Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Method Development for Pilot Production of Astragaloside Vii(Izmir Institute of Technology, 2019) Kurt, Mustafa Ünver; Bedir, ErdalBased on the promising adjuvant properties comparable to most widely used adjuvants Alum and Quillaja saponins (including QS-21), our team has decided to carry out advance studies to develop Astragaloside VII (AST VII) as a new vaccine adjuvant and/or an immunotherapeutic agent. To do so, one of the most important steps is establishing efficient isolation and purification processes to obtain AST VII at large scale. In this thesis, starting from laboratory scale to semi-pilot scale, it was aimed to optimize the production steps of AST VII from Astragalus trojanus. Factor screening (1 categorical and 3 numerical) and optimization studies were performed using experimental design, based on which methanol (MeOH) as solvent, 1:20 (g/mL) as plant:solvent ratio, 0.5-1.0 mm as plant particle size and 8-10 hours for extraction time were selected yielding 0.36 percent g AST VII/g plant. To enrich AST VII in extract, pre-purification studies were performed such as liquid-liquid extraction, resin fractionation and precipitation. The results showed that the resin (D-101) fractionation employing water, 20 percent EtOH and EtOH was superior. To enrich AST VII up to 85-90 percent purity, several chromatographic steps using normal (employing EtOAc:MeOH:Water and Chloroform:MeOH:Water systems) and reversed phase (C18; employing MeOH:Water systems) silica gel were used. In last step, a precipitation method was developed using MeOH and acetone affording 98 percent purity. Developed method at lab scale (3.5 g) was successfully transferred to semi-pilot scale (about 100 g) with minor modifications, and a crucial step towards large-scale isolation (kg) of AST VII was accomplished.Master Thesis Dual Therapy Opportunities for Tuberculosis and Prostate Cancer Based on Crystal Engineering Principles(Izmir Institute of Technology, 2019) Arpacıoğlu, Merve; Şanlı Mohamed, GülşahWithin this work, it is aimed to facilitate dual therapy opportunities for the treatment of tuberculosis and prostate cancer separately based on crystal engineering principles. In this regard, a quinolone family member antibiotic namely Sparfloxacin (SPX) is combined with another antibiotic called 4-aminosalicylic acid (4-ASA). Due to the drug-drug combination, it is expected to have superior biological activity since both drugs are stated and offered to be used in the treatment of tuberculosis. In addition to that, Apalutamide (APA) a recent drug in the treatment of castration-sensitive metastatic prostate cancer (CSMPC), is metallo-encapsulated by using zeolitic imidazolate framework-8 (ZIF-8). Owing to pH-sensitive decomposition, the new formulation is expected to combine the benefit of free zinc release and APA release to the targeted cancerous section. Both new compounds are synthesized characterized by various methods such as single crystal X-ray diffraction (SCXRD), powder X-ray diffraction (PXRD), Fourier-transform infrared radiation (FTIR), thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and high-performance liquid chromatography (HPLC).Master Thesis Directed Evolution of a Cytochrome P450 Enzyme To Increase Peroxidation Activity(Izmir Institute of Technology, 2019) Başlar, Muhammet Semih; Sürmeli Eraltuğ, Nur BaşakDirected evolution, mimicking the natural selection, is a powerful tool to create novel enzymes. Evolution of natural enzymes to achieve desired properties are performed in iterative rounds of random mutagenesis followed by a screening/selection method. Enzyme activity can be enhanced with substituting the active site amino acids in the enzyme. CYP119, a member of cytochrome P450 protein family, is a thermophilic enzyme extracted from Sulfolobus acidocaldarius that exhibits monooxygenase, peroxidase and oxidoreductase activity. These properties give CYP119 a potential to be used in production of fine chemicals and pharmaceuticals. Herein, two different mutant libraries of CYP119, containing substituted amino acids at Thr213-Thr214 and Val151- Phe153 positions, constructed via combinatorial active site saturation test (CAST), and screened for improved peroxidation activity. Additionally, fluorescence based Amplex Red peroxidation activity assay using hydrogen peroxide as cofactor of CYP119 was optimized. Screening of mutant libraries resulted four improved CYP119 mutant enzymes from Thr213-Thr214 mutant library. Val151-Phe153 