Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Development of Keratin Based Hydrogel Systems(Izmir Institute of Technology, 2022) Yalçın Göl, Damla; Top, AybenIn this study, keratin proteins from Merino sheep wool were obtained via oxidative extraction (Chapter 2), sulfitolysis extraction (Chapter 3) and sulfitolysis with reductive extraction methods (Chapter 4). Keratin proteins were characterized XRD and FTIR spectroscopy and thermal analysis. In the SDS-PAGE gel results of the keratins diffusive protein bands between ~23 kDa and >170 kDa and a discrete band at about 12 kDa were observed confirming highly polydisperse nature of the protein samples. Then, keratin-based hydrogel systems were obtained via different methodologies. In Chapter 2, oxidized keratins (keratoses) were crosslinked with THPC to form keratose hydrogels. Effect of the amount of the crosslinking agent on the viscoelastic, swelling, and morphological properties of hydrogels was investigated. In Chapter 3, the keratin hydrogels were obtained via reformation of disulfide bridge and self-assembly of the keratin chains. In Chapter 4, keratins reduced with DTT were crosslinked with 2000 Da PEG-(C2H4-mal)2 and 6000 Da PEG-(C2H4-mal)2 to prepare PEG-hydrogels. Storage moduli of the hydrogels were obtained in the range of 63 ± 22 and 2613 ± 254 Pa and were shown to be tuned by the amount and chain length of the crosslinker. The highest swelling ratios were obtained for the THPC crosslinked hydrogels whereas the highest pore size was observed in PEG-keratin hydrogels. Cytocompatibility of the keratin based hydrogel systems was confirmed using L929 mouse fibroblast cells by applying CCK-8 tests. Of these hydrogels, PEG-keratin hydrogels were found to support cell proliferation with a higher rate than empty TCPS wells up to 4 days. These results demonstrate that low-cost keratin-based hydrogels can be used in a variety of biomedical applications, such as drug delivery systems for cancer therapy, and scaffolds in wound healing and soft tissue engineering.Master Thesis Peptide Hydrogels Containing Cell Attachment Molecules(Izmir Institute of Technology, 2019) Uysal, Berk; Top, Ayben; Top, AybenIn this study, peptides with sequences and notations as KLELKLELKLEL (KLEL), KLDVKLDVKLDV (KLDV), KLDVKLDVKLKV (KLKV1), KLKVKLDVKLKV (KLKV2), KLKVKLKVKLKV (KLKV3) were synthesized using solid phase peptide synthesis (SPPS) method based on Fmoc chemistry. Reverse phase HPLC and MALDI-TOF mass spectroscopy characterization methods were used to assess the purity of the peptides. Three different synthesis procedures were tested, and it was found that employing DMF:DMSO at 1:1 ratio as a solvent increased purity of the resultant peptide. FTIR results indicated the presence of expected β-sheet secondary structure, as well as an interference band from TFA salts for all of the peptides. All the peptides formed hydrogels at pH 7.4 with 1 wt% concentration in deionized water (DIW). AFM results of these hydrogels indicated that KLKV1 and KLKV2 had fibrous morphology with a width of 5-20 nm and 7-18 nm respectively. KLDV and KLKV3 peptide hydrogels, on the other hand, exhibited globular structures, having sizes with 15-50 nm and 8-15 nm, respectively. Storage moduli (G’) of these hydrogels in DIW were obtained as ~860 ± 150 Pa, ~260 ± 60 Pa, ~210 ± 30 Pa and ~1850 ± 200 Pa for KLDV, KLKV1, KLKV2 and KLKV3 respectively. Of these peptides, only HCl salt of KLDV and KLKV1 peptides more readily formed hydrogels in PBS but at 1.5 wt% concentration. G’ values of these KLDV and KLKV1 hydrogels were determined as ~1810 ± 850 Pa and ~700 ± 230 Pa, respectively. Cell proliferation tests (CCK-8 assay) of KLDV and KLKV1 hydrogels were performed by using L929 mouse fibroblast cells. Empty wells (TCPS) were used as a control group. Cell proliferation was observed to be comparable for both select hydrogels and empty wells, suggesting possible applications of these hydrogels in tissue engineering.Master Thesis Development of Endosome Disruptive Peptide and Peg Conjugate Based Doxorubicin Delivery System(Izmir Institute of Technology, 2019) Özkıyıcı, Selin; Top, AybenIn this study, it was aimed to develop a drug carrier system including a TAT-derived cell penetrating peptide in order to provide fast transport of anticancer drugs from endosomal compartments to nucleus. The drug delivery system, denoted as mPEGpeptide- oxime-DOX, was based on polyethylene glycol, endosome disruptive peptide (G2RQR3QR3G2S), and doxorubicin (DOX) conjugate. Control drug delivery system, lack of the peptide (mPEG-oxime-DOX) was also synthesized to assess the effect of the peptide on the physiochemical and drug release