Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Computational investigation of allostery in hydroxyacid oxidase(01. Izmir Institute of Technology, 2024) Gökoğlu, Gizem; Uyar, Arzu; Ceylan, ÇağatayUnderstanding allosteric regulation in proteins may enable the development of effective new-generation therapeutics for disease treatments. Computational methods are gaining importance daily due to their fast and effective nature in examining protein dynamics and discovering potential allosteric binding sites. In this context, computational methods were used to determine the potential binding sites of the human hydroxyacid oxidase 1 (hHAO1) protein, which is effective in the rare disease Primary hyperoxaluria type 1 (PH1). Then, potential binding sites were predicted in monomeric and homotetrameric crystal and modeled structures of hHAO1 using the Fragment–Based Mapping (FTMap) web server and the Essential Site Scanning Analysis (ESSA) method. In ESSA, first, the default parameters cut-off 10 Å and mode 10 were applied. Then, to examine the effect of the cut-off value (distance) (Å), the parameters cut-off 7 Å and cumulative mode 20, and cut-off 13 Å and cumulative mode 20 were applied in ESSA. The modeled structures for hHAO1 were successful in terms of conformation and predicted binding sites. In the results of hHAO1, more binding sites were determined in the monomeric forms of hHAO1 than in tetrameric forms. FTMap and especially ESSA successfully predicted allosteric and potential new binding sites for hHAO1. This thesis might contribute to the improvement of computational methods and the development of allosteric therapeutics for hHAO1 and PH1.Master Thesis Development of Polymeric Carriers for Mrna Delivery(01. Izmir Institute of Technology, 2023) Savaş, Müge; Bulmuş Zareie, Esma VolgaDevelopment of efficient delivery systems is a constraint on the delivery of messenger RNA (mRNA). In gene therapy, it is crucial that mRNA molecules are efficiently delivered into cells. The thesis study has focused on the development of a relatively new polymer, P(OEGMA)-b-P(AEAEMA) as a new mRNA delivery system. P(OEGMA)-b-P(AEAEMA) block copolymers with changing block lengths (P(OEGMA)42-b-P(AEAEMA)48 and P(OEGMA)42-b-P(AEAEMA)71) were first synthesized via RAFT polymerization. The synthesized block copolymers efficiently complexed with eGFP encoded mRNA as shown by gel electrophoresis. The hydrodynamic size of polyplexes was less than 150 nm, as determined by dynamic light scattering measurements. Zeta potential measurements showed that the surface charge of the polymer-mRNA complexes was slightly negative. The eGFP-encoding mRNA transfection ability of the copolymers was investigated via fluorescence microscopy and Image J analyses. The block copolymers showed mRNA transfection efficiency on human embryonic kidney (HEK293T) and mouse fibroblast (L929) cell lines higher than the golden standard polymer, branched PEI. P(OEGMA)42-b-P(AEAEMA)48 and P(OEGMA)42-b-P(AEAEMA)71 showed a transfection efficiency of 54 and 64% of the positive control (cells transfected with Lipofectamine-mRNA complexes), respectively, on human embryonic kidney (HEK293T) cell line. On mouse fibroblast (L929) cell line, P(OEGMA)42-b-P(AEAEMA)48 and P(OEGMA)42-b-P(AEAEMA)71 block copolymers showed a transfection efficiency of 61 and 56% of the positive control, respectively. The polymer complexes showed tolerable (>70%) or no cytotoxicity on the cells in the tested range. In summary, P(OEGMA)-b-P(AEAEMA) block copolymers have shown promising mRNA transfection ability on both HEK293T and L929 cell lines.Master Thesis Bioinformatic Approaches To Investigate Hiv Capsid-Nanobody Interaction(01. Izmir Institute of Technology, 2023) Atik, Şeref Berk; Taşkent Sezgin, HümeyraInfection with HIV is still a global pandemic. Since the discovery of this highly mutagenic virus, nearly 40 million people have passed away as a result of HIV-related health problems. Currently, 38.4 million people are HIV-positive. Following infection, the viral genome gets integrated into the host cell genome. The infected person carries the virus for the rest of their life and can spread it to others through bodily fluids. Because there is no treatment for HIV, the World Health Organization recommends that infected people be diagnosed early through comprehensive screening to restrict