mutant library did not yield any improved peroxidation activity mutants which indicated amino acid substitutions at 151- 153 positions do not have any effect on peroxidation activity of CYP119. Furthermore, effect of substituted amino acids at predetermined positions were analyzed. Substrate, Amplex Red, makes single or double hydrogen bond when molecular docking was performed on improved mutant enzymes also distance of nitrogen atom in Amplex Red to heme iron is closer than wild type CYP119 in improved mutant enzymes. Thus, increasing the peroxidation activity of mutant CYP119 enzymes.Master Thesis Cloning of Sericin-Like Proteins for Recombinant Production in Bacteria(Izmir Institute of Technology, 2019) Bostan, Fatmanur; Sürmeli Eraltuğ, Nur Başak; Bulmuş, VolgaSilk consists of two main proteins called fibroin and sericin. While fibroin is used in textile production and various biomaterial applications, sericin is considered as waste material in the textile industry. Sericin is a multi-component protein with an indefinite structure and it has been shown to be biocompatible and has biological activity. Because of the positive effects on keratinocytes and fibroblasts have led to the development of sericin-based biomaterials for the repair of skin tissue. Sericin from silkworm cocoons can be obtained by chemical treatment, enzymatic treatment and boiling in water. Although sericin can be separated from fibroin by chemical, enzymatic and boiling in water treatment methods, all these treatment methods are not enough to obtain recovery of high-quality sericin. Moreover, in these treatment methods, the exposure of sericin protein to high temperature causes even sericin protein obtained by the same method to indicate different characteristics. The fact that the obtained sericin demonstrate such major changes in the structure according to treatment methods bring inconsistencies in the quality of sericin produced as a biomaterial. The aim of the study is to produce native sequence of sericin that forms a tetramer contain each containing 38 amino acids with recombinant production in E.coli and to characterize structural properties Thus, obtaining sericin protein from the bacteria with recombinant methods will solve these problems in question The results indicate that for the first time, the conformational properties of recombinant sericin were obtained similar to the native sericin structure.Master Thesis Bioinformatics Based Approach To Design a Thermophilic P450 Fot Industrial Biocatalysis(Izmir Institute of Technology, 2019) Kestevur Doğru, Ekin; Sürmeli Eraltuğ, Nur BaşakEnzyme catalyzed biosynthesis of steroidal drugs is an important process for pharmaceutical manufacturing. Cytochrome P450 (P450) monooxygenases are important for hydroxylation of steroid structures because they can catalyze the oxidation of inactive carbon bonds with high selectivity and efficiency. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius, which has the potential to be used as biocatalyst for industrial production since it shows activity at high temperature and low pH conditions. In this work we aim to use CYP119 for selective hydroxylation of progesterone, which is not the original substrate of CYP119, for production of precursor molecules of important hormones like cortisone and aldosterone. Crystal structure of CYP119 (PDB ID: 1F4T) was used for selecting residues that were mutated according to structural alignment with other CYPs that can catalyze progesterone hydroxylation naturally. Progesterone-docking performed with CYP119 to identify residues that create clashes with substrate. Finally selected 12 residues (Leu69, Val151, Phe153, Leu155, Leu205, Ile208, Ala209, Thr213, Thr214, Val254, Thr257, Leu354) were mutated with PyRosetta program to Gly, Glu, Phe, Met, Ala, His, Arg and Ile. Progesterone-docking performed with using DockMCM Protocol of PyRosetta. We used two different starting coordinates of progesterone for docking and results were eliminated according to their energy scores. Best mutants were used for creating double/triple mutants and second round of docking and elimination process were performed with using double/triple mutant enzymes. Final number of 11 mutants with best scores were selected and their possible products were identified.Master Thesis Increasing Doxorubicin (dox) Release From Liposomes(Izmir Institute of Technology, 2019) Hanoğlu, Berçem Dilan; Özdemir, Ekrem; Altun, Zekiye SultanCancer is the second most common cause of death in the world and its incidence is