Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Investigation on the Bioactive Secondary Metabolites of the Endophytic Fungus Penicillium Roseopurpureum(01. Izmir Institute of Technology, 2022) Dizmen, Berivan; Bedir, ErdalIn recent years, endophytic fungi have been considered as significant resources of new bioactive secondary metabolites, so they are predicted to have an important impact in drug discovery and development. In our preliminary study, the chemical diversity and cytotoxic activity of an endophytic fungus, namely Penicilium roseopurpureum 1E4BS1 isolated from Astragalus angustifolius, have been demonstrated. Based on these data, it was aimed to obtain bioactive secondary metabolites of P. roseopurpureum 1E4BS1 within the scope of this thesis. Firstly, a fermentation study was carried out in a rotary shaker at 180 rpm, 25 ºC for 20 days, and the obtained broth was extracted with EtOAc. Secondly, nine metabolites were isolated from the EtOAc extract by chromatographic methods, and the structures of compounds were elucidated by spectral methods (1D-, 2D NMR, and MS). The structure elucidation studies revealed that seven metabolites had anthraquinone backbone, whereas two compounds were found to be derivatives of curvularin from macrolide group. A chemical structure search confirmed that five of the metabolites were new for nature. Cytotoxic activity of the compounds and the EtOAc extract was tested against three cancer (DU145, LnCaP, and PC3) and normal (RPWE-1) cell lines by MTT cell viability assay. The metabolite PR-EB-01 exhibited the highest activity with IC50 values of 26.0, 37.2, 24.7, and 30.9 μM against LNCaP, PC3, DU145, and RPWE-1 cells, respectively.Master Thesis Effects of Centella Asiatica Saponins on Telomerase Activation and Wound Healing(Izmir Institute of Technology, 2021) Demirbaş, Devran; Bedir, Erdal; Büyükkileci, Ali OğuzCentella asiatica L. is a well-known plant species endemic to Southeast Asia that has noteworthy biological effects. Triterpenoid saponins, comprising more than 80% of the content, are suggested to be the chief compounds responsible for the biological effects. A recent study has described that the extract of Centella asiatica exhibits telomerase activation. In line with these developments, as part of our studies on natural products demonstrating anti-aging properties, we decided to engage Centella asiatica and its components. Within the scope of this thesis, four major compounds, viz. madecassoside, asiaticoside, madecassic acid, and asiatic acid were isolated from the standardized extract of Centella asiatica, and their structures were elucidated by spectroscopic methods. Using in vitro methods, the effects of the extract and purified compounds on cell proliferation under standard culture and oxidative stress (H2O2) conditions, wound healing, and human Telomerase Reverse Transcriptase (hTERT) protein level were investigated. Our experiments were conducted on MRC-5 and HEKn cell lines. It was observed that the standardized extract of Centella asiatica increased the proliferation of the MRC-5 cells meaningfully between 5 to 100 µg/ml. Moreover, the extract showed protective effects on MRC-5 cells at 500 and 1000 ng/ml under oxidative stress conditions. Madecassoside, madecassic acid, asiaticoside, and asiatic acid exhibited the highest proliferative effects on MRC-5 cells at concentrations of 1000 nM (28%), 2 nM (66%), 300 nM (61%), and 300 nM (56%), respectively. Asiatic acid and the extract accelerated cell migration in wound areas that were made on MRC-5 cells up to 32% and 36% in the range of 10 to 300 nM or ng/ml, respectively. The immunoblotting assay studies showed that madecassoside and asiaticoside were increased the expression of hTERT protein level on HEKn cell line by 3.16-fold and 5.62-fold, respectively, at 30 nM concentration. Furthermore, the extract was observed to increase the protein level by 2.62-fold at 300 ng/ml.Master Thesis Docking Studies on Selected Sapogenins Targeting Androgen and Estrogen Receptors(01. Izmir Institute of Technology, 2021) Çömlekçi, Yiğit Ege; Bedir, ErdalNatural products have been used in the treatment of various diseases in history, and they are still in use today. Sapogenins are natural products derived from plant and animal sources that also have numerous biological activities. Furthermore, some sapogenins have been found to be active in common cancers such as prostate and estrogen alpha-mediated breast cancer and exert their effects via the androgen and estrogen receptors. For this reason, identifying alternative ligands for these receptors may aid in enhancing the benefits or avoiding adverse effects. Traditional or advanced molecular screening techniques are available with their respective applications. However, these