the virus's spread. As a result, there is still a need to create practical, sensitive diagnostic tools, particularly for use in the field of HIV infection testing. In this study, the interaction between HIV-1 capsid protein, the first antigen found in the blood during the acute phase of HIV infection, and a nanobody (Nb, a single domain antibody) known to bind to capsid is investigated at the molecular level through computational methods. Because the structure of HIV-1 CA binding-Nb is unknown, all-atom models of the Nb structure were constructed using comparative methods, deep-learning-based methods, and hybrid methods (SwissModel, trRosetta, Robetta, AlphaFold2), and promising models were chosen. In the second stage, molecular docking was used to produce HIV-1 capsid- nanobody complex structures, which were then tested for stability and native-likeness using standard molecular dynamics simulations. Understanding the molecular details of the HIV-1 capsid-nanobody complex, we believe, will provide essential data for using this antigen-antibody pair inan immunosensor system for HIV-1 infection diagnosis.Master Thesis Crude Pectinolytic Enzymes Production in Fed-Batch Shake Flask Cultivation(01. Izmir Institute of Technology, 2023) Esen, Büşra Nur; Uzuner, Sibel; Uzuner, Sibel; Taşkent Sezgin, HümeyraThe use of waste in the production of enzymes, which is one of the products with high added value, is one of the right strategies to reduce the production cost of the product and sustainability movement. In this study, the production of polygalacturonase (PGase) and pectin lyase (PLase) enzymes from Bacillus subtilis ATCC 6633 in fed batch submerged fermentation, the conditions and composition of the fermentation medium and the effects of pretreatment methods (thermal, thermo-chemical, microwave assisted dilute acid (MW- DA)) on the conversion of fermentable sugar from black carrot pulp were investigated. The MW-DA was chosen the best with higher fermentable sugar content (FSC). The three different powers (300, 600, 850 W) and 3 different treatment time (30, 60, 90 s) were examined by Taguchi design. The highest FSC was found at 300 Watt for 30 seconds. MW-DA followed by ES produced the most fermentable sugar (0.493 g/g, 87.3% conversion). The amount of fermentable sugar was enhanced from 15.8% to 87.3% when MW-DA treatment is combined with enzymatic saccharification (ES). Yeast extract, whey and pea protein were examined as nitrogen sources. According to the enzyme activity results obtained, the fermentation medium was modified with pea protein. Certain concentrations (2.5%, 5%, 10%, 15%) were fed to the fermentation medium. The highest PGase activity was determined at the 15% feed concentration and 72th hours (164.34±2.26 U/L) whereas the highest PLase activity was obtained at 72th hours (188.22±1.72 U/L) at 5% feed concentration.Master Thesis Cell Separation in Microfluidic Devices(01. Izmir Institute of Technology, 2022) Öksüz, Cemre; Tekin, Hüseyin CumhurCell separation is used to separate homogeneous and individual cell classes from a heterogeneous cell population. The efficiency and purity of these separated cells are of great importance in personalized medicine, regenerative medicine, disease monitoring and drug testing as well as in the therapeutic and diagnostic research. In this thesis, different microfluidic approaches were presented for cell separation. With this regard, a closed channel vacuum-integrated microfluidic chip was developed using an air permeability of a Polydimethylsiloxane and density-based separation of microparticles was performed. Besides, a centrifugal microfluidic system, Spinochip, was developed with one reservoir as inlet and outlet for the first time and different fluid manipulations were shown in the system. The system was applied to clinical tests of hematocrit measurements and white blood cell estimation using real patient samples. The developed system offered correlated results with clinical results. In addition to closed channel microfluidics, negative-magnetophoresis microfluidic chip was demonstrated for the size-based separation of microparticles and cells. In this regard, capturing rate of breast cancer cells (MCF-7) and human monocyte cells (U937) was investigated. The results showed that the approaches presented here could promote to the microfluidic studies for size-based