increasing day by day. Doxorubicin (DOX) is an anthracycline group drug frequently used in many cancer treatments including breast cancer. However, free DOX has many harmful side effects and need to be encapsulated into nanocarrier such as liposomes. Although liposomal DOX has many advantages over its free form, liposomal DOX has undesirable side effects such as hand and foot syndrome. In this thesis, it was aimed to develop a more effective liposomal DOX delivery and release systems. Liposomes were prepared with alkaline solutions containing tris, sodium carbonate, ammonium chloride, and ammonium sulfate. DOX loading into liposomes and the percentage of release from liposomes were examined. A loading efficiency of about 80% was achieved, while the release was found to be below 13% at room temperature. The release of DOX was found to be enhanced from liposomes in the presence of ammonia (NH3), whose content was dependent on pH. Temperature was also found an important parameter and enhances DOX release at higher temperatures than the phase transition temperature of the lipid. A two-component liposomal system was proposed where ammonia (NH3) would be released from one liposome and enhance the DOX release from other liposomes. It was found that temperature, pH, and ammonia (NH3) concentration affected DOX release from liposomes. As a result, DOX was successfully loaded into liposomes and ready to study their effect on breast cancer cells.Master Thesis Surface Modification of Chitosan Films/Meshes for Biomaterial Applications(Izmir Institute of Technology, 2019) Işıklı, Berçin; Tıhmınlıoğlu, FundaModification of surface of biomaterials is a great interest for many years due to first contact of surface of materials with the biological fluids. This thesis aims to investigate surface modification effect on the chemical, surface wettability, protein adsorption as well biodegradability properties of dense chitosan (Ch) and asymmetric chitosan films (ACh). The surfaces of chitosan dense and asymmetric films were modified by ion implantation technique using carbon and carbon-nitrogen hybrid ions at a fluence of 1x1015 ions/cm2 and ion energy of 20kV. Chemical compositions of the film surfaces were analyzed by Fourier transform infrared spectroscopy (FTIR-ATR). Surface hydrophobicity measurements were conducted by static contact angle measurements. Protein adsorption on unmodified and modified surfaces on films was investigated as a function of time at various pH conditions. After ion implantation on chitosan films, both C and C-Nitrogen ion implantation, the surfaces become rougher and hydrophobic having moderate wettability (����� values in the range of 72-85°) and in good agreement with FTIR-ATR data findings. It was found pH dependence of the amount of protein adsorbed on the dense chitosan films as a function of time for both un-implanted and implanted films. BSA and fibrinogen were more adsorbed on the chitosan films at pH 5. The amount of BSA and fibrinogen protein adsorption was 0.97 and 1.33 gprotein/gfilm, respectively for 60 min incubation period. Protein adsorption enhanced for C and C+N2 ion implanted samples for BSA and fibrinogen, respectively due to the hydrophobic protein surface interaction effect. In vitro degradation results showed that ACh films degrade much faster (mass loss 57 %) than Ch films (40 %) due to the porous structure at the end of 3 weeks. However, the ion implanted Ch samples degraded much slower having mass loss of 30% and 17.7% for C+N2 and C implanted samples, respectively at the end of 3 weeks compared to un-implanted Ch films as 40 %. The results are in good agreement with water sorption and surface hydrophobicity of the implanted films. This study demonstrated that surface modification, as well as structure, changes the protein sorption, wettability and biodegradation properties of the chitosan films.Master Thesis Naphthoquinones From Natural Sources and Their Bioactivities(Izmir Institute of Technology, 2019) Kul, Demet; Bedir, ErdalOnosma L. is a large heterogenic genus of Boraginaceae family includes about 230 species distributed mainly in Central Asia and the Mediterranean region. According to ‘Flora of Turkey, Onosma genus is represented with 104 species and 108 taxa and 52% of which are endemic. Phytochemical studies have revealed that Onosma species possess various constituents including alkaloids, naphthoquinones, polyphenols, phytosterols, terpenoids and fatty acids. Naphthoquinones are naturally widespread secondary metabolites deriving from