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2016 - 20207

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Author: search.filters.author.Sürmeli, Nur BaşakHas files: Yes

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Now showing 1 - 7 of 7
  • Master Thesis
    Chemical Characterization of Caldanaerobacter Subterraneus Subsp. Tengcongensis Heme-Nitric Oxide/Oxygen Binding Protein
    (01. Izmir Institute of Technology, 2020) Erdal, Merve; Sürmeli, Nur Başak; Sürmeli, Nur Başak
    Hemoproteins, which contain the heme prosthetic group , take part in different biological processes in many stages of life. Their ability to catalyze important biosynthesis reactions makes them good candidates for understanding and elucidating complex mechanisms for biocatalysis. In this study, the catalytic properties of thermophilic Thermoanaerobacter tencogensis nitric oxide/oxygen binding protein, a heme protein reshaped by rational design, were investigated and chemical characterization was carried out. The peroxidase activity of the enzyme was investigated by the oxidation reactions of guaiacol, amplex red and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS). Kinetic parameters of the reactions were determined. These obtained results demonstrated that, in the presence of H2O2, wild type and Y140H TtH-NOX proteins are able to catalyze oxidation reactions of guaiacol, Amplex red and ABTS. Comparison of the kinetic parameters showed that Y140H mutant catalyzed the guaiacol and ABTS oxidation 3-fold and 15 -fold faster than wild type enzyme, respectively. The stability of TtH-NOX proteins were investigated in the presence of organic solvents. Results were demonstrated that WT TtH-NOX was more stable than Y140H mutant in the presence of organic solvents In addition to these, for the first time, thermophilic TtH-NOX proteins were immobilized with a novel enzyme immobilization method and organic-inorganic hybrid nanostrucrures were obtained. Copper ion incorporated TtH-NOX-based hybrid nanoflowers were synthesized at different pH values. SEM and EDX analysis of TtH-NOX-based hybrid nanoflowers proved that free TtH-NOXs were immobilized successfully.
  • Master Thesis
    Evaluation of Biophysical Aspedts of Cancer Using Lab-On Chip Devices
    (Izmir Institute of Technology, 2019) Tahmaz, İsmail; Pesen Okvur, Devrim; Sürmeli, Nur Başak
    Breast cancer metastasis is really crucial point from cancer related deaths. As cancer cells from primary tumor are travelling through blood, they hang on to blood vessel and finally they exit from blood vessel into secondary site where is extracellular matrix and/or tissue/organ. This process commonly known as extravasation. Cancer cells sometimes can be highly aggressive when it exposed to hypoxia referred low oxygen amount by activating HIF1α. This transcription factor is activated in malignant cells, normal cells and endothelial cells in blood vessel when oxygen amount decreased to certain levels and it induce several genes expression such as VEGF, LOX, Angiopoietin-like-4 etc. In this study we investigated effect of HIF1α which is hypoxia indicator on breast cancer extravasation by comparing to normal oxygen level. This study represents both anemic hypoxia physiologically and lead to understand underlying mechanism of extravasation into extracellular matrix related to low oxygen circulating through blood. In addition to HIF1α effects, dynamic perfusion mimicking blood flow was applied to determine effects on extravasation. For this purpose, lab-on-a chip device was utilized for real time visualization. In conclusion, although hypoxia is giving permission MDAMB231 to extravasate because of reshaping of vascular geometry, less extravasated cancer cells observed in matrix during hypoxia under both static and flow condition when compared to normoxic and static conditions. Moreover, it was shown that flow triggers extravasation distance in normoxia against static condition and normal breast epithelial cells extravasated away in hypoxia comparing breast cancer cells by means of flow.
