Phd Degree / Doktora
Permanent URI for this collectionhttps://hdl.handle.net/11147/2869
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Doctoral Thesis Multi-organ-on-a-chip for cancer drug testing(Izmir Institute of Technology, 2022) Mohammed, Abdurehman Eshete; Pesen Okvur, Devrim; Erdal Bağrıyanık, Şerife EsraCancer 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.Doctoral Thesis Magnetic Levitation of Cells From Bone Marrow Origin(Izmir Institute of Technology, 2021) Anıl İnevi, Müge; Özçivici, Engin; Güven, SinanMagnetic 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.Doctoral Thesis Engineering Target Tissue in Lab-On Devices for Predicting Homing Choices of Metastatic Cancer(Izmir Institute of Technology, 2020) Batı Ayaz, Gizem; Pesen Okvur, Devrim; Yavuz, OktayThe 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.Doctoral Thesis Molecular and Cellular Level Adaptations of Bone Marrow Mesenchymal Progenitor Cells To Chemical and Physical Signals(Izmir Institute of Technology, 2020) Baskan Erbilgiç, Öznur; Özçivici, Engin; Atabey, Safiye NeşeMechanical 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.Doctoral Thesis Biochemical and Mechanical Cues for Osteogenic Induction of Stem Cells on Paper Based Scaffolds(Izmir Institute of Technology, 2019) Karadaş, Özge; Özçivici, Engin; Özhan Baykan, Hatice GüneşTissue 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.Doctoral Thesis Increasing Stability of Microbubbles Under Ultrasound(Izmir Institute of Technology, 2019) Ayaz, İlyas Umur; Özdemir, Ekrem; Seçil, MustafaMicrobubbles 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.Doctoral Thesis The Effects of Engineered Silica Nanoparticles on the Cellular Behaviours of Human Hepatocellular Carcinoma Cell Lines(Izmir Institute of Technology, 2018) Tüncel Çerik, Özge; Özçelik, Serdar; Atabey, Safiye NeşePhysicochemical 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.Doctoral Thesis Invetigation of Mechanical Vibration Effects on Breast Cancer Cells(Izmir Institute of Technology, 2018) Olçum Uzan, Melis; Özçivici, Engin; Erdal Bağrıyanık, Şerife EsraIn 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.Doctoral Thesis The Investigation of Anticancer Properties of (r)-4'-methylklavuzon in Liver Cancer Cells and Liver Cancer Stem Cells(Izmir Institute of Technology, 2017) Delman, Murat; Çağır, Ali; Erdal, EsraHepatocellular 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.Doctoral Thesis Pre-Clinical Trial Treatment of Hepatocellular Carcinoma on Cirrhosis in a Rat Model(İzmir Institute of Technology, 2017) Zeybek Kuyucu, Ayça; Şanlı Mohamed, GülşahHepatocellular 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.