Doktora Tezleri

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Automatic identification of evolutionary and sequence relationships in large scale protein data using computational and graph-theoretical analyses
(Izmir Institute of Technology, 2012) Doğan, Tunca; Karaçalı, Bilge
In this study, computational methods are developed for the automatic identification of functional/evolutionary relationships between biomolecular sequences in large and diverse datasets. Different approaches were considered during the development and optimization of the methods. The first approach focused on the expression of gene and protein sequences in high dimensional vector spaces via non-linear embedding. This allowed statistical learning algorithms to be applied on the resulting embeddings in order to cluster and/or classify the sequences. The second approach revised the pairwise similarities between sequences following multiple sequence alignment in order to eliminate the unreliable connections due to remote homology and/or poor alignment. This is achieved by thresholding the pairwise connectivity map over 2 parameters: the inferred evolutionary distances and the number of gapless positions in each pairwise alignment. The resulting connectivity map was disjoint and consisted of clusters of similar proteins. The third and the final approach sought to associate the amino acid sequences with each other over highly conserved/shared sequence segments, as shared sequence segments imply conserved functional or structural attributes. An automated method was developed to identify these segments in large and diverse collections of amino acid sequences, using a combination of sequence alignment, residue conservation scoring and graph-theoretical approaches. The method produces a table of associations between the input sequences and the identified conserved regions that can reveal both new members to the known protein families and entirely new lines. The methods were applied to a dataset composed of 17793 human proteins sequences in order to obtain a global functional relation map. On this map, functional and evolutionary properties of human proteins could be found based on their relationships to the ones bearing functional annotations. The results revealed that conserved regions corresponded strongly to annotated structural domains. This suggests the method can also be useful in identifying novel domains on protein sequences.
Biochemical and mechanical cues for osteogenic induction of stem cells on paper based scaffolds
(Izmir Institute of Technology, 2019-12) Karadaş, Özge; Özçivici, Engin; Özhan Baykan, Hatice Güneş
Tissue engineering aims to produce functional constructs with living cells that can fully integrate with the tissue when inserted into the body. Design of the scaffold and the choice of cell type that will be used for production of the tissue engineering construct are very important for the success of the application. For bone tissue engineering, incorporation of substances with antimicrobial properties can supply additional benefits. This dissertation seeks answers for two discrete questions in different chapters: Do carnosol and carnosic acid, phenolic antimicrobial compounds extracted from plants have cytotoxic effect on bone tissue derived cells and do the culture conditions (monolayer or 3D) effect the response of cells (Chapter 2); and how do application of a single type of mechanical force (vibration) and a combination of two forces (vibration plus fluid shear) affect the osteogenesis of tissue engineering constructs (Chapters 3 and 4)? The results of this research demonstrated that carnosol and carnosic acid had bacteriostatic effect at 60 µg/mL but this concentration value was highly cytotoxic for bone tissue derived cells. Nevertheless, when the same cells were incubated under 3D culture conditions their cytotoxic tolerance was higher. The supportive role of mechanical forces on osteogenic differentiation of stem cells on 3D scaffolds prepared by using filter paper, on the other hand, was demonstrated with the increase in osteoblastic gene expression, immunocytochemical staining and detection of mineralization by Alizarin red S staining and quantification. In conclusion this research showed the importance of biochemical and biomechanical cues on osteogenesis.
Characterization of conjugated polyelectrolytes for nucleic acid sensing, gene delivery and imaging
(Izmir Institute of Technology, 2022-07) Yücel, Müge; Yıldız, Ümit Hakan
In this thesis, cationic derivatives of poly(3-alkylmethoxythiophene) (PT) which are a class of conjugated polyelectrolytes (CPE), have been synthesized. PT has been polymerized via FeCl3 oxidative polymerization, were treated in a set of solvents to elaborate coil conformation of polymer chain in different physicochemical environment. Spectroscopic and scattering techniques have ascertained that ethylene glycol is a good solvent for PT regarding Flory-Huggins theory. The smaller interaction parameter of PT with respect to ethylene glycol than water drives a thermodynamically driven ultra-small particle (Pdot) formation in aqueous phase by a rapid nanophase separation between PTrich ethylene glycol and PT-poor water phase. All CPEs have been then employed to prepare single polymer chain polymer dots (Pdot) by “nanophase separation” method. As a next step, Pdots have been characterized in terms of optical and colloidal properties that they possess in the backbone conformations altered by solvation effect. Regarding their colloidal characteristic, translocation of Pdot into cancerous cells was analyzed compared to healthy cells by 2D cell culture and co-culture studies. It has reported that Pdots have ability to penetrate through nuclear envelope in hepatocellular carcinoma whereas accumulate around nucleus of healthy liver cells in cytoplasm. Additionally, Pdots were studied in breast cancer cell lines to understand the behavior of Pdot staining in 2D cell culture of invasive and non-invasive breast cancer types. The findings suggest that Pdots are prone to penetrate into the invasive cancerous cells attributed to the greater deformations on nucleus membrane of triple negative breast cancer cells. In a next application, the enhanced photophysical property of PT exhibited in ethylene glycol media allows PT to be utilized as a fluorescent probe for determination of single nucleotide polymorphism by a non-amplification-based protocol. Fluorescence emission at specific wavelengths resulted from very distinct conformations of PT chain is the key elements for the SNP detection assay. The evaluation of optical data obtained from the probe with principal component analysis proves the separation of healthy individuals from patients with an overall 96% accuracy.