  • Master Thesis
    Mimicking the Tumor Microenvironment in Lab-On Devices
    (Izmir Institute of Technology, 2019) Bilgen, Müge; Pesen Okvur, Devrim; Sürmeli, Nur Başak
    Breast cancer is one of the cancers with the highest incidence and mortality rates in women in the world. The leading cause of death for cancer patients is tumor metastasis. Cancer cells can extravasate the blood vessel, go through the distant organs and form the metastasis. Tumor microenvironment comprises of cancer and normal cells, extracellular matrix, soluble biological and chemical factors. Biochemical aspects of the interactions of cancer cells with the constituents of the microenvironment are widely studied whereas biophysical studies are at limited numbers. There is increasing evidence that extracellular matrix can change the mechanics and function of cancer and stroma cells. It has been observed that cancer cells show different responses to soft and stiff tissues they are in direct contact with than normal cells. New cell culture setups should be developed to better understand the interactions of cancer cells with their microenvironment. To develop a three dimensional (3D) in vitro model will allow the study of stiffness which is one of the mechanical features of extracellular matrix features first, 3D (dimensional) Controlled in vitro Microenvironments (CivMs) that mimic a blood vessel and its neighboring tissue in vivo will be fabricated using UV lithography. Monolayer which was formed by endothelial cells play a role in pathophysiological processes, so it shows a barrier role between both blood and tissues. To form a blood vessel bEnd.3 cell line was used. Collagen which is the most abundant protein in connective tissues were used to mimic extracellular matrix. pH value of collagen was changed and represented two different stiffness value. Here, the in vitro model we define as controlled in vitro microenvironments (CivM) is a lab-on-a-chip (LOC) application. In this microenvironment; MDA-MB-231 cells which are known to be invasive and MCF10A which is normal mammary epithelial cells were used as control. LOC devices were used to investigate cancer cell extravasation which is the prominent step of metastasis and extracellular matrix relation.
  • Master Thesis
    Optimization of Expression and Isolation of a Thermophilic P450 Enzyme
    (Izmir Institute of Technology, 2018) Aslantaş, Yaprak; Sürmeli, Nur Başak; Şanlı Mohamed, Gülşah
    Cytochrome P450 enzymes (CYP or P450) are monooxygenases that catalyze the oxidation of hydrocarbons with high efficiency and selectivity, and many other reactions like hydroxylation, epoxidation, reduction, demethylation. CYP119, is a thermophilic P450 from Sulfolobus acidocaldarius. Thanks to thermophilic properties, CYP119 has potential to be widely used as a biocatalyst in production of fine chemicals and pharmaceuticals. However, production and purification of CYP119s is quite difficult and time consuming. Here, through recombinant protein production techniques, the optimum production and purification of heat-tolerant CYP119 has been successfully carried out. N-terminal and C-terminal histidine tags were cloned to CYP119. Protein expression was induced in Escherichia coli BL21 (DE3) cells with isopropyl β-D-1-thiogalactopyranoside (IPTG). δ-aminolevulinic acid (ALA) was also used to increase the heme biosynthesis. Different IPTG and ALA concentrations, expression temperature and duration were used to optimize production. CYP119 was isolated and purified with Ni-NTA affinity column. The thermostability of purified N (N-His-CYP119) and C (C-His-CYP119) terminal His-tagged were compared with wild type CYP119 (Wt-CYP119). Oxidation reaction of CYP119 and variants carried out and compared at 25 °C and 65 °C. Also, epoxidation of styrene was performed with N-His-CYP119 in different temperatures. The effects of histidine tags on stability and activity of the CYP119s were observed. Here, conditions for the production of CYP119 were optimized and the histidine tags were found to cause changes in stability and function of proteins. This project will lead to increase in the production of the important enzyme CYP119, which will increase its utilization in the industry.
