Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
Browse
3 results
Search Results
Master Thesis Investigation of the Effect of 4'-alkylıklavuzon Derivatives on Nucleotide Synthesis and Nucleocytoplasmic Transport(Izmir Institute of Technology, 2016) Kutluer, Meltem; Köksal, Mustafa; Çağır, Ali; Çağır, Ali; Köksal, MustafaIn anti-cancer agent development studies one of the most significant issue is to get an agent that specifically targets cancer cells without any effects on healthy cells. Goniothalamin, that is a styryl lactone isolated from Goniothalamus plant species, is an anti-cancer agent that has selective anti-proliferative activity on cancer cell lines. Klavuzon and derivatives, which can be thought as analogs of goniothalamin, are more cytotoxic in cancer cells compared to goniothalamin. Previous structure activity relationship studies implies that α,β-unsaturated δ-lactone moiety is the source of the biological activity. Since it behaves as Michael acceptor, in this thesis possible irreversible inhibitions of two separate intracellular targets are investigated. In the first part, thymineless death caused by possible thymidylate synthase inhibition has been studied. Anti-proliferative effect of 4’-methylklavuzon in HuH-7 cancer cell line was tested by using MTT. Viability of klavuzon treated cells did not changed significantly in the absence and presence of varying concentration of additional thymidine supplement, and it is concluded that thymineless death is not a crucial mechanism for klavuzon derivatives. In the second part, 4’-methylklavuzon and its derivatives were tested on HeLa cell line to investigate inhibitory effect on the nucleocytoplasmic transport. Immunocytochemistry was used to demonstrate nucleocytoplasmic localization of Riok2 protein which is transferred from nuclei to cytoplasm by CRM1 nuclear export protein. Successfully, all tested klavuzon derivatives inhibit CRM1 nuclear export protein. Potency of the inhibition depends on the size of the alkyl substituent at 4’- position of klavuzon.Master Thesis Purification and Biochemical Characterization of Xylanase Expressed in Thermophilic Geobacillus Sp.(Izmir Institute of Technology, 2015) Algan, Müge; Köksal, Mustafa; Şanlı Mohamed, Gülşah; Şanlı Mohamed, Gülşah; Köksal, MustafaXylanase is an enzyme that catalyzes the degradation of the linear polysaccharide β-1,4-xylan into xylose and breaks down the hemicellulose structure of plant cell wall. The xylanolytic property of the enzyme makes it preferable for many biotechnological applications in industry. This enzyme is possibly produced by some bacterial and fungal microorganisms. In this study, briefly, xylanase enzyme was expressed in thermophillic Geobacillus sp. and purified by cold acetone precipitation and gel filtration chromatography. Molecular weight of our xylanase was found as 40.1 kDa by SDS-PAGE and this protein band was verified by Native-PAGE activity staining. Finally, it was characterized using biochemical methods. For characterization studies, Km and Vmax values were calculated from Lineweaver-Burk plot as 10.2 mg/ml and 31.7 U/ml, respectively. The optima temperature and pH for enzyme activity were investigated using beechwood xylan as substrate and found as 55°C and 8.0, respectively. Furthermore, effects of some metal ions, various chemical reagents and organic solvents on enzyme activity were also determined and we observed that Ca2+, Mn2+ and Co2+ affected the activity positively while Zn2+, Cd2+, Fe3+, EDTA, SDS, CHAPS and DTT shielded the activity. And only β-mercaptoethanol caused a significant change amoung organic solvents. Lastly, that the enzyme has a long shelf-life was confirmed assaying the samples taken from enzyme stocks stored at +4°C and room temperature for six weeks.Master Thesis Engineering of Geranyl Diphospate C-Methyltransferase for the Development of New Diterpenoid Precursors(Izmir Institute of Technology, 2014) Akıl, Caner; Köksal, Mustafa; Köksal, MustafaTerpenoids constitute the most diverse family of natural products. They are involved in several biological functions and are used in medical and industrial applications. The key to their diverse biological activities is their structural diversity. Terpenoids are synthesized in three stages, all of which contribute to generation of structural diversity. In the terpenoid biosynthetic pathways, terpene synthases generate larger linear terpenoid precursors from smaller units via condensation reactions, terpene cyclases transform precursors via cyclization reactions, and then tailoring enzymes modify terpenoid products via addition of functional groups. Recently discovered geranyl diphosphate C-methyltransferase (GPPMT) from Streptomyces coelicolor A3(2) is able to modify a linear monoterpenoid precursor, geranyl diphoshate (GPP), to produce a non-canonical terpenoid precursor, 2-methylgeranyl diphosphate. Modification of GPP by GPPMT is the first example of modification of a canonical linear isoprenoid precursor in nature. This study aims to achieve enzymatic synthesis of novel methylated non-canonical diterpenoid precursors, such as 2-methylgeranylgeranyl diphosphate (2MGGPP) by engineering GPPMT. The novel non-canonical precursors may later be utilized by cyclases to enhance the diversity of the terpenome. For example, taxadiene synthase could utilize 2MGGPP to generate variants of taxadiene, the precursor of the leading anti-cancer drug paclitaxel (Taxol®). Candidate mutants predicted to use GGPP as substrate were selected via in silico analysis of GPPMT structure. These mutations were introduced using the Quick-change site-directed mutagenesis. Mutant genes were expressed in E.coli strains. Mutant proteins were purified by Fast Protein Liquid Chromatography. Catalytic activities of mutants against canonical terpenoid precursors were determined by SAM methyltransferase assay.