Development of conducting polymer-based fluorescence on/off biosensor for biomolecule analysis
(01. Izmir Institute of Technology, 2022-11) Arslantaş, Duygu; Arslan Yıldız, Ahu
Sensitive and selective detection of biomolecules and cells is essential for early diagnosis of diseases, prognosis monitoring, and effective therapy. This thesis aimed to develop a novel fluorescence ‘‘turn-on/off’’ biosensor for biomolecules and cells detection. In this study, cationic polythiophene derivative poly(1,4-dimethyl-1-(3-((4- methylthiophen-3-yl)oxy)propyl)piperazin-1-ium bromide) (PT–Pip) was used as an efficient fluorescence transduction element to discriminate proteins, mammalian cells, and amino acids for the first time. Initially, pH–dependent spectroscopic characterization of the PT–Pip was performed to monitor the conformational and optical changes. The pH sensitivity of the PT–Pip was demonstrated for the first time. Afterwards, the fluorescence ‘‘turn–off’’ phenomena were investigated in detail using citrate–capped gold nanoparticles as an efficient fluorescence quencher. Further, the interaction of target analytes such as proteins, mammalian cells, and amino acids with pre–quenched non–covalent PT–Pip–AuNP complexes was examined. Disruption of the binding equilibrium between PT–Pip and AuNP by analytes resulted in the selective displacement of PT–Pip, which generated signal output as a fluorescence ‘‘turn–on’’ mode. Consequently, for the sensitive detection of biomolecules and cells, chemical tongue sensor arrays were developed utilizing differential sensing approaches. PCA was used for the statistical evaluation of the multi–dimentional fluorescence response patterns. As a result, unique fingerprints were rapidly obtained by the direct sensing of proteins, ratiometric sensing of mammalian cells, and indirect sensing of amino acids. The combination of a differential sensing strategy with an appropriate multivariate statistical technique enabled the selective and sensitive detection and identification of proteins, mammalian cells, and amino acids.
Development of magnetic levitation-based sensitive assays
(Izmir Institute of Technology, 2020-07) Yaman, Sena; Tekin, Hüseyin Cumhur; Ergon, Mahmut Cem; Izmir Institute of Technology
Magnetic levitation (MagLev), in which an object is levitated with no support other than magnetic force and buoyancy force, is a powerful tool employed in many applications regarding the characterization of materials, biosensing of macromolecules, separating of cells, and monitoring of cellular events. Levitation of an object in MagLev depends on magnetic susceptibility and density of that object relative to its surrounding medium. In this thesis, MagLev-based miniaturized and affordable assay formats for biomolecule detection and cell separation were investigated. In this regard, a novel biomarker method detection in MagLev was developed using polymer microspheres as three-dimensional (3D) assay surfaces to capture target proteins and magnetic nanoparticles to label the captured target on the microspheres. Levitation heights of the microspheres conjugated to the protein were distinctly different than those of without protein. Thus, the magnetic susceptibility change of microspheres was precisely measured to convert the levitation height of microspheres into protein concentration. The principle developed for a biotinylated target protein was then investigated by designing sandwich immunoassays using model protein biomarkers: mouse immunoglobulin G and human cardiac troponin I. The developed assays enabled a protein detection range of femtogram-microgram per milliliter. In addition to biomolecule detection, using a lensless holographic microscopy-integrated MagLev platform, three different cell lines, bone marrow stem cells (D1 ORL UVA), breast cancer cells (MDA-MB-231), and human monocyte cells (U-937), were distinguished based on their density. The results revealed that the methods developed here could contribute to the magnetic MagLev-based sensitive and inexpensive bioanalytical applications.