  • Master Thesis
    Generation of Mutant Libraries for Directed Evolution of a Thermophilic P450 Enzyme
    (Izmir Institute of Technology, 2018) Haklı, Emre; Sürmeli, Nur Başak; Bedir, Erdal
    Directed evolution, inspires from natural selection, is a frequently utilized approach in protein engineering for designing enzymes. It allows iterative evolution of existing proteins towards the ones with desired characteristics by the application of random mutagenesis in the laboratory. However, library construction constitutes the most fundamental part of directed evolution. Application of different construction methods affects both the number and diversity of variants created and the screening/selection techniques used. Early procedures including error-prone PCR, mutator strains, chemical mutagens and gene shuffling have been successful in whole gene mutagenesis yet have been required more screening/selection effort by leading larger libraries. On the other hand, recent approaches such as use of degenerate primers and site saturation mutagenesis have decreased the screening/selection effort by allowing random mutagenesis of amino acids located at specific positions in the polypeptide chain. Especially, active site residues of biocatalysts were chosen as targets and the catalytic efficiencies were enhanced. CYP119, a member of cytochrome P450 protein family, from Sulfolobus Acidocaldarius is a thermostable enzyme capable of catalyzing peroxidation, monooxygenation and oxidoreduction reactions. Here, a library of mutants consist of CYP119 variants was created via application of combinatorial active site saturation test (CAST) in amino acid positions 213 – 214 and an effective fluorescence-based method was developed to screen the library for increased peroxidase activity while utilizing hydrogen peroxide as oxidant. After screening of mutant library, a variant with Thr213Arg – Thr214Ile substitutions showed 1.32-fold increased peroxidase activity in the catalysis of Amplex Red compared to wild type CYP119.
  • Master Thesis
    Developing Mobile, Electronic and Positioning Applications for Emergency Situations Inside the Hospital
    (Izmir Institute of Technology, 2018) Keskin, Özge Sevin; Sürmeli, Nur Başak; Karabudak, Engin; Karabudak, Engin; Sürmeli, Nur Başak
    The aim of this project is to prevent mortality by providing emergency response on time. The communication inside the hospital is provided via developing application for Android and Ios operating system. The content of application is suitable for everyone’s use. The emergency call button makes it easy to call, and if the button is disabled, the heart rate rhythm starts for emergency intervention. This application is associated with bluetooth low energy systems in terms of help to locate the patient. Beacon systems provide data transfer through bluetooth by this means location is detected without internet. The project will be tested primarily at the laboratory following these steps. If it succeeds statistically, the project will be tried at the Tepecik Education and Research Hospital. The success of the project will be reviewed depends on the scenarios in the hospital.
  • Master Thesis
    Cloning and Purification of Biosynthetic Enzymes
    (Izmir Institute of Technology, 2016) Gürelme, Ebru; Sürmeli, Nur Başak; Şanlı Mohamed, Gülşah
    Bioproducts have become prominent with their sustainable, eco-friendly and renewable features. In recent years, research and development studies focused on production of biodiesel and pharmaceuticals. Biodiesel can be synthesized in the form of fatty acid ethyl ester (FAEE) by in vivo activity. This synthesis is catalyzed by wax ester synthases (WS). This study aims cloning and purification of wax ester synthases from Psychrobacter arcticus 273-4 (PaWES) and Mus musculus C57BL/6 (MmWES). PaWES was cloned and expressed by Escherichia coli BL21(DE3) strain, at the proper conditions with using pET expression system. It was purified with approximately 1 mg yield. Cloning of the second wax ester synthase (MmWES) was achieved to Saccha-romyces cerevisiae and it was purified with lower than 1 mg yield. The other aim of this study is related to taxadien-5α-ol-O-acetyltransferase from Taxus cuspidata (TcT5AT). This enzyme belongs to the biosynthesis pathway of Taxol, which is the most commonly used chemotherapy drug. Cloning and purification studies of this enzyme were successfully performed. It was expressed by Escherichia coli BL21(DE3) Star strain and purified with the yield of 23 mg. Immobilized Metal Affinity Chromatog-raphy (IMAC) is used for all three enzymes as a purification strategy. This project can pave the way for structural studies of all biosynthetic enzymes mentioned above. In summary, the findings of this study will circuitously help for solving the relationship between function and structure of these enzymes. It may lead to increased generation of Taxol and FAEE based biodiesel.