Phd Degree / Doktora

Permanent URI for this collectionhttps://hdl.handle.net/11147/2869

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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; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
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.
Development and Characterization of Novel Bioink by Using Decellularized Extracellular Matrix for Bone Tissue Engineering Applications
(01. Izmir Institute of Technology, 2023-05) Kara Özenler, Aylin; Tıhmınlıoğlu, Funda; Havıtçıoğlu, Hasan; 03.02. Department of Chemical Engineering; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
Bone tissue engineering has focused on the development of functional scaffolds that can organize bone regeneration with appropriate structures and properties. Three-dimensional (3D) printing technology enables the development of personalized scaffolds. In addition, biological scaffolds obtained by decellularization have various advantages for developing natural-based scaffolds. The development of printable, patient-specific bioinks derived from decellularized extracellular matrix could provide 3D fabrication of tissues and organs with high potential to mimic native tissues. The presented thesis study demonstrates the development of various bioink compositions for bone tissue engineering applications. In this regard, bone tissues were decellularized with a novel method and then characterized in order to verify the removal of whole cellular components for eliminating immunological reactions. After the pulverization of tissues, decellularized bone (DB) particles were used as an additive within various ink combinations (alginate-, gelatin- and alginate-gelatin-based). Thus, various bioink formulations were developed containing DB particles, biopolymers and mesenchymal stem cells (MSC). All prepared bioinks were bioprinted, then the viability, proliferation and differentiation capacity of the cells inside the structures as well as the physical, rheological, and printability properties of the inks were assessed. The results revealed that all bioink combinations were suitable for bioprinting and the addition of DB particles improved cell proliferation and osteogenic differentiation in all bioink formulations. Alginate-based bioinks exhibited the greatest printability and shape fidelity, gelatin-based bioinks showed the highest cell proliferation and attachment, also, gelatin incorporation into alginate-based bioinks improved the biological activity of cells. In conclusion, cytocompatible, functional composite bioinks developed in this thesis study are of value for bone tissue engineering research in future to explore their functions in the living system and show complete bone regeneration while maintaining their stability for a long time.
Development and Use of Contactless Magnetic Manipulation Methodologies for the Formation of 3d Cardiac Models
(01. Izmir Institute of Technology, 2022-12) Önbaş, Rabia; Arslan Yıldız, Ahu; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
In this thesis, two contactless magnetic manipulation methodologies were introduced, which are magnetic levitation (MagLev) and biopatterning techniques. The optimization steps of both techniques were completed with NIH/3T3 mouse fibroblast cells. Later, 3D cardiac models were developed using H9c2 rat cardiomyocytes. For the MagLev technique, tunable 3D spheroids were obtained with changing initial cell seeding number, gadobutrol concentrations, and culturing time. For the biopatterning approach, a new bio-ink formulation, which comprises alginate, magnetic nanoparticles, and cells, was developed. Further, biopatterned cellular structures were fabricated in different shapes such as discs, rings, and rectangles under an external magnetic field. Later, characterization was done successfully via immunostaining of collagen I, F-actin, and DAPI. Moreover, cardiac-specific markers; cardiac troponin T and MYH6 were analyzed for both 3D cardiac spheroids and patterned 3D cardiac structures. Finally, doxorubicin was applied to evaluate the drug responses. IC50 values were calculated as 14.7 μM and 8.1 μM for 3D cardiac spheroids and 3D cellular structures respectively, while standard 2D cell culture was 3.5 μM which indicated 3D cardiac models were more resistant to drug exposure. In the last part of thesis, patterned 3D cardiac structures were fabricated using co-cultured hiPSC-derived cardiomyocytes and cardiac fibroblast cells via biopatterning methodology. Characterization was carried out successfully by immunostaining of α-actinin, collagen I, Cx-43, Troponin T, and DAPI. Taken together, to fabricate 3D cell culture models, MagLev and biopatterning-based contactless manipulation methodologies may be good alternatives to conventional 2D cell culture methods for tissue engineering applications, especially for drug screening.