Development of novel chitosan nanocomposites as a controlled drug release system for Helicobacter pylori treatment
(İzmir Institute of Technology, 2016-01) Güneş, Suna Seda; Tıhmınlıoğlu, Funda; Yılmaz, Özlem
Helicobacter pylori is one of the most common bacterial infection and responsible for gastroduodenal diseases in humans. Due to increasing failure rate of currently used antibiotic therapies, newer drugs and therapeutic approaches are needed. Therefore the use of encapsulated cinnamon bark oil is a promising approach for H. pylori eradication. The main objective of this dissertation was to develop a novel drug delivery system using chitosan and nanoclay containing cinnamon bark oil to be released in a controlled manner in gastrointestinal system for H. pylori eradication as an alternative or complementary to conventional antibiotic treatment. Minimum inhibition concentration (MIC) value of cinnamon bark oil was determined. Cinnamon bark oil loaded chitosan nanocomposite microspheres were produced by spray drying. The prepared microspheres were characterized for particle size & morphology, encapsulation efficiency, surface charge, mucoadhesion, degradation, swelling and drug release. Antimicrobial activity of the microspheres against H. pylori were investigated. In vitro cell viability of fibroblast and gastric epithelial cells were evaluated. In vitro cellular uptake and binding studies of microspheres were investigated by using gastric epithelial cells. MIC value of cinnamon bark oil was found as 8 μg/mL. Nanoclay incorporation decreased the biodegradation of nanocomposite microspheres and improved the release of cinnamon bark oil. Drug release mechanism was anomalous diffusion which refers to combination of diffusion and erosion controlled. The prepared microspheres showed strong mucoadhesive property. Oil released from prepared microspheres inhibited H. pylori growth. Although cinnamon oil showed cytotoxic effect above 31 μg/mL, the oil encapsulated chitosan microspheres did not show any cytotoxic effect on NIH3T3 and MKN45 cells. The prepared microspheres were able to get internalized into MKN45 cells and had great incorporation activity. The results of this study demonstrated that cinnamon bark oil loaded chitosan nanocomposites may serve as biocompatible and effective gastroretentive drug carrier for the treatment of H. pylori infection.
Development of novel polymeric carriers for gene therapy
(Izmir Institute of Technology, 2021-06) Zelçak, Aykut; Bulmuş Zareie, Esma Volga; Izmir Institute of Technology
The development of effective delivery systems is a limiting step in gene therapy. In this work, new linear block copolymers and star polymers were synthesized, and their siRNA delivery abilities were investigated. For this aim, diblock copolymers consisting of alternative "stealth" polymer blocks (PEG, P(OEGMA) (Poly(oligo(ethylene glycol) methyl ether methacrylate)) or P(OEtOxMA) (Poly(oligo(2-ethyl-2-oxazoline) methacrylate))); and same cationic polymer block (P(AEAEMA) (Poly(2-((2-aminoethyl)amino)ethyl methacrylate))), have been prepared via RAFT polymerization or combination of CROP and RAFT polymerizations. Additionally, to demonstrate the effect of polymeric architecture, P(OEGMA)/P(AEAEMA) miktoarm star polymers have also been synthesized via RAFT polymerization. Polymers were characterized by SEC, NMR and DLS. siRNA complexation was investigated by gel electrophoresis, DLS, SEM and TEM. Compared to star polymers, linear block copolymers could bind the siRNA molecules easier and tighter due to their more flexible natures and sterically accessible amine groups. The diameter of star polymer-siRNA complexes at N/P of 50 was found to be approximately 20 nm. Compared to this, linear block copolymers formed smaller particles (≈ 10 nm) at the same N/P ratio. The viability of linear block copolymer-treated cells was found to be 50% or better at the polymer concentration of 5 µM. In contrast, star polymers showed more detrimental effects at the same polymer concentrations. P(OEGMA)43-b-P(AEAEMA)45-siRNA complexes at N/P of 50 were taken up by 63.5% and 74.1% of H460 and Mda-mb-231 cells, respectively. In contrast, P(AEAEMA)40-b-P(OEtOxMA)38 complexes showed much lower uptake profile at the same conditions. Remarkably, P(OEGMA)43-b-P(AEAEMA)45-siRNA complexes showed potent gene silencing effect on Mda-mb-231 cells as shown by luciferase and RT-qPCR assays. Overall, it has been found that "stealth" polymers and polymeric architecture have a very significant effect on siRNA delivery.
