Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Detection of Dna Methylation of Multiple Tumor Supressor P16ink4a Gene by Polythiophene Based Optical Sensor(Izmir Institute of Technology, 2018) Kaya, Hakan; Yıldız, Ümit Hakan; Elmacı Irmak, NuranDNA methylation is epigenetic events commonly occurs in mammalian genome starting from formation of embryo to the end of life. Especially, hypermethylation in tumor suppressor genes, corresponds the cancer growth and detection of DNA methylation in these genes crucial for the diagnosis of cancer. Water soluble polythiophenes are frequently used for the detection of biomolecules through the optoelectronic properties. In this study, detection of DNA methylation of multiple tumor suppressor p16INK4A gene via polythiophene based optical sensor was achieved. Newly designed, synthesized and characterized poly(1-(3-((4-methylthiophen-3-yl) oxy) propyl)-1,4diazabicyclo [2.2.2] octan-1-ium bromide) was used during characterization of DNA sequences and detection of DNA methylation. The target sequence position is +137 to +156 in p16INK4A gene which have three potential CG dinucleotide to be methylated. Detection of DNA methylation based on sodium bisulfite treatment, complementary sequence of unmethylated ssDNA and the conformational change of water soluble polythiophene. In our fluorometric analysis, unmethylated sequence/complementary successfully hybridized and dsDNA Io/I ratio is under the 1.40 while the methylated sequence/complementary hybridization failed due to different base content and remain as ssDNA and, Io/I ratio is higher than 1,60. The novelty of work is detection mechanism is PCR and FRET free with a range of 300 ng to 700 ng sample requirement. Characterization of homopurines, homopyrimidines, methylated and unmethylated sequence with cationic polythiophenes also accomplished. PolyG (10), polyG (20) and polyA (10) yielded a no signal in UV-VIS region while the polyA (20) yielded a 100 nm red shift. Furthermore, PolyC (10), PolyC (20), PolyT (10), PolyT (20) yielded three vibrionic peaks at 505 nm, 545 nm and 595 nm with different intensities and unique isosbestic points. All 10 bases long homopyrimidine and homopurine have a unique quencher character with cationic polythiophene. Lastly, conformational change of polythiophene investigated with computational methods and heptamer used as a model.Master Thesis Sericin-Polymer Conjugates: Preparation and Physicochemical Characterization(Izmir Institute of Technology, 2017) Gül, Abdulkadir; Bulmuş Zareie, Esma VolgaSericin is a protein derived from silkworm, Bombyx mori, and has several useful properties as a natural biomaterial such as antioxidant character, moisturizing ability, hydrogel forming property and most importantly immunogenic inertness. The aim of this thesis is to prepare and physicochemically characterize sericin-polymer conjugates as potential natural-synthetic hybrid biomaterials with enhanced properties for drug delivery and tissue engineering applications. For this purpose, three polymers having the same degree of polymerization (n~42) and varying chemical nature, i.e. poly(oligoethylene glycol methacrylate), P(OEGMA) hydrophilic and neutral, poly(hydroxyethylmethacrylate) P(HEMA) less hydrophilic and neutral, and poly(dimethylaminoethyl methacrylate) P(DMAEMA) hydrophilic and cationic after quaternization, were first synthesized via reversible addition fragmentation chain transfer (RAFT) polymerization. Each polymer was characterized via nuclear magnetic resonance (1H-NMR) and gel permeation chromatography (GPC). Separately, molecular weight and isoelectric point of sericin were characterized using various techniques including Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and High-Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE). Polymers were then covalently conjugated to sericin using NHS/EDC chemistry. The conjugates were characterized using SDS-PAGE, GPC and DLS (Dynamic Light Scattering). The SDS-PAGE and GPC results showed the successful preparation of the conjugates. DLS revealed that the hydrodynamic size of P(OEGMA) and P(DMAEMA) polymers and their conjugates were between 1 and 10 nm as they are soluble in PBS and do not form aggregates. Unlike the other two polymers, although the size of P(HEMA) polymer was observed to be 3.24 ± 0.62 nm, the DLS measurements of P(HEMA) conjugates indicated the presence of self-organization and aggregation of Sericin-P(HEMA) conjugates in aqeuous solution. Consequently, the size of sericin-P(HEMA) conjugates were found to be 530 ± 60.83 and 223.3 ± 25.2, respectively.Master Thesis Design and Construction of Portable Localized Surface Plasmon Resonance Device for Detection of Biological Molecules(Izmir Institute of Technology, 2017) Gül, Aytaç; Bulmuş Zareie, Esma VolgaPoint-of-care devices giving rapid results in non-laboratory settings have become important for biosensor applicattions in a wide range of fields including medical, food, agriculture and pharmaceutical. This work aims to portable device based on localized surface plasmon resonance spectroscopy (LSPR-S) as a potential biosensor platform that can be used in non-laboratory settings for rapid detection of biological molecules at hifh sensivity. The thesis can be divided into two parts: In the first part, the design and consctruction of the device including both the mechanical and electronic parts are presented. The mechanical section includes the integration of the parts to build the device and microchannels designed with the aid of a three-dimensional drawing program Solid Works 2015. The second step of the construction process was the installation of electronic components onto the device. The electronicpart consists of a light source, fiber optic cables, a spectrometer and a temperature sensor. In conclusion, a portable LSPR-S device with an integrated microchannel system has been produced, which potentially allows analysing low volumes of sample without the need to label the molecules. The second part of the thesis covers the studies towards the preparation and application of sensing platforms for the LSPR-S device constructed to enable the rapid detection of biplogical molecules at high sensitivity. These included the preparation of gold nanorods and nanoparticles-based LSPR-S detection of model antibody-antigen and bacteria-bacteriophage interactions, respectively. Studies conducted in this section have led to the conclusion that LSPR-S-based