some higher plants, fungi and bacteria, and have shown significant biological activities such as cytotoxic, antibacterial, antifungal and wound healing. In this thesis, bioassay-guided isolation studies were performed on Onosma aksoyii and Onosma isaurica to obtain naphthoquinone type cytotoxic compounds and investigate their topoisomerase inhibitory properties. Isolation studies were guided by MTT assay using three human cancer cell lines (HeLa, HCC-1937, DU-145) and a nontumor cell line (MRC-5). whereas the enzyme inhibition tests were against human topoisomerases IIα and IIβ. Six compounds, one of which was new (OA-PE-D1), were isolated using chromatographic methods and their structures were elucidated by spectral methods (1D, 2D NMR, and MS). The known compounds were acetylshikonin, β,β- dimethylacrylshikonin, arnebidin, arnebifuranone and shikonofuran E. The cytotoxicity screenings showed that these compounds had IC50 values ranging from 6.485 μM to 32 μM. According to topoisomerase inhibition studies, OA-PE-D1 and β,β- dimethylacrylshikonin showed promising inhibitory effects on topoisomerase IIβ at dose of 1 mg/mL.Master Thesis Modification of Graphene Surfaces for Detection of Biomicroparticles(Izmir Institute of Technology, 2019) Yeşiltaş, Gözde; Bulmuş, VolgaPathogens present in the food we consume and the water we drink pose a major threat to human health. Another major health concern is the metastasis of cancer in which cancer cells spread to new areas of the body, often by way of the lymph system or bloodstream. To minimize the burden on health and economy, the detection of biomicroparticles such as pathogens or circulating cancer cells in a highly sensitive and practical manner is higly desirable. This thesis aims to develop a method to create graphene-based biosensor substrate for detection of biomicroparticles such as bacteria, viruses or mammalian cells. For this aim, graphene surface was first functionalized using a linker molecule. The effect of solvent type on functionalization was investigated via Raman spectroscopy and X-Ray spectroscopy (XPS). AntiCD2 antibodies (Ab), as a model antibody, were then conjugated to the functionalized graphene via NHS/EDC chemistry. The Ab conjugation was verified by Raman spectroscopy and XPS analyses. Finally, Jurkat cells, as model biomicroparticles, were recognized and captured by Abconjugated graphene surface, as evidenced by optical microscopy. The temperature, medium, and method for interaction of cells with graphene surfaces as well as the specificity of the Ab- functionalized graphene surface were investigated. The results overall showed the specific and efficient recognition of model cell line by Abconjugated graphene surfaces.Master Thesis The Effect of Metal Doping on Tio2 for Photocatalytic Applications(Izmir Institute of Technology, 2019) Alduran, Yeşim; Özyüzer, Lütfi; Öztürk, OrhanRecently, the photocatalysis method has been an active research area as a promising solution for environmental cleaning method, leading to self-cleaning and sterilization of solar cell surfaces to produce water dissociation reaction. Titanium dioxide (TiO2) is the most suitable semiconductor for photocatalytic applications due to its high oxidation potential and high efficiency when irradiated by ultraviolet light (UV). Undoped and Ruthenium (Ru+) doped TiO2 thin films were prepared using magnetron sputtering technique. All thin films were grown on SLG different ratios like 1 sec, 3 secs, 5 secs and 7 secs to set shutter position in magnetron sputter target. Transparent substrate SLG is coated with nearly 50 nm TiO2 thin films without compromising any optical properties. Samples were heat treated for two hours at 500°C to get the anatase phase crystal structure. The crystallization peaks of TiO2 are proved to get the anatase phase. Photocatalytic activity of TiO2 thin films are determined after 1, 3, 5 and 24 hours with organic pollution as a methylene blue dye degradation under UV light. The degradation of methylene blue was investigated kinetically and photocatalytic activity rate constants of the photocatalysts were calculated. All thin films could not reach super hydrophilicity state. Undoped TiO2 contact angle 47.309o and Ru doped TiO2 63.218o were evaluated. The photocatalytic degradation percentage of Methylene Blue was reached 87%, after 24 hours of UV irradiation, when using Ru-doped TiO2 thin film. Consequently, the anatase phase of Ru-doped TiO2 thin films are found best photocatalytic activity in self-cleaning performance.