cell separation.Master Thesis Dynamics and Bioinformatis of Microbial Spoilage Ecology of Kefir(Izmir Institute of Technology, 2022) Memon, Ayşe; Baysal, Ayşe Handan; Sezgin, EfeIn this study, it was aimed to characterize the microbiological properties, bacterial composition, and microbial stability of 5 different commercial milk kefir beverage products during refrigerated storage. In order to determine the microbiological load and compositions, total mesophilic aerobic bacteria, yeast and molds, lactic acid bacteria, Lactobacilli, Lactococci, total coliforms, and E.coli were investigated by cultural conventional analysis for milk kefir beverages. According to the obtained data, microbiological and hygienic characteristics of the samples were found acceptable. The bacterial load of the kefir beverage samples ranged between 7.086 and 8.794 log10 cfu.ml-1 for viable total aerobic mesophilic bacteria (TAMB), 6.792 and 8.382 og10 cfu.ml-1 for lactic bacteria (LAB), <10 and 6.322 log10 cfu.ml-1for Lactobacillus, 5.857 and 8.146 log10 cfu.ml-1 Lactococcus, 5.176 and 7.218 log10 cfu.ml-1 for yeasts, negative for molds, coliform bacteria and negative for E. coli. Principal component analysis (PCA) of the compounds separated the kefir beverages according to the storage time and kefir brands. Strong relationship were found between storage time and PC1 and between kefir brands and PC2. To date, information on microbial properties, bacterial composition, and constancy of commercial kefir is scant, and to the best of our knowledge, this is the first research to contribute information on kefir beverages in microbial properties, bacterial composition, and their stability during refrigerated storage by evaluating Fourier Transform Infrared Spectroscopy (FTIR) spectra analysis and Bioinformatics besides cultural conventional analysis.Master Thesis Magnetic-Based Cell Manipulation in Microfluidic Devices(01. Izmir Institute of Technology, 2022) Özçelik, Özge Solmaz; Tekin, Hüseyin CumhurCell manipulation is the concept of altering cell movement. Different manipulation techniques have been demonstrated with microfluidic systems for various studies such as tissue engineering, circulating tumor cell (CTC) filtering, and other biomedical applications. For instance, cell patterning and filtering studies are being developed through different manipulation approaches in microfluidic platforms where one of these approaches is the magnetophoresis principle method. Positive and negative magnetophoresis can be utilized generally through labeling or non-labeling, respectively. In this thesis, two different cell manipulation platforms using negative magnetophoresis were developed for cell patterning and cell filtration applications. These platforms allow several advantages such as simple fabrication, easy control, and low cost. Compared to other devices, the developed microfluidic platforms do not require any labeling process for cells for magnetic manipulation. In the patterning platform, microparticle and cell patterns were formed inside a simple microfluidic channel with different tilted angles in <1.5 hours. Furthermore, in the filtration platform, large microparticles were separated from small microparticles with 98.25% trapping efficiency. Live/dead cell separation of human monocyte macrophage cells (U937) under different flow rates was also investigated. The suggested platforms could be useful for label-free magnetic cell patterning and filtering in biomedical applications.Master Thesis Developing a Lamp Pcr Based Diagnostic Test for Crimean Congo Hemorrhagic Fever Virus(Izmir Institute of Technology, 2022) Üstün, Selcen; Taşkent Sezgin, HümeyraCrimean-Congo hemorrhagic fever infection is one of the most common tickborne viral infections, non-infectious in animals and fatal to humans with a mortality rate around 40%. The high mortality rate and lack of vaccines or drug treatment point to the potential danger of infection. The aim of this thesis was to detect 21 complete S-segment Crimean-Congo hemorrhagic fever virus strains originating in Turkey using Fluorimetric Loop Mediated Isothermal Amplification (LAMP) method, which is one of the PCRbased isothermal amplification methods for detecting viral infections. Conserved regions of the 21 strains of the CCHFV observed in Turkey were determined by sequence alignment and LAMP primers