Master Degree / Yüksek Lisans Tezleri

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Access type: Open accessAuthor: search.filters.author.Taşkent Sezgin, Hümeyra

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  • Master Thesis
    Bioinformatic Approaches To Investigate Hiv Capsid-Nanobody Interaction
    (01. Izmir Institute of Technology, 2023) Atik, Şeref Berk; Taşkent Sezgin, Hümeyra
    Infection 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, Hümeyra
    The 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
    Development of a Nucleic Acid-Based Isothermal Diagnostic Test for Border Disease Which Causes Losses in Animal Husbandry
    (01. Izmir Institute of Technology, 2022) Ayaz Kök, Sanem; Taşkent Sezgin, Hümeyra; Meşe Özçivici, Gülistan
    Border disease is viral infection of ruminants, and it is associated with abortions, stillbirth, and birth of persistently infected (PI) lambs. It has a great potential to cause an outbreak and it is declared as one of the notifiable diseases of ruminants by World Organization for Animal Health (OIE). Border disease poses a threat against ruminant farming industry by causing major economic losses. Since there is no treatment or vaccine against border disease virus (BDV), early diagnosis and early isolation of infected animals is necessary. RT-qPCR is the gold-standard method for BDV identification, but it can only be applied by trained personnel in a laboratory with expensive instruments. There is a need for a point-of-care (POC) test, specifically designed for BDV. This thesis study aimed to develop a nucleic acid-based loop mediated isothermal amplification (LAMP) technique for BDV identification. LAMP is a nucleic acid identification technique that can be performed using 4-6 primers at a constant temperature with a Bst DNA polymerase. Firstly, multiple alignment of BDV sequences across the world was performed and most conserved region of genome was detected as 5’UTR. Then, three LAMP primer sets 1, 2a and 2b were designed to target 5’UTR. Designed primer sets were optimized in terms of temperature, fluorescent dye, primer mix, Mg2+ and enzyme concentration. After designation of optimum conditions, limit of detection (LOD) was determined for each primer set and their performances were compared. All primer sets have LOD equals to 2x104 copies/μl. Overall, primer set 1 and 2b has higher sensitivity and specificity compared to primer set 2a, therefore they are more suitable to be used for BDV identification with LAMP.
  • Master Thesis
    Developing a Lamp Pcr Based Diagnostic Test for Crimean Congo Hemorrhagic Fever Virus
    (Izmir Institute of Technology, 2022) Üstün, Selcen; Taşkent Sezgin, Hümeyra
    Crimean-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, Hümeyra
    Human 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.