applications have some limitations, such as being complicated and costly or requiring a significant amount of time due to the large number of molecules involved. With advancements in technology, in-silico methods such as molecular docking have developed into a highly accurate and cost-effective method for high throughput screening. Additionally, rapid and high-quality in-silico visualization of docked ligands and their interactions serves as a preliminary step toward determining structure-activity relationships. The molecular docking method was used in this study to identify novel androgen and estrogen receptor ligands, and to evaluate the structure-activity relationship of sapogenin molecules, which were selected from our research group's molecule library. Moreover, the Swiss Target Prediction web service was used to determine the probability of bindings prior to molecular docking. The molecular docking results demonstrated that nine of the selected sapogenins were more bindable to the androgen receptor than testosterone, whereas another nine were found to be more bindable to the estrogen receptor than estradiol. Additionally, immunoblotting was utilized to validate the activity of several molecules by examining their effects on PSA levels for androgen receptor binding.Master Thesis Induction of Secondary Metabolism of Some Marine Derived Streptomyces Species, and Isolation and Identification of Their Bioactive Secondary Metabolites(Izmir Institute of Technology, 2020) Gezer, Emre; Bedir, Erdal; Büyükkileci, Ali OğuzSecondary metabolites are natural products with low molecular weight produced by different organisms. These metabolites have a wide variety of bioactivities because of their adaptive roles in the nature. These properties make secondary metabolites important source in drug discovery studies. Streptomyces genus, on the other hand, attracts attention due to their ability to produce many secondary metabolites for the treatment of various diseases, especially infectious diseases and cancer. However, secondary metabolism is not fully expressed under standard laboratory conditions as in nature. This phenomenon limits the discovery of new/novel bioactive molecules from the microbial sources. In this study, a previously studied marine derived actinobacterium, namely Streptomyces cacaoi, was investigated further to discover new antimicrobial metabolites via medium and temperature optimization using Box Behnken design. As a result, GPM medium containing 2.25% glycerol, 1% peptone water, 0.2% CaCO3, 0.1% MgCl2 in distilled water was found to provide the highest chemical diversity with potent bioactivity at 30oC. In subsequent studies, inductive effects of some microorganisms and inorganic compounds on secondary metabolism were also determined. Using optimized conditions, a larger fermentation study was undertaken (25 L) followed by extraction and isolation procedures. Sixteen metabolites were purified by chromatographic methods, and structures of the isolates were elucidated by spectral methods. Thirteen compounds, five of which were new, were members of polyketide-type polyether antibiotics. The structures of other molecules were determined as cyclo(Thr-Trp), 6-hydroxy-6-methyloctanoic acid, and 5-hydroxy-1,6-diazacycloundec-5-en-2-one, and all were found to be new. In antimicrobial tests, most polyethers were found to be active against Gram-positive bacteria. In particular, two new polyethers SC-EG-05 and SC-EG-07 showed higher antimicrobial activity than widely used antibiotic vancomycinMaster 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 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 Preparation of Some Semi-Synthetic Saponin Analogs and Investigation of Their Mechanism of Action on Necrotic Cell Death(Izmir Institute of Technology, 2019) Üner, Göklem; Bedir, Erdal; Ballar, PetekSince antitumor potency of saponins is relatively weak, researchers focus on semi-synthetic modification of saponins to obtain highly potent structures. With the same motivation, we prepared cytotoxic sapogenol (AG-08), from cycloastragenol. Our preliminary studies revealed that AG-08 was inducing necrotic cell death together with autophagic inhibition. Furthermore, immunoblotting experiments suggested that AG-08 promoted cleavage of various proteins. A continuation study was performed in this thesis with aims of: i) verifying previous studies; ii) identifying molecular mechanism of AG-08; iii) preparing further analogs of AG-08 and deduce structure activity relationships(SAR). Accordingly, necrotic cell death and autophagic inhibition via AG-08 was verified, and cytotoxicity of AG-08 on 13 cell lines was examined. Furthermore, inhibitors of calpain-1, general caspases, cathepsin B/L/S, and caspase-8 were found to partially alleviate cell