Development of a Plasmonic Biosensor for Detection of Exosomes
(Izmir Institute of Technology, 2020-07) Taykoz, Damla; Bulmuş Zareie, Esma Volga; Tekin, Hüseyin Cumhur; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
The aim of this work was to develop Localized Surface Plasmon Resonance (LSPR) surfaces for quantitative detection of exosomes from different sources. For this aim, gold nanorods (AuNRs) with a mean diameter of 40 nm with an aspect ratio of 2.9 were first synthesized and characterized. The self-assembly of AuNRs on glass wafers were optimized through several experiments. In parallel, PEGylation of cetrimonium bromide (CTAB) stabilized AuNRs was investigated using PEGs with three different molecular weights via LSPR, zeta potential and XPS techniques. PEGylated AuNRs were further self-assembled on silanized microscope slides as confirmed. Surface functionalization of AuNR patterned slides was performed using alkane thiol molecules having carboxylic acid and hydroxyl functional groups and confirmed via XPS, FTIR and zeta potential. Specific antibodies (Ab) were conjugated to the surface following two different methods, i.e. click and NHS/EDC chemistry. To perform click chemistry strategy, ImmuneLink® molecules were conjugated with Abs and the final conjugate was used to functionalize surfaces prepared beforehand using azide bearing molecules. The functionalization procedure was confirmed via XPS FTIR and LSPR spectroscopy. The orientation of the antibodies on the AuNRs patterned surfaces was investigated with LSPR in comparison with conventional EDC/NHS chemistry. The click-chemistry strategy proved to provide conjugation of antibodies through their Fc regions exposing Fab regions better for antigen recognition. Finally, surfaces functionalized with a variety of antibodies were used to detect first a pregnancy-associated protein, PLAP, and then exosomes obtained from human semen samples with pre-determined exosome concentrations. The LoD of the biosensor surfaces was found to be between 103-104 exosomes/mL and 5 ng/mL (0.3 pM) PLAP. Human breast cancer cell culture samples having an unknown concentration of exosomes were further analyzed using the newly developed LSPR biochips and the exosome concentration was determined as 108 exosomes/mL for MCF-7 cell line and 107 exosomes/mL for MDA-MB-231 cell line.
Development of a Telemedicine Platform for Remote Monitoring of Patients
(01. Izmir Institute of Technology, 2023-04) Tarım, Ergün Alperay; Tekin, Hüseyin Cumhur; Mevsim, Vildan; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
Telemedicine supports patients and healthcare professionals to provide remote medical services and to ensure that medical operations, services, and applications. Telemedicine provides mobile health services, doctor-patient communication, relationship and consultation, medical clinic, and operation service for the diagnosis, monitoring, treatment, and rehabilitation by remote health services. In this thesis, telemedicine-based applications and integrated devices are developed to assist in the diagnosis, monitoring, and treatment processes of specific diseases. In this regard, a WebRTC-based telemedicine application has been developed to ensure the transfer of health data and doctor-patient communication for monitoring diseases. The developed telemedicine application performance of providing doctor-patient communication and its effect on the management of chronic heart failure disease were examined. Thereupon, medical diagnosis and wearable devices have been developed that can be integrated into the telemedicine application that has been proven for disease monitoring. As part of this study, vital health data, biomarkers, and pathogen analysis were performed by developed devices to diagnose and monitor three chronic diseases, namely sleep apnea, chronic kidney disease, and COVID-19. First, a wearable device platform has been developed that can analyze breathing patterns via diaphragm acceleration and breath temperature and diagnose and monitor sleep apnea. Afterward, electromechanical LOC platforms used for colorimetric determination of serum creatinine levels for the diagnosis of chronic kidney disease by two methods were characterized and presented in detail. Finally, a real-time LAMP-based electromechanical device used to detect SARS-CoV-2 viral RNA has been produced, and a colorimetric rapid test system has been developed and tested for COVID-19 diagnosis. In this way, devices and systems integrated into the telemedicine platform have been developed for use in different diseases and medical applications. With these developed platforms, disease diagnosis, treatment and rehabilitation, remote monitoring and patient management are provided with inexpensive, portable, user-friendly, easy-to-use solutions without the need for professional service providers.