The effects of engineered silica nanoparticles on the cellular behaviours of human hepatocellular carcinoma cell lines
(Izmir Institute of Technology, 2018-07) Tüncel Çerik, Özge; Özçelik, Serdar; Atabey, Safiye Neşe
Physicochemical properties of the silica nanoparticles have vital roles in determining the physiological behaviours of the cells. Applications of nanoparticle treatments have some outcomes as a response of the cells in living systems as mitochondrial disruption, oxidative stress, reactive oxidative species (ROS) generation, altered cell cycle regulation and DNA damage. In this study 10 and 100 nm sized SiNPs were prepared and physicochemically characterized in the second part. Well characterized silica nanoparticles were used to assess the cytotoxicity and genotoxicity of the hepatocellular carcinoma cell lines as HuH-7 and SK-HEP-1 and lymphocytes. The cell cycle analysis was performed for engineered SiNPs to elucidate the DNA damage in the third part. In the fourth part mitochondrial responses of the cells were determined by real time confocal microscopy at single cell level. An image analysis method for evaluating the cellular responses by mitochondrial staining was developed. DCF stained cells were analyzed in order to assess the production of ROS in the cells. Localization of the SiNPs were determined by lysosomal and mitochondrial staining. Pearson correlation coefficients of the images were used for evaluating the colocalization of organelles with SiNPs. Lastly, diffusion coefficients of the SiNPs in the cells were determined by quantitative confocal microscopy. The SiNPs were found as non-toxic up to 200 μg/ml for 5 days. The SiNPs did not induce the formation of micronuclei in lymphocytes. The SiNPs were not cause an arrest in cell cycle progression. Mitochondrial potentials were not changed after SiNP exposure as well. They were mostly internalized at 30 minutes in both cell line in lysosomal parts without increasing ROS in the cells. It can be concluded that the SiNPs can be safely used for targeted delivery of organic compounds, biological molecules or drugs in medicine, and may be utilized as a probe system in biological studies.
Engineering target tissue in lab-on-a-chip devices for predicting homing choices of metastatic cancer
(Izmir Institute of Technology, 2020-12) Batı Ayaz, Gizem; Pesen Okvur, Devrim; Yavuz, Oktay; Izmir Institute of Technology
The metastatic cascade of cancer results in the extravasation of the tumor to other parts of the body. Metastasis is the leading cause of cancer related deaths. Breast cancer is the most common cancer in women, and lung is one of the organs with the most metastasis. For this reason, it is critical to engineer a tissue microenvironment that includes complex cell-cell interactions with co-culture of endothelial, epithelial and stromal cells, and the invasion and extravasation steps of metastasis can be observed for early diagnosis of metastasis. Vascularization is the critical step for engineering the tissues. The in vitro models used today are insufficient to create the tissue environment closest to in vivo conditions. Recently developed lab-on-a-chip platforms provide suitable environments for mimicking the in vivo structure in tissue engineering studies. In this research: -Different lab-on-a-chip devices fabricated to engineer breast and lung target tissues. -For the first time, epithelial, fibroblast and endothelial cells were tri-cultured and breast and lung tissue environments were engineering with microvasculature. -Different gel, media and cell numbers have been optimized for engineering of breast and lung tissue environments with microvascularization. -Different matrix environments have been optimized to observe invasion and/or extravasation steps separately or together.
Increasing stability of microbubbles under ultrasound
(Izmir Institute of Technology, 2019-12) Ayaz, İlyas Umur; Özdemir, Ekrem; Seçil, Mustafa
Microbubbles are used as effective contrast agents in ultrasound imaging. However, low stability of the microbubbles limits their use for prolonged period of time in medical applications. The aim of this dissertation is to increase the stability of microbubbles under ultrasound. The stability and acoustic response of microbubbles were investigated under ultrasound as a function of their shell composition. Microbubbles were fabricated using combinations of phospholipid (DSPC) and an emulsifier (PEG40St) in different molar ratios. It was found that adding the emulsifier decreased the microbubble stability under ultrasound; however, the echogenicity of microbubbles was shown to increase with increasing emulsifier content. A method was developed to estimate the concentration of microbubbles with ultrasound. Hydrostatic pressure studies showed that the microbubbles recovered their spherical structures at low pressure pulses, in contrast, disappeared in a very short time at high pressure pulses. B-mode ultrasound intensity of microbubbles was investigated at different ultrasound powers under Doppler ultrasonography, and for the first time, a model was developed to relate the intensity to effective bubble concentration. We calculated acoustic energy thresholds and explained a possible mechanism for the destruction of microbubbles under ultrasound. The effect of shell loadings on the acoustic response and stability of microbubbles were investigated under ultrasound. It was found that both the echogenicity and stability of microbubbles increased with increasing mass of the loadings on microbubble shell. In-vivo studies showed that the acoustic performance of in-house made microbubbles was comparable to that of commercial standard Vevo MicroMarker® contrast agents.