for these conserved regions were designed. DNA of CCHFV Ank-2 (GeneBank accession number: MK309333) strain was used and optimum LAMP reaction conditions were determined by changing the temperature, primer amount and MgSO4 concentration. LAMP results were compared with qPCR, which is considered the gold standard. The detection limit for LAMP and qPCR was 2x106 copies/µl. In the study, the CCHFV LAMP primer set-1 gave similar results to the qPCR primer set. The CCHFV LAMP primer set-4 performed better by turning positive 9 minutes earlier than the qPCR primer set. This results indicate LAMP is an alternative method for detecting CCHFV but reveals the necessity of improving the sensitivity of the test.Master Thesis Investigation of the Interaction and Olgiomerization of Hiv Capsid and Single Domain Antibody as a Biotechnological Drug Against Hiv(Izmir Institute of Technology, 2022) Güney, Seniha; Taşkent Sezgin, HümeyraHuman immunodeficiency virus (HIV) causes AIDS which is still a global public health threat. Current drugs against HIV infection cannot eradicate the virus therefore, research on new drug targets continues. HIV capsid protein, which has a highly conserved sequence and is sensitive to mutations, has critical roles in the virus lifecycle, making it a high-potential drug target. A nanobody is the antigen-binding domain of heavy-chain only antibodies of camelids. Small size, thermal stability and ease of production makes nanobodies ideal antibody fragments for therapeutic and diagnostic purposes. In the literature, a nanobody binding to the HIV-1 capsid-N terminal domain (NTD) has been reported. The aim of this thesis is to examine the potential of this nanobody as a biotechnological drug candidate against the HIV-1 and HIV-2 capsid proteins. In the study, HIV-1 capsid was expressed, purified and biophysically characterized. Thermal and chemical denaturation of the protein were done, the melting temperature and unfolding free-energy values of the protein were determined. In-vitro oligomerization of the HIV-1 capsid was performed and observed that the protein self-oligomerized over time. Pure HIV-2 capsid protein could not be produced recombinantly. Thereupon, HIV1 capsid-NTD and HIV-2 capsid-NTD proteins were expressed and purified. Secondary structure of HIV-1 capsid, HIV-1 capsid-NTD and nanobody were analyzed with circular dichroism (CD) spectroscopy and the results matched with the literature. Isothermal titration calorimetry (ITC) experiments were done to examine the HIV-1 capsidnanobody interaction, but good binding was not observed between the two proteins. Future work requires repeating ITC experiments.Master Thesis Cloning, Expression and Characterization of Sericin Like Octamer-Repeat Protein(01. Izmir Institute of Technology, 2021) Şahin, Alper; Sürmeli Eraltuğ, Nur BaşakSilk proteins are natural polymers which are synthesized by insects like silkworms and spiders. There are two proteins in the silk obtained from the cocoon of B. mori, these are silk fibroin and silk sericin. While the silk fibroin is the main part of the cocoon with a high commercial importance, sericin is the "glue" protein that holds fibroins together. Sericin has wide variety of applications from cosmetics to biomaterial research. Because of its high fibrillar structure, it can be used in wound healing or drug delivery studies. It can be obtained from the degumming process of cocoons which includes chemical treatment like alkali treatment and boiling, or biological treatment like enzymatic digestion. However, sericin obtained by these processes are not consistent in structure, and the sequence of the protein obtained depends on the extraction methodology and conditions. However, proteins obtained by recombinant production can be standardized, and repeat-chain length can be adjusted as desired. In this thesis study, a sericin like protein which includes eight repeats of the 38 amino acid sequence of the natural sericin was cloned and expressed in E. coli. The resulting protein, Ser-8mer, was analyzed in terms of fibril structure and secondary structure. It was found out that the protein has beta sheet conformation in contrast to commercial sericin with random coils. And as a result of this conformation, it forms insoluble self-assembled fibril structures which shows a promising contribution to the biomaterial research.