death, whereas cathepsin D/E inhibitor were not able to do. Additionally, lysosomal impairment due to loss of acidic nature was demonstrated. Later data and effect of cathepsin inhibitor on AG-08 mediated cell death suggest lysosomal membrane permeabilization. In synthesis part, 15 AG-08 analogs were prepared, three of which were cytotoxic. Additionally, active analogs triggered similar cell death mechanism with AG-08. SAR evaluation reveals that presence of tosyl, and tetrahydrofuran ring are required for activity, while double bond at C-6 is not essential. Consequently, this thesis provides important data on mechanism of necrotic cell death and autophagic inhibition via AG-08 treatment as well as relationship between structure and activity. However, further studies are warranted to clarify complete mechanism of AG-08 and substantiate structure activity relationship deductions.Master Thesis Fungal Biotransformation of Novel 20(27)- Octanor Cycloastragenol and Biological Activities of the Purified Metabolites(Izmir Institute of Technology, 2019) Duman, Seda; Bedir, Erdal; Çağır, AliBiotransformation is the chemical modifications performed by enzymes or living organisms. The difficulty and high cost of enzyme isolation and purification makes it more advantageous to use whole cell systems as biocatalysts. Microbial biocatalysts are particularly interesting in the modification of complex molecules such as steroids and triterpenoids, which can catalyze stereo- and regio- selective reactions that are difficult or impossible to perform with chemical reactions. Specially, ability of endophytic microorganisms to produce specific enzymes for adaptation to their environment by tolerating toxic defense metabolites, makes them interesting for biotransformation studies. Telomeres are nucleotide structures located at the end of chromatids shortening with each cell division. Telomerase is a reverse transcriptase enzyme, and it helps to replenish telomere ends that are truncated by aging and stress factors. The telomerase activators (TA) with their potentials are suggested encouraging agents for healthy aging, and they are projected to generate a huge market in the future. Cycloastragenol is the only natural product in the sector marketed as a potent telomerase activator. In this study, by using endophytic fungi, the biotransformation studies were performed on 20(27)-octanor cycloastragenol. As a result, 14 biotransformation products, were isolated by chromatographic studies, and the structures of the metabolites were established by spectral methods. Based on the literature survey, 13 compounds turned out to be new for nature. Seven metabolites were screened for telomerase activation. In these screenings, metabolites showed telomerase activation ranging from 5.43 to 12.36 fold at doses ranging from 0.1 to 30 nM compared to the control cells treated with DMSO.Master Thesis Investigating Immunomodulator Mechanisms of Astragalus Saponins(Izmir Institute of Technology, 2018) Yakuboğulları, Nilgün; Yakuboğulları, Nilgün; Bedir, Erdal; Sağ, DuyguAdjuvants are chemical/biological substances that are used in vaccines to increase immunogenicity of antigens. Astragaloside VII (AST VII), a triterpenoid saponin isolated from Astragalus species, stimulates Th1 mediated immune response with antigen specific antibody response. The main goals of this thesis are synthesis of immunologically active analogs of AST VII and identifying immunomodulatory mechanism of actions of AST VII. The impact of AST VII and its synthesized analogs (dicarboxylic AST VII: DC-AST VII and dodecylamine conjugated AST VII: DAC-AST VII) on the cytokine release profile of human whole blood cells (hWB), dendritic cell maturation and subsequently T cell activation were analyzed by using flow cytometry and ELISA. IL-1 and IL-17A cytokines were substantially induced on hWB following treatments of the compounds. The most potent compounds were: DAC-AST VII (3.32 fold) for production of IL-1, AST VII (5.05 fold) for production of IL-17A. AST VII was more effective than DAC-AST VII (7.52 fold versus 1.34) in IL-1 production in BMDCs (bone marrow derived dendritic cells). The co-stimulation with AST VII and LPS enhanced dendritic cell maturation and activation by upregulating MHC II, CD86 and CD80, as well as IL-12 induction. All compounds were able to activate CD4+ and CD8+ T cells via increasing CD44 expression. Inflammasome activation may have a role in AST VII induced IL-1 secretion, dendritic cell maturation and T cell activation. However, more detailed molecular mechanism studies are warranted to substantiate our findings and to put forward signaling pathways involved.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, ErdalDirected 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.