The investigation of anticancer properties of (R)-4'-methylklavuzon in liver cancer cells and liver cancer stem cells
(Izmir Institute of Technology, 2017-12) Delman, Murat; Çağır, Ali; Erdal, Esra
Hepatocellular carcinoma (HCC) is the fifth most seen cancer type and the third leading cause of death from cancers. HCC is a fatal disease and HCC patients have a 5-year survival rate of 14%. Discovery and identification of mechanisms of action for new therapeutic agents are required for a better treatment of HCC. One of the most important target in cancer treatment is the epigenetic acetylation of histones. Histone deacetylases (HDAC) and sirtuins provide chromatin compaction and transcriptional repression by removing acetyl groups from histone proteins and nonhistone proteins. Re-acetylation of chromatin and re-expression of tumor suppressor genes with the discovery of novel HDAC and/or sirtuin inhibitors are therapeutic targets in cancer research. In this study, (R)-4’-methylklavuzon was found to be cytotoxic in HuH-7 cells with IC50 values of 1.25 μM for HuH-7 parental cells, 2.5 μM for EpCAM+/CD133+ HuH-7 cells and 1.25 μM for EpCAM-/CD133- HuH-7 cells. It was observed that (R)-4’-methylklavuzon causes cell cycle arrest at G1 phase at 1.00 μM concentration in three cell populations, it induces apoptosis at 10 μM concentration at the end of 24 hours incubation. (R)-4’-methylklavuzon does not inhibit Class I/II HDACs in vitro whereas it causes inhibition of endogenous HDACs and/or sirtuins inside the cells sorted by MACS and FACS at 0.10 μM concentration. (R)-4’-methylklavuzon upregulates p21 expression significantly in HuH-7 cell populations to cause G1 arrest. It causes 45% inhibition in p53/MDM2 complex formation when examined with pure p53 and MDM2 proteins. Drug candidate causes 46% SIRT1 inhibition at 100 μM concentration in vitro whereas there was no inhibition of HDAC1 enzyme at the same concentration. SIRT1 protein levels in HuH-7 parental cells were upregulated to 240% within 24 hours of incubation with 3.00 μM of drug candidate. It was found that (R)-4’-methylklavuzon can also inhibit CRM1 protein providing increased retention of tumor suppressor proteins in the nucleus. p53 was overexpressed at 0.10 and 1.00 μM concentrations within 6 and 24 hours in HepG2 cells but slightly overexpressed in HuH-7 parental cells.
Investigation of hemostatic biomaterials containing plant extracts
(Izmir Institute of Technology, 2014) Uslu, Mehmet Emin; Bayraktar, Oğuz; Başal Bayraktar, Güldemet; Akhisaroğlu, Mustafa
Haemostasis in other words a process which causes bleeding to stop is very important in injury. Recent researches were focused on discovery of haemostatic agents and developing biomaterials which transfer them to the injury side. Therefore plant extracts and three dimensional biomaterials were widely investigated. In this research the extract of Equisetum arvense was investigated as a coagulatory agent. Silk fibroin-hyaluronic acid mixture was used for the preparation of three dimensional sponge like biomaterials. The prepared sponge forms could also be used as scaffold for wound healing. In this research effect of extraction parameters on extract composition and bioactivity was investigated. Effect of extraction parameters were analyzed on 6 factors at 3 levels. It was seen that extraction parameters had high influence on both composition and bioactivity of the prepared extract. Although it was reported that the extract of equisetum arvense had anticoagulant activity, in this research it was shown that changing parameters caused variation of extract bioactivity from anticoagulant to coagulant as a result of changing extract composition. Addition of extract into silk fibroin-hyaluronic acid mixture caused proteins to precipitate as a result of interaction between protein and phenolic compounds. As a result of this precipitation significant decrease in the mechanical strength of biomaterial was observed. In order to minimize this interaction, plant extract was added into the silk fibroin after mixing with hyaluronic acid solution. As a result, mechanical strength and pore size of the biomaterial were increased and pore distribution became more regular. Also biomaterial gained tubular network on both vertical and horizontal dimensions. This would help the proliferation and migration of the fibroblast cells and moreover prevent the formation of scar tissue.