properties of the drug carrier. As the first synthesis step, mPEG-OH was converted to mPEG-aldehyde form using DMSO-acetic anhydride oxidation reaction and aldehyde functionalization was determined by using FTIR and NMR spectroscopy. The peptide and mPEG-peptide were synthesized using solid phase synthesis protocol, and their purities were confirmed using HPLC and MALDI-TOF mass spectroscopy analyses. Prior to DOX conjugation, hydroxyl group of serine residue in the mPEG-peptide system was oxidized to aldehyde. The anticancer drug was attached to the carrier molecules via amine-aldehyde reaction forming an acid cleavable oxime bond. Drug release, size distribution, and stability of the PEG-peptideoxime- DOX system were evaluated and compared with those results of the control drug delivery system. For mPEG-oxime-DOX, a pH programmed DOX release with the respective % DOX release values of ~68 % and ~28 % at pH 5.0 and pH 7.4 was observed. For mPEG-peptide-oxime-DOX, on the other hand, quite low DOX release (~10-15 %) was obtained for both pH values suggesting possible interaction between DOX and the peptide. Mean size value of the mPEG-oxime-DOX was measured as ~24 nm. However, mPEG-peptide-oxime-DOX, had quite lower hydrodynamic diameter values (~3nm and ~6 nm at pH 5.0 and pH 7.4, respectively) possibly due to repulsions between the arginines in the peptide domain. Observation of the morphology and evaluation of the cytotoxicity of these drug delivery systems are underway.Master Thesis Effects of Grid Design on Lead-Acid Battery Performance(Izmir Institute of Technology, 2017) İşler, Tuğçe; Ebil, Özgenç; Top, AybenIn today’s world, approximately 88 percent of the total energy demand is supplied by fossil fuels; however, it has become clear that; other energy sources are needed due to limited fossil fuels. The demand for energy can most effectively be filled by renewable energy sources as installed energy storage capacity is growing rapidly. If renewable energy sources advance enough to fulfill the high demand, earth-friendly, clean and sustainable energy will help to protect the environment, thus ensuring a healthier life for future generations. Energy storage systems are essential in this endeavor, and in order to become more prevalent, storage systems for renewable energy sources must supply electricity without interruption as much as possible. As an electrochemical storage, a battery with a high level of performance, high energy density and life cycle could offer a viable solution for electricity storage provided that battery cost should be economically viable. This thesis aims to improve the geometry of the grid used in lead acid batteries in order to obtain a more uniform current and potential distribution, and minimize the potential drop for improved battery performance. A 3D mathematical model was developed using finite element method to evaluate the behavior of the grid under various conditions. Five different porous grid geometries were simulated under different loads and optimum grid geometry was identified. The 3D mathematical model of the lead-acid battery based on finite element method was simulated under certain conditions in order to evaluate the effect of grid geometry on battery performance.Master Thesis Hydrogels and Self-Assemled Nanostructures Based on Wool Keratose(Izmir Institute of Technology, 2017) Pakkaner, Efecan; Top, AybenIn this study, water soluble keratose proteins were extracted from “Ovis aries” wool using peracetic acid oxidation with a yield of 35 ± 5 %. Wool samples and the extracted keratose proteins were characterized by using FT-IR, XRD, SEM and TGA techniques. α-keratose fractions (MW = 43-53 kDa) along with cleaved fragments of α-keratoses with molecular weights between 23 and 33 kDa were identified in the extracted protein mixture using SDS-PAGE analysis. DLS and AFM experiments indicated self-assembled globular nanoparticles with diameters of 20-40 nm formed at 5 and 10 mg/ml keratose concentrations. On the other hand, at 10 % w/v keratose concentration interconnected keratose hydrogels with pore sizes of 6 ± 4 and 7 ± 4 μm were obtained upon incubation at 37 and 50 °C, respectively. Storage moduli (G’) of these physical hydrogels were increased from ~100 to ~1000 Pa, as gelation temperature was increased from 37 to 50 °C. Hydrogels were also obtained at 7.5 % w/v keratose concentration by the addition of a crosslinker, THPC. Amine group:crosslinker ratio was used as 1:1, 1:2 and 1:4. As the amount of crosslinker increased, network transformed from fibrous to more planar structures exhibiting a significant decrease in average pore size from 24 to 11 μm. G’ values of the crosslinked