Development of Computational Models To Predict the Toxicity of Advanced Materials
(01. Izmir Institute of Technology, 2023) Bilgi, Eyüp; Karakuş, Ceyda Öksel; Bedir, Erdal; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
The aim of this study is to harness computational power to enhance existing knowledge on NM safety and to optimize the use of existing nanotoxicity data. The primary goal is to support the safe(r)-by-design concept, necessitating early integration of safety considerations into NM design through structural manipulation strategies. This thesis focuses on three case studies: zinc oxide, silver, and gold NP, using data manually collected from the literature. Analyses with zinc oxide and silver NP revealed a correlation between their toxicity and both internal (intrinsic properties, size, shape, surface charge) and external (cell and analysis-related properties) factors. For zinc oxide, it was found that coating had significant influence on cell viability, with a critical threshold identified at 20 µg/ml concentration and 10 nm size. Similarly, for silver NPs, concentration, size, and exposure time were significant factors. Coating with organic macromolecules increased cell viability, whereas green-synthesized NPs (using bacteria, plant extracts, algae) decreased it. The gold NP study highlighted that ensemble methods were more effective in elucidating complex relationships, with cellular uptake linked to particle size, zeta potential, concentration, and exposure time. Overall, this thesis contributes to safer-by-design strategies, crucial for developing commercially viable and safe NMs. The findings advocate for a broader toxicity evaluation approach, considering various physicochemical aspects and experimental procedures. The complex interactions observed suggest that advanced algorithms are necessary for accurate modeling, supporting the optimization of experimental parameters in NP engineering for biomedical applications.
Development of Conducting Polymer-Based Fluorescence On/Off Biosensor for Biomolecule Analysis
(01. Izmir Institute of Technology, 2022-11) Arslantaş, Duygu; Arslan Yıldız, Ahu; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
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
(01. Izmir Institute of Technology, 2020-07) Yaman, Sena; Tekin, Hüseyin Cumhur; Ergon, Mahmut Cem; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. 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 Polymeric Carriers for Gene Therapy
(01. Izmir Institute of Technology, 2021-06) Zelçak, Aykut; Bulmuş Zareie, Esma Volga; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. 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.
Development of Optical Sensor Platforms for Exosome Detection
(01. Izmir Institute of Technology, 2024) Sözmen, Alper Baran; Yıldız, Ahu Arslan; Akan, Pınar; 01. Izmir Institute of Technology; 03.01. Department of Bioengineering; 03. Faculty of Engineering
Bu tez, eksozom tespiti için optik sensör platformlarının kullanılması yoluyla kanserin erken teşhisi ve izlenmesi için yeni bir yaklaşım önermektedir. Kanser prognozu, teşhisi ve izlenmesine yönelik mevcut teknolojiler, özellikle erken evrelerdeki etkinlikleri ve invazivlikleri açısından önemli sınırlamalarla karşı karşıyadır. Bu zorlukların üstesinden gelmek için bu çalışma, iyi tanımlanmış membran protein profili nedeniyle Küçük Hücreli Dışı Akciğer Kanserine (NSCLC) vurgu yaparak, kanserli ekzozomal membran proteinlerini tespit edebilen gelecekteki sıvı biyopsi uygulamaları için biyosensör platformları geliştirmeye odaklanmaktadır. Araştırma, lokalize yüzey plazmon rezonansı (LSPR) ve manyetik kaldırma (MagLev) prensiplerini kullanan iki optik biyosensör platformunun üretimini, optimizasyonunu ve karakterizasyonunu içermektedir. Biyosensör platformları başlangıçta bir model protein olan Bovine Serum Albumin (BSA) ve sonrasında EpCAM, CD151 ve CD81 Eksozomal Membran Proteinleri (ExoMP'ler) ile test edilmiştir. Bu Sırasıyla eksozomal kanser biyobelirteçleri, eksozomal NSCLC biyobelirteçleri ve eksozomal biyobelirteçler olarak yaygın şekilde kullanıldıkları için bu ExoMP'ler hedef olarak seçilmiştir. A549 NSCLC ve MRC5 sağlıklı akciğer fibroblast hücre hatları, geliştirilen optik biyosensör platformlarının eksozom algılama, tanıma ve miktar belirleme yeteneklerini analiz etmek için in-vitro eksozom kaynakları olarak kullanılmıştır. Her iki platform da kanserden türetilen eksozomları sağlıklı eksozomlardan istatistiksel anlamlılıkla başarılı bir şekilde ayırt edebilmiştir. Genel olarak, bu araştırma, sıvı biyopsi teknikleri yoluyla erken teşhis ve izleme için umut verici bir yaklaşım sağlayarak kanser teşhisi ve kişiselleştirilmiş tıbbın ilerlemesine katkıda bulunmaktadır. Geliştirilen platformlar, daha fazla geliştirme ve araştırma ile kanser prognozu ve teşhisine katkıda bulunma potansiyeline sahiptir.