Invetigation of mechanical vibration effects on breast cancer cells
(Izmir Institute of Technology, 2018-07) Olçum Uzan, Melis; Özçivici, Engin; Erdal Bağrıyanık, Şerife Esra
In this doctoral dissertation, low magnitude mechanical signals (LMMS, <1g in magnitude) were used to test the stress shielding model hypothesized on breast cancer cells. The hypothesis was that the breast cancer cells will be sensitive to mechanical vibrations and will respond to these vibrations. It was similarly used to test the adipogenic differentiation of Lamin A/C knockdown (by siRNA) bone marrow-derived mesenchymal stem cells. It is known that Lamin A/C plays a role in the nucleus and intracellular organization in these cells and affects gene expression by chromatin regulation. The hypothesis was that if these cells are deprived of the organization for the nucleus, they will be sensitive to mechanical vibrations, but that the mechanical vibrations cannot restore the effect of lamin A/C on gene regulation. We investigated the effects of high-frequency low-density mechanical signals (LMMS) on cell proliferation, apoptosis, cell cycle, protein expression, differentiation, cytoskeleton and phenotypic change processes. According to findings, LMMS caused cell cycle arrest in the aggressive type of breast cancer cells and slowed proliferation. Non-aggressive breast cancer has not responded to LMMS. In mammary epithelial cells, LMMS has not shown an effect that triggers proliferation. In the mesenchymal stem cell model, Lamin A/C knockdown accelerated adipogenic differentiation. Although LMMS in these cells decreased the rate of adipogenic differentiation, it was not sufficient to restore the baseline.
Magnetic levitation of cells from bone marrow origin
(Izmir Institute of Technology, 2021-07) Anıl İnevi, Müge; Özçivici, Engin; Güven, Sinan; Izmir Institute of Technology
Magnetic levitation via negative magnetophoresis is a new label-free technology that is important in cell- and tissue-level bioengineering applications. Biofabrication applications of the technology is an area that still needs to be developed. In this doctoral thesis, 3D cellular structures with contrable size and cellular arrangement were formed and cultured with magnetic levitation using bone marrow-derived stem cells in both a miniature system that provides levitation between two magnets and a ring magnet-based large-scale system. First, a miniaturized magnetic levitation system that allows real-time imaging was produced and comprehensive protocols were described for its use for both single-cell level analysis and cell culture. With this setup, complex in situ 3D cellular aggregates were formed and their culture was maintained by levitation. Then, a new system that provides levitation on a single ring magnet was produced and used for biofabrication for the first time to overcome the reservoir volume constraint in the existing system and thus to create larger and symmetrical 3D cellular clusters. With the elimination of the upper limit in the system, the volume of the chamber was increased and the medium and biological structure transfer became easily applicable. It has been shown that this ring magnet-based magnetic levitation setup is suitable for cell culture, formation of millimeter-sized cellular structures with various cell types, and that pre- formed cellular structures can be combined by levitation. The low-cost and easy-to-use systems presented in this thesis have the potential to be applied in many areas such as tissue engineering and drug testing.
Molecular and cellular level adaptations of bone marrow mesenchymal progenitor cells to chemical and physical signals
(Izmir Institute of Technology, 2020-12) Baskan Erbilgiç, Öznur; Özçivici, Engin; Atabey, Safiye Neşe; Izmir Institute of Technology
Mechanical forces are the integral determinants in cell and tissue homeostasis and regeneration, and they can affect numerous biological process from proliferation to fate determination. Mechanical forces that possess low magnitude and high frequency characteristics are also known as low intensity vibrations (LIVs). These signals were studied widely on many cell types for regenerative purposes, however most of these studies select components of LIV signals (e.g. magnitude, frequency, duration, etc.) arbitrarily. Here, we addressed the effect of LIV applied frequency, LIV daily exposure time and fate induction on the viability of preadipocyte 3T3-L1 cells. For this, we performed a frequency sweep that was ranging from 30 to 120 Hz with 15 Hz increments applied for 5, 10 or 20 minutes during quiescent growth or adipogenesis for up to 10 days. Results suggest that the applied frequency and fate induction was an important determinant of cell viability, lipid droplet physiology, triglyceride concentration, cell density and adipogenic-specific gene expression while daily exposure time had no effect. These findings contribute to the effort of optimizing a relevant mechanical stimulus that can inhibit adipogenesis. On the other hand, random and aligned PAN/PPy nanofibers were investigated as a scaffold material for osteogenic differentiation of D1 ORL UVA mouse bone marrow mesenchymal stem cells. Cells were able to attach and grow on nanofibers confirmed by cell viability results. Stem cells that were cultured with osteogenic induction were able to mineralize on electrospun nanofibers based on alizarin red and Von Kossa dye staining. For aligned PPy nanofibers, mineralization occurred in the fiber alignment direction. Consequently, PAN/PPy nanofibrous mats in both random and aligned forms would be potential candidates for bone tissue engineering.
