Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Secondary Metabolites From Marine Derived Actinobacteria and Their Bioactivities(01. Izmir Institute of Technology, 2024) Can, Özge; Bedir, ErdalAktinobakteriler, antibakteriyel ve antikanser bileşikler dahil olmak üzere birçok biyoaktif ikincil metabolit üretebilmesi açısından dikkat çekmektedir. Bu tez kapsamında, İzmir (Türkiye) Ilıca Körfezi'nden Aktinobakteri olduğu öngörülen bakteriler izole edilerek antimikrobiyal potansiyelleri ortaya konmuştur. On iki izolatın fermantasyon sıvıları etil asetat ile ekstre edilerek, disk difüzyon testi ile seçilen patojenlere karşı (Staphylococcus aureus, Escherichia coli ve Candida albicans) taranmıştır. İzolatların çoğunluğu en az bir patojene karşı antimikrobiyal aktivite göstermiştir. Elde edilen izolatların morfolojik özelliklerine dayanarak Streptomyces üyeleri oldukları düşünülmüştür. Antimikrobiyal potansiyel ve ikincil metabolit profillerine dayanarak, 35M1 izolatı ileri çalışmalar için seçilmiştir. İlgili izolatın tüm genom dizilim (WGS) analizi sonucu türü Streptomyces sp. 35M1 olarak netleştirilmiş ve DDBJ/ENA/GenBank sistemine kaydedilmiştir (Erişim numarası: JBCLWP010000000). Preparatif ölçekli fermantasyonu takiben biyoaktivite rehberli izolasyon çalışmaları gerçekleştirilmiş, beş bileşik saflaştırılarak karakterize edilmiştir. Bu bileşikler; Actinomycin D (ActD), Actinomycin X2 (ActX2), Actinomycin XOβ (ActXOβ), Collismycin A (ColA) ve Collismycin C (ColC) olarak tanımlanmıştır. Sonrasında, ilgili moleküllerin antibiyofilm etkileri araştırılmış ve ActD ile ActX2'nin Metisiline dirençli Staphylococcus aureus, Listeria monocytogenes ve Staphylococcus epidermidis 'e karşı biyofilm inhibitör etkileri olduğu ortaya konmuştur. Son olarak, yeterli miktarda bulunan bileşikler ile (ActD, ActX2 ve ActXOβ) bir sinerjizm çalışması yapılmıştır. ActD ile Nalidiksik asit ve ActX2 ile Ampisilin, Nalidiksik asit ve Rifampisin kombinasyonları, gram-negatif E. coli 'ye karşı sinerjik etki göstermiş ve bu durum literatürde ilk kez rapor edilmiştir.Master Thesis Development of Microfluidic Devices for Investigating Small Molecule Induced Chemotaxis of Dendritic Cells(01. Izmir Institute of Technology, 2023) Khurram, Muhammad Maaz; Bedir, Erdal; Tekin, Hüseyin CumhurMicrofluidics is the core branch of science and technology in which interdisciplinary research is conducted with a low amount of samples in microchannels ranging from 10-100 μm. The main objective of this thesis is to design and fabricate a chemotaxis microfluidic device (CMD) from the poly-methyl methacrylate (PMMA) substrate to analyze the immune cell behavior against cancer cells. The patterns of the three-layered CMD were generated using laser ablation. During the fabrication, Power (P) and Speed (S) values were varied to determine the optimal P-S combination. Then, the structural properties of microfluidic channels in the CMD were examined via microscope. The mechanical properties and liquid handling abilities of CMDs were also investigated through tensile and leakage tests, respectively. Moreover, cell viability of DC2.4 dendritic cells (DCs) and B16-F10 murine melanoma (B16-F10) cells in CMDs sterilized through either autoclaving or UV treatment were determined to test the suitability of CMDs via Live/Dead Assay. The highest cell viability for DCs and B16-F10 was obtained in autoclaved CMDs. For the maturation of DCs before seeding into CMD, DCs were stimulated with lipopolysaccharide (LPS) and Astragaloside VII (AST-VII) at various concentrations. While the cytotoxicity of LPS and AST-VII were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, the expression levels of specific chemokine receptors were also analyzed through flow cytometry. Lastly, stimulated DCs and B16-F10 were simultaneously cultured in the CMD, and the migratory behavior of DCs against B16-F10 was time-dependently studied. Consequently, CMD that provided cost-effective and rapid analysis of intercellular interactions was successfully developed.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 Phytochemical Studies on the Mastic Gum of Pistacia Lentiscus Var. Chia Collected From Karaburun Peninsula and Bioactivities of Isolates(01. Izmir Institute of Technology, 2021) Demir, Mehmet; Bedir, Erdal; Ballar Kırmızıbayrak, PetekPistacia lentiscus L. is an evergreen shrub or tree-like plant named as 'mastic tree'. The phytochemical studies of Chios mastic gum have revealed that it is a complex natural product comprising several organic ingredients like natural polymers, essential oils and triterpenic compounds. Triterpenes are the main components of mastic gum, and they are thought to be responsible for important biological activities such as anti-cancer, anti-ulcer, cytotoxic, antidiabetic, and anti-inflammatory. Within the scope of this thesis, mastic gum collected from the mastic trees (Pistacia lentiscus var. chia) grown on the Karaburun peninsula of İzmir was investigated to reveal its phytochemical composition and neuroprotective effects of the isolates. Isolation and purification studies utilizing chromatographic methods are followed by structural elucidation of the isolates by spectroscopic methods (1D NMR, 2D NMR, MS and X-RAY). Detailed analysis of the obtained spectra helped us to establish structures of 14 molecules possessing triterpenic skeleton, and two of them turned out to be undescribed compounds. The bioactivity of selected compounds was screened for their neuroprotective effects against H2O2 induced oxidative stress on differentiated and undifferentiated SH-SY5Y cells. Two compounds showed neuroprotective effects against H2O2-induced cell death on undifferentiated SH-SY5Y cells at 5 and 10 µM concentrations, while the other compounds either showed little or no effect. This study provides promising new insights into the use of mastic gum or its components in the treatment of neurodegenerative diseases.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.