biosensor platforms developed in thisis are promising solutions to overcome current challenges in biosensor applications.Master Thesis Automatic, Fast and Accurate Sequence Decontamination(Izmir Institute of Technology, 2016) Bağcı, Caner; Allmer, Jens; Tekir, SelmaThe introduction of massively parallel sequencing technologies was a revolutionary step in genomics. Their decreasing cost and powerful features have put them more and more on demand in the last decade. It is now possible to sequence even complete genomes of organisms, using massively parallel sequencing technologies even for small laboratories around the world. However, the power of this powerful technology comes with its challenges. The challenges are both in technological and computational side of the work. In this work, one of these computational challenges is addressed and a novel algorithm is offered to solve the problem. Sequencing by synthesis is one of the methods used in many different massively parallel sequencing instruments. This method utilizes the biological process of DNA replication and with the help of different means of detection, it allows sequencing a DNA molecule while it is replicated. Since DNA polymerase requires a primer to start the replication reaction, short oligonucleotide adapters are used in sequencing by synthesis methods to initiate the reaction. However, certain circumstances allow these adapters to contaminate final sequence reads. Several tools have been offered to trim adapters from reads; but all depend on the prior knowledge of the adapter sequence by the bioinformatician. In this work, an algorithm is offered to detect and trim adapters only using the sequences of reads, without relying on prior knowledge of adapter sequences. The algorithm was shown to perform better or on the same grounds with existing methods in terms of speed and efficiency.Master Thesis Invadopodia Formation on Nanometer Scale Protein Patterns(Izmir Institute of Technology, 2014) Batı, Gizem; Pesen Okvur, Devrim; Özyüzer, LütfiHow the positions of invadopodium in the cell are determined and if they have an adhesivefunction are not known. Using fluorescence microscopy and antibodies that recognize actin, cortactin and MT1-MMP proteins, invadopodia formed by breast cancer cells plated on protein nanopatterns of different geometeries and components after stimulation with epidermal growth factor which is known to induce invadopodia formation, were examined. Invadopodia formation was studied for the first time on nanometer scale, single and double active component, protein patterns with equal distance and gradient spacings. The results show that: • On K-casein-fibronectin nanopatterns, invadopodia prefer to form on K-casein which blocks cell adhesion rather than on fibronectin nanodots which promote cell adhesion. • On Laminin-fibronectin nanopatterns, invadopodia prefer to form on laminin rather than on fibronectin nanodots. • On gradient patterns, invadopodia prefer areas with wide spacings. These results support the hypotheses that the positions where invadopodia form can be determined by surface protein nanopatterns and that cell adhesion is not required at points where invadopodia will form.Master Thesis Molecular Characterization of Adult Stem Cells' Adaptations To Mechanical Signals During Adipogenic Commitment(Izmir Institute of Technology, 2015) Baskan, Öznur; Özçivici, Engin; Baskan, Öznur; Yalçın Özuysal, Özden; Özçivici, Engin; Yalçın, ÖzdenPrevalence of obesity have increased across the years based on technological developments that supported nutritional availability and sedentary lifestyles. Restoring mechanical activity with physical exercise suppresses obesity, and mechanical loading can also be delivered passively with whole body vibrations with high frequency and low magnitude. Anabolic effects of high frequency low magnitude mechanical vibrations on adult stem cells are well identified whereas sensing mechanism of cells and their response to mechanical stimuli is largely unknown. Here, we hypothesed that daily bouts of low intensity vibrations will affect molecular, physical and ultrastructural profile of the cells and the effect will interact with the adipogenic induction. To test this hypothesis mouse bone marrow stem cell line D1 ORL UVA were subjected to mechanical vibrations (0.15g, 90 Hz, 15min/d) for 7 days to both during quiescence and adipogenic commitment. Ultrastructural changes were identified on cellular and molecular levels. Atomic force microscopy was used to characterize the changes on cell surface and significant increase was observed in cell surface height. Moreover, in order to identify the changes in cytoskeleton structure and physical properties, actin were stained with phalloidin and imaged with inverted microscope. To quantify phalloidin amount, signal intensities and physical features of the cells were measured. It was observed that mechanical stimulation and adipogenic induction affect actin content and the physical structure of the cells significantly. Molecular level analysis of cytoskeleton elements and adipogenic markers were performed with Real time PCR. Dramatic increases in adipogenic markers were detected with adipogenic induction. These results indicate that mesenchymal stem cells responds to mechanical vibrations by altering their molecular and ultrastructure during both quiescence and adipogenesis.Master Thesis Cell Adhesion on Nanomater Scale Fibronectin Patterns: a Comparision of Breast Cancer Cells and Normal Breast Epithelial Cells(Izmir Institute of Technology, 2014) Horzum, Utku; Pesen Okvur, DevrimCell adhesion to extracellular matrix is an important process for both health and disease states. Surface protein patterns are topographically flat, and do not introduce other chemical, topographical or rigidity related functionality and, more importantly, that mimic the organization of the in vivo extracellular matrix are desirable. Previous work showed that vinculin and cytoskeletal organization are modulated by the size and shape of surface nanopatterns. However, a comparative and quantitative analysis on normal and cancerous cell morphology and focal adhesions as a function of micrometer scale spacings of protein nanopatterns was absent. Here, electron beam lithography was used to pattern fibronectin (FN) nanodots with micrometer scale spacings on a K-casein background (single active) on indium tin oxide (ITO) coated glass which, unlike silicon, is transparent and