Multi-organ-on-a-chip for cancer drug testing
(Izmir Institute of Technology, 2022-05) Mohammed, Abdurehman Eshete; Pesen Okvur, Devrim; Erdal Bağrıyanık, Şerife Esra
Cancer is one of the devastating and fatal severe diseases worldwide that kills millions of people every year. Globally cancer is the second leading cause of death after cardiovascular disease and was responsible for 10 million deaths in 2020. Breast cancer is one of the predominant cancers in females and is the cause of more than half a million females death each year. The primary cause of cancer patients' death is cancer metastasis. Triple-negative BREAST cancer (TNBC) is mainly treated by chemotherapy. In the current drug discovery and development processes, the efficacy and toxicity of chemotherapies identify using 2D and animal testing but not simulating the in vivo microenvironment. This research designed multiorgan-on-a-chip with liver and breast cell line compartments, and drug PKPD modeling was done by Monolix software. In this research, a unique multiorgan-on-a-chip (MOC) was designed and fabricated, generated experimental PK and PD data using the new MOC device, and modeled and simulated PK and PD using the experimental data. To conclude, we developed a new multiorgan-on-a-chip (MOC) platform used for PKPD modeling and PKPD simulations that would be helpful in the preclinical research to evaluate the effectiveness and toxicity of drugs. In the future, using calceinAM, a fluorescent cell viability dye, generating PD data for each cell type and determining side effects of doxorubicin in each cell line is essential. Adding more organs to the MOC, such as heart tissue, to study the cytotoxicity of doxorubicin in different organs gives more efficient data for PKPD modeling.
Natural and synthetic silica incorporated chitosan composite scaffolds for bone tissue engineering applications
(İzmir Institute of Technology, 2016-07) Tamburacı, Sedef; Tıhmınlıoğlu, Funda; Havıtçıoğlu, Hasan
Recently bone tissue engineering studies have focused on the development of 3D scaffolds that can organize the tissue regeneration in natural way with appropriate porosity and reinforced the structure. Natural polymer-based composites have been focused with more attention than synthetic polymer composites for bone tissue engineering applications because of their biocompatibility and biodegradability. In this work, the goal was to combine the useful biomaterial properties of both chitosan and silica to design biocomposite organic/inorganic biomaterials for bone tissue engineering applications. The composite scaffolds were fabricated by freeze drying method bu using two different silicas; natural silica; Diatomite and synthetic silica, octa (tetramethylammonium) polyhedral oligomeric silsesquioxanes (OctaTMA-POSS). The effects of silica type and loading on the mechanical, morphological, chemical, surface properties, wettability and biocompatibility of composite scaffolds were investigated and characterized by using SEM, AFM, contact angle analysis, swelling study, protein adsorption assay, biodegradation and biomineralization tests. WST-1 cytotoxicity, cell proliferation with rezasurin and alkaline phosphatase activity assays were performed to determine biological activity of the composite scaffolds. In vitro biomineralization on scaffolds was determined by Von Kossa and Alizarin red staining. POSS and diatomite incorporation increased the surface roughness. Chitosansilica composites exhibited 82-90% porosity. Wet chitosan-silica composite scaffolds exhibited higher compression moduli compared to pure chitosan scaffold in 67.3- 81.4kPa and 78.1 to 107.6kPa range respectively. Average pore size range of chitosandiatomite and chitosan-POSS composite scaffolds was obtained as 15-180μm and 220- 300μm, respectively. Results indicated that chitosan-silica composites did not show any cytotoxic effect on 3T3, MG-63 and Saos-2 cell lines. Chitosan-silica composites were found to be favorable for osteoblast proliferation. Diatomite and POSS incorporation showed promising effects with enhancing ALP activity on hFob cells. Therefore, these composite scaffolds could be used for bone tissue engineering applications.