Development of Plasmonic Nanostructures for Photothermal Therapy of Prostate and Breast Cancer
(Izmir Institute of Technology, 2019-12) Tomak, Aysel; Bulmuş, Volga; Şahin, Hasan; 03.01. Department of Bioengineering; 04.04. Department of Photonics; 03. Faculty of Engineering; 04. Faculty of Science; 01. Izmir Institute of Technology
The aim of this thesis is to synthesize gold nanorods (AuNRs) and lipid-stabilized nanobubbles containing AuNRs and investigate the potential of these plasmonic nanostructures as photothermal therapy agents for breast and prostate cancer through in vitro cell culture experiments. For this aim, firstly, AuNRs were synthesized at varying aspect ratios (ARs) and characterized via several techniques including UV-Vis/NIR spectroscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM), inductively coupled plasma-mass spectroscopy (ICP-MS), electrophoretic light scattering (ELS) and X-ray photoelectron spectroscopy (XPS). The surface of AuNRs was modified with a biocompatible polymer, poly(ethylene glycol) (PEG), via ligand exchange method. Cytotoxicity, cell uptake and photothermal effects of AuNRs were investigated via in vitro cell culture experiments using human prostate cancer (DU 145) and epithelial (RWPE-1), breast cancer (MCF7) and epithelial (MCF 10A) cell lines. It was concluded that AuNRs (AR=4.0) were superior than AuNRs (AR=7.0) in terms of cell viability and photothermal effect. Separately, a non-commercial antibody (Ab) targeting a specific sialic acid derivative on the plasma membrane of DU 145 and MCF7 cancer cells was conjugated to AuNRs. Conjugations were characterized with the same techniques and investigated via in vitro cytotoxicity and cell uptake experiments. The Ab-conjugated AuNRs displayed the capability of selective targeting prostate cancer cells. Additionally, lipid-stabilized AuNRs and lipid-stabilized nanobubbles containing AuNRs (AuNBs) were synthesized for the first time and characterized using UV-Vis/NIR spectroscopy, SEM, ICP-MS and ELS techniques. Lipid-stabilized AuNRs were successfully synthesized using varying lipid mixtures instead of cationic, toxic surfactant. Separately, AuNBs were synthesized by combining PEG modified AuNRs with DPPC: DSPE-PEG lipid film under sonication and gas stream. AuNBs showed the same or significantly lower toxicity depending on the cell types and the same photothermal effect with respect to AuNRs (AR=4.0) upon irradiation under laser at 808 nm.
Effects of Telomerase Activators on Monoclonal Antibody-Producing Cell Lines and Stem Cells, and Their Utilization in Industrial Productions
(01. Izmir Institute of Technology, 2024-03) Kuru, Gülten; Bedir, Erdal; 03.01. Department of Bioengineering; 01. Izmir Institute of Technology; 03. Faculty of Engineering
Aging is a physiological and multifactorial biological process of functional decline in any living organism. Telomere shortening, high levels of reactive oxygen species (ROS), and cellular senescence are the primary physiological changes that accompany aging. While there has been an increase in human life expectancy in recent decades, there has not been a concomitant increase in healthy aging. Degenerative diseases, including musculoskeletal disorders such as osteoporosis and osteoarthritis, have been found to be directly linked to aging. Age-related degenerative diseases are devastating diseases that cause millions of deaths worldwide each year and place an economic and psychological burden on society. Due to the ever-increasing number of patients, there is a huge demand for novel therapeutic approaches to treat degenerative diseases. Two main approaches are at the forefront of technology for the treatment of degenerative diseases: stem cell transplantation (regenerative medicine) and monoclonal antibody-based therapy. Indeed, it is well known that there is a strong correlation between disease pathology and telomeres. In fact, the possible therapeutic effects of telomerase activation have been evaluated in diverse backgrounds to cure and prevent various diseases. Within the scope of this thesis, we aim to investigate the effects of telomerase activator novel molecules from Astragalus sp., obtained in our previous studies by biotransformation of cycloastragenol (CG) via the plant's endophytic fungi on the health span/lifespan of mesenchymal stem cells (MSCs) during in vitro expansion and their osteogenic differentiation. Additionally, the efficacy of these compounds was investigated in the monoclonal antibody (mAb) production process in terms of mAb productivity. Based on the outcomes of the study, novel telomerase activators deriving from natural resources of our country have significant potential in stem cell research, thus regenerative medicine, since promising results were obtained for the clinical use of these novel molecules. Our data also suggest that molecules simultaneously promote osteogenic differentiation and telomerase activation.
Hazard Assesment and Reduction of Nanomaterials
(01. Izmir Institute of Technology, 2025) Dincay, Selin Çeşmeli; Karakuş, Ceyda Öksel; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
Demir oksit NP`ler çeşitli alanlardaki, özellikle tanı ve tedavi uygulamaları, kullanımları sayesinde biyomedikal araştırmalarda popüler hale gelmiştir. Ancak, yüzey kaplamasız demir oksit NP`ler toksik etkiler gösterebilmektedir. Bu nedenle, bu NP`lerin biyouyumluluklarını optimize ederken işlevselliklerini koruma konusu büyük öneme sahiptir. Bu çalışmada, işlevselleştirilmiş demir oksit NP`lerin sentezi, karakterizasyonu ve hem in vitro hem de in vivo toksisite değerlendirmeleri yapılarak, tıbbi alandaki güvenli kullanımlarını desteklemek amaçlanmıştır. Bu amaçla, çıplak, dekstran-kaplı, askorbik asit-kaplı ve oleik asit-kaplı olmak üzere dört tip demir oksit NP üretilmiş ve Taramalı Elektron Mikroskopi (SEM), Geçirimli Elektron Mikroskopi (TEM), X ışını difraksiyonu (XRD) ve Fourier-dönüşümlü kızılötesi spektroskopisi (FTIR) gibi yöntemler kullanılarak kimyasal ve yapısal bütünlükleri doğrulanmıştır. Çalışmanın sonuçları, IONP'lerin yüzeyini değiştirmek için kullanılan kaplama malzemelerinin hücreler ve partiküller arasındaki etkileşimleri önemli ölçüde etkilediğini göstermiştir. 2 boyutlu (2B) hücre kültürleri, farklılaşmış monolayerler ve sferoidlerde farklı hücreler (HepG2, CaCo-2 ve HEK293) kullanılarak yapılan sitotoksisite çalışmalarına (WST-1, resazurin ve Annexin V) göre dekstran- ve askorbik asit-kaplı IONP`lerin biyoaktivitesi yüzey kaplamasız NP`lere kıyasla önemli ölçüde artırmıştır. Bu çalışmanın bulguları, biyomedikal uygulamalar için daha güvenli ve etkili NP`ler geliştirilmesinde yüzey işlevselleştirmenin kritik bir öneme sahip olduğunu vurgulamaktadır. Sonuç olarak, bu tez, IONParaştırmalarını içeren literatüre katkıda bulunmakta ve öne çıkan tanı ve tedavi yöntemlerinde kullanılmak üzere ileri düzey NP`lerin oluşturulmasına yardımcı olabilecek değerli bilgiler sağlamaktadır.
Hedeflenebilir Oleandrin Taşıyıcısı Siklodekstrin Temelli Nanoyapıların Sentezi ve Sitotoksik Etkilerinin Araştırılması
(2025) Doğan, Gamze; Bedir, Erdal; Altürk, Rükan Genç; 01. Izmir Institute of Technology; 03. Faculty of Engineering; 03.01. Department of Bioengineering
Kemoterapötik ilaçların yan etkilerini önlemek ve pankreas kanseri tedavi etkinliğini arttırmak için hedefe yönelik ilaç dağıtım stratejileri büyük önem kazanmıştır. Nerium oleander (Zakkum) ekstreleri, tümör hücrelerine karşı sitotoksik aktivite gösteren oleandrin içermektedir. Bu molekül yüksek sitotoksisiteye sahip olmasına rağmen terapötik indeksinin düşük olması ve istenmeyen dokularda birikmesi nedeniyle hedefe yönelik ilaç taşıyıcı formülasyonlarının geliştirilmesi gereklidir. Bu tez, oleandrinin biyoyararlanımını artırmak ve sağlıklı hücrelere yönelik sitotoksisitesini azaltmak için siklodekstrinin konukçu-konuk etkileşimi ile azobenzenin foto-izomerizasyon yeteneğini birleştiren, 200-400 nm aralığında hidrodinamik çapa sahip, UV ışığına duyarlı, anti-EGFR ve anti-CA19-9 konjuge akıllı nanokapsül geliştirmeyi amaçlamıştır. Elde edilen siklodekstrin bazlı nanokapsüller (CD-NK'ler) FT-IR spektroskopisi, SEM ve TEM mikroskopisi ve Zeta Sizer ile karakterize edilmiştir. Sitotoksik aktiviteler, pankreas kanseri (PANC-1, MIA PaCa-2) ve sağlıklı hücre hatları (HEK-293, MRC-5 ve HUVEC) üzerinde MTT analizi ile belirlenmiştir. Ole CD-NK için IC50 değeri tüm hücre gruplarında en az 4 kat artarken, sağlıklı hücrelerde bu oranın kanser hücrelerine göre arttığı gözlenmiştir. Her iki pankreas kanseri hücresi de antikorla konjuge Ole CD-NK'ler uygulandığında, IC50 değerlerinde azalma gözlemlenmiştir. 2D in vitro hücre kültürü çalışmalarının ardından mikroakışkan çip ve sferoid kültür üzerinde 3D hücre kültüründe sitotoksisite çalışmaları, 0,5 µg/ml anti-EGFR Ole CD-NK uygulamasının pankreas kanseri hücrelerinin canlılığında önemli azalmaya neden olduğunu göstermiştir. Sentezlenen Ole CD-NK nanoformülasyonlarının zebra balığı embriyosu ve larva toksisitesi de öncül in vivo çalışmalar olarak gerçekleştirilmiştir.
Invetigation of Mechanical Vibration Effects on Breast Cancer Cells
(Izmir Institute of Technology, 2018-07) Olçum Uzan, Melis; Özçivici, Engin; Erdal Bağrıyanık, Şerife Esra; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
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; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. 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.
Magnetic Manipulation of Cells for Tissue Engineering and Diagnostic Applications
(01. Izmir Institute of Technology, 2025) Özkan, İlayda; Özçivici, Engin; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
Bu tez kapsamında, negatif magnetoferez prensibine dayalı manyetik levitasyon tekniği, iki farklı yaklaşım için kullanılmıştır. İlk olarak, manyetik levitasyon sistemi, doku iskelesiz üç boyutlu doku modelleri oluşturmak için bir biyofabrikasyon yöntemi olarak kullanılmıştır. İlk yaklaşımda, in vivo dokuyu daha iyi taklit edebilen, üç boyutlu heterojen küresel modeller geliştirmek ve iyileştirmek amaçlanmıştır. Tek halka mıknatıs tabanlı levitasyon sisteminde çeşitli konfigürasyonlarda heterojen meme kanseri küreleri elde edilmiştir. İki farklı hücre tipinin lokalizasyonunda sferoid yapı içerisindeki farklı hücre yükleme parametrelerinin etkisi incelenmiştir. Ek olarak, hücre dışı matriks birikimini artırarak, manyetik levitasyon ile oluşturulan doku iskelesiz sferoid modellerin in vivo yapıyı taklit edebilme kapasitesini artırmak için makromoleküler kalabalıklaştırma yöntemi entegre edilmiştir. İkinci olarak, nörogelişimsel bozukluklarda teşhis amaçlı olarak manyetik levitasyonun kullanımı araştırılmıştır. Araştırmada, sağlıklı bireylerden ve nörogelişimsel bozukluğu olan bireylerden elde edilen fibroblastlar, sinir progenitör hücreleri ve indüklenmiş pluripotent kök hücreler arasındaki farkı belirlemek amacıyla hücrelerin özkütle profilleri analiz edilmiştir. Ayrıca, farklı tipte lizozomal depo hastalıklarının hücre özkütlesi üzerindeki etkisi fare modellerinden izole edilen primer nöroglial hücreler kullanılarak incelenmiştir. Bu tezde, hem doku mühendisliği uygulamaları hem de hücre bazlı tanı çalışmalarında hızlı, maliyet etkin ve güvenli bir yöntem olarak manyetik levitasyon tekniğinin potansiyeli gösterilmiştir.
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; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. 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.
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; 03.01. Department of Bioengineering; 04.01. Department of Chemistry; 03. Faculty of Engineering; 04. Faculty of Science; 01. Izmir Institute of Technology
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 of Vaccine Formulations for Melanoma Using Potent Adjuvant Candidate Astragaloside Vii and Investigation of Anti-Tumor Activities of Formulations in Mouse Cancer Models
(01. Izmir Institute of Technology, 2024) Özefe, Nilgün Yakuboğulları; Bedir, Erdal; Sağ, Duygu; 03.01. Department of Bioengineering; 03. Faculty of Engineering; 01. Izmir Institute of Technology
Kanser, genomdaki nokta mutasyonların birikmesi sonucu ortaya çıkan ve yapısal değişikliklerle ilerleyen bir hastalıktır. Kanser immünoterapisinin ana kategorilerinden biri, vücudun kansere karşı kendi bağışıklık sistemini harekete geçiren kanser aşısıdır. Geleneksel tedaviler güvenlik sorunları ve bağışıklık sisteminin uygun olmayan modülasyonu nedenleriyle etkili olmadığından, nanotıp temelli yaklaşımların uygulanması bu sorunların çözümü için bir potansiyel oluşturmaktadır. Bir taşıyıcı malzeme içinde immünostimülatör ajanların/adjuvanların formülasyonları, hedef hücreler tarafından alımı sağlar, sistemik etkiyi değiştirir, güvenli bir profil sağlar ve immünoterapötiklerin terapötik etkinliğini arttırır. Bu bakış açısıyla bu tez kapsamında, Astragalus polisakkariti temelli bir nanotaşıyıcıya MPLA/Astragaloside-VII entegre edilerek yeni bir adjuvan sistemi (MA-NP) tasarlanmış ve geliştirilmiştir. MA-NP'nin in vitro ve in vivo immünomodülatör özellikleri ve ardından iki fare melanoma modelinde profilaktik ve terapötik etkinliği araştırılmıştır. Biyouyumlu, 20-50 nm boyutunda, negatif yüklü, dendritik hücreler tarafından etkin bir şekilde alınabilen MA-NP başarılı bir şekilde üretilmiştir. Çoklu peptitler ile formülize edilen MA-NP, doğal ve kazanılmış bağışıklık hücreleri aktive etmiş, öncelikli olarak merkezi bellek CD8+ T hücre yanıtı gösteren antijen spesifik sitotoksik T hücre popülasyonunu arttırmış, fonksiyonel IFN-+CD8+ T hücrelerini indüklemiş, tümör içi CD4+, CD8+ T hücre, dendritik hücre ve M1 makrofajlarını arttırmış ve güçlü bir şekilde tümör büyümesini inhibe etmiştir. Ayrıca MA-NP ile oluşturulan nanoaşı, anti-PD1 antikorları ile birlikte farelere uygulandığında yerleşik B16-F10 tümörlerini ortadan kaldırmıştır. Bu bulgular, kanser aşılarında kullanılabilecek yeni bir saponin temelli adjuvan sistemini ve kanser immünoterapi yaklaşımını geliştirmek için umut verici bir kombine terapiyi ortaya koymaktadır.

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