hydrogels were obtained between ~1 and ~5 kPa tuned by the crosslinking amount. Cell interaction properties of a select physical hydrogel prepared at 37 °C was tested using CCK-8 assay. It was observed that the keratose hydrogel supported L929 mouse fibroblast cell proliferation as much as collagen, which suggests that these keratose hydrogels can be promising candidates in soft tissue engineering applications.Master Thesis Peg-Peptide Drug Carrier Systems With Enzymatic Degradation Units(Izmir Institute of Technology, 2016) Yüksel, Nesligül; Top, AybenIn this study, it was aimed to develop drug delivery systems with high drug release rate, capable of overcoming multidrug resistance of cancer cells. The first generation drug delivery system (DDS) denoted as mPEG-AT3-DOX was prepared by methoxy polyethylene glycol (mPEG) and peptide conjugation, and the model anticancer drug, DOX, was attached to the mPEG-peptide carrier system using stable amide linkage. mPEG was used to increase blood circulation time of the DDS. Designed peptide (AT3 = CG3H6R2ALALG3E) controls release of the drug via RRALAL sequence, which is a substrate for a lysosomal enzyme, cathepsin B, overexpressed in most of the tumor cells. pH responsive histidines and reactive amino acids (glutamic acid and cysteine) for drug and mPEG conjugations were also added to the peptide sequence. The peptide synthesized by using Fmoc chemistry was conjugated to mPEG-maleimide via Michael addition reaction. DOX was attached to the carboxylic acid group present in the carrier system (mPEG-AT3) via amide linkage. Mass spectroscopy and HPLC were used to assess the purity of the AT3 and mPEG-AT3. At pH 7.4, mPEG-AT3-DOX exhibited bimodal size (hydrodynamic diameter) distribution at about 15 and 30 nm independent of pH. % DOX release from mPEG-AT3-DOX was observed to be below 10 % at neutral pH and pH 5.0 in the absence of cathepsin B, and increased to 17 ± 2 % in the presence of cathepsin B. Complete degradation of AT3 peptide within three hours in the presence of cathepsin B suggests lower than expected DOX release is due to aggregation tendency of the DDS. Cytotoxicity of the conjugates was evaluated using the lung cancer (A549) and prostate cancer (PC3) cell lines. At the end of 24 hours the absolute IC50 values of free DOX and mPEG-AT3-DOX were obtained as 1.37 ± 0.05 and 1.33 ± 0.11 for the A549 cell line, 1.51 + 0.07 and 1.63 + 0.80 μg equivalent DOX / ml for the PC3 cell line, respectively. Considering, these results, the second generation DDS will be designed to have more pronounced pH sensitive property by increasing the number of histidines in the peptide domain.Master Thesis Development of Peg and Peg-Peptide Based Drug Delivery Systems(Izmir Institute of Technology, 2016) Balcı, Beste; Top, AybenIn this study, two types of drug delivery systems (DDS) were prepared; mPEG (methoxy polyethylene glycol)-HYD (hydrazide)-DOX and mPEG-peptide-(HYD)-DOX. In the design of the conjugates, mPEG was used to increase the blood circulation time. HYD provided an acid cleavable bond between the carrier molecule and DOX, whereas peptide containing histidines imparted pH responsiveness of the molecule. Doxorubicin (DOX) was selected as a model anti-cancer drug. DDS were synthesized using two steps; hydrazide functionalization of carboxylic acid of the carrier molecule followed by DOX conjugation. Hydrazide form of the carrier molecules denoted as HYD1 and HYD2 were obtained using adipic acid dihydrazide (AADH) and carbohydrazide (CH), respectively. To increase DOX conjugation, trifluoroacetic acid (TFA) and DOX amounts were changed and the reactions were carried out at the conditions giving the highest DOX conjugation (mPEG-HYD:DOX:TFA= 2.5mg:2mg:20μL per 1 mL of DMSO). The peptide (AT1=CGGGHHHHHHGGGE) was synthesized using solid phase peptide synthesis (SPPS) and PEGylated using mPEG-maleimide to obtain mPEG-AT1 conjugate. The purity of AT1 and mPEG-AT1 were confirmed using mass spectroscopy and high performance liquid chromatography (HPLC). DOX conjugation percentages were obtained as 62 7, 60 3 and 35 + 3 for mPEG-HYD1-DOX, mPEG-HYD2-DOX and mPEG-AT1-HYD1-DOX, respectively. Drug release studies indicated modest pH responsiveness of the carrier molecules obtained using AADH. On the other hand, mPEG-HYD2-DOX released 13% of drug at the end of the 72h independent of pH. For mPEG-AT1-DOX, drug release percentage values were obtained as 15% and 30% at pH 7.4 and 5.0 respectively. Cytotoxicity of the conjugates of DDS was determined using lung cancer (A-549) cell lines. DOX equivalent IC50 values were determined as 20, 40 and 5 for mPEG-HYD1-DOX, mPEG-HYD2-DOX and mPEG-AT1-DOX respectively.