Pre-clinical trial treatment of hepatocellular carcinoma on cirrhosis in a rat model
(İzmir Institute of Technology, 2017-01) Zeybek Kuyucu, Ayça; Şanlı Mohamed, Gülşah
Hepatocellular carcinoma (HCC) is the second most common cause of cancer related mortality worldwide. AKT pathway has been found activated in 50% of HCC cases, making it promising target. Therefore we assess efficacy of the allosteric AKT inhibitor or the combination of Sorafenib with AKT inhibitor compared to untreated control and to standard treatment, Sorafenib, in vitro and in vivo. AKT inhibitor blocked phosphorylation of AKT in vitro and strongly inhibited cell growth and migration with significantly higher potency than Sorafenib. Similarly, apoptotic cell was strongly increased by AKT inhibitor in vitro. To mimic human advanced HCC, we used diethylnitrosamine-induced cirrhotic rat model with fully developed HCC. MRI analyses showed that AKT inhibitor significantly reduced overall tumor size. Furthermore, number of tumors was decreased by AKT inhibitor, which was associated with increased apoptosis and decreased proliferation. Tumor contrast enhancement was significantly decreased in the AKT inhibitor group. Moreover, on tumor tissue sections, we observed a vascular normalization and a significant decrease in fibrosis in surrounding liver of animals treated with AKT inhibitor. Finally, pAKT/AKT levels in AKT inhibitor treated tumors were reduced, followed by down regulation of actors of AKT downstream signaling pathway: pmTOR, pPRAS40, pPLCγ1 and pS6K1. In conclusion, we demonstrated that AKT inhibitor blocks AKT phosphorylation in vitro and in vivo. In HCC-rat model, AKT inhibitor was well tolerated, showed anti-fibrotic effect and had stronger antitumor effect than Sorafenib. Our results confirm the importance of targeting AKT in HCC.
Preparation and characterization of polymeric scaffolds for nerve tissue engineering applications
(Izmir Institute of Technology, 2014) Büyüköz, Melda; Alsoy Altınkaya, Sacide; Erdal, Şerife Esra
The major goal in tissue engineering is to develop three-dimensional biomimetic scaffolds which can provide an optimal environment for cell adhesion, proliferation, differentiation and guide new tissue formation. In this study macroporous, nanofibrous gelatin scaffolds in the form of a disc and channeled conduit were prepared for nerve tissue engineering applications. Alginate microspheres have been integrated into the scaffolds to deliver nerve growth factor (NGF) to differentiate PC12 cells. Methods combining thermally induced phase separation technique with porogen leaching and injection molding were used to manifacture disc shaped and channeled nanofibrous scaffolds, respectively. Microcarriers loaded with NGF were fabricated by water-in-oil emulsification technique and attached in the scaffold by chemical crosslinking with carbodiimide reaction. The relationship among processing parameter, porosity, pore size, interpore connectivity and the mechanical properties were investigated. In addition release kinetics of NGF from the particles were determined and viability, proliferation and differentiation of PC12 cells in the scaffolds were evaluated. The fiber sizes of nanofibrous scaffolds were found similar to the size of natural collagen fiber bundles. In nanofibrous scaffolds, the dimensional stability and in vitro degredation rates improved when compared to solid walled scaffolds. The release rate of NGF from the particles was controlled by the alginate concentration and poly(L-lysine) coating. Integrating NGF into the nanofibrous gelatin scaffold in encapsulated form reduced amount of NGF and time required for the differentiation of PC12 compared to free NGF directly added to the cells.
Preparation and characterization of wound dressing contact layer
(Izmir Institute of Technology, 2015-07) Erdoğan, İpek; Bayraktar, Oğuz; Atabey, Atay; Başal Bayraktar, Güldemet
Wound dressings provide therapeutic and protective features and promotes natural healing process when applied to a wound area. Being non-toxic and immunologically inert, natural biopolymers have potential in fabrication of wound dressings. Growth factors and antibiotics can also be used in functionalization of wound dressings as well as plant extracts. Olive leaf extract has gained attraction due to its dual antimicrobial and antioxidant effect. By clearing pathogenic microorganisms and scavenging against increased amount of reactive oxygen species in the wound area, it has high potential in wound healing. In this study, olive leaf extract incorporated zein fibers were prepared as a model of wound dressing contact layer. In this regard, crude olive leaf extract was fractionated and characterized in terms of antioxidant capacity, total phenol content and antimicrobial activity. Crude extract and its fractions were also subjected to wound scratch assay in the presence of hydrogen peroxide. Oleuropein, as the most abundant component in crude extract, was found to promote cell migration better and close the wound area at a higher rate than other components. On the other hand, crude olive leaf extract exhibited higher percentage of wound closure than its fractions within the same time period, which may be attributed to synergistic effect of unidentified phenolics. Crude olive leaf extract also provided crosslinking effect when incorporated into zein fibers, as well as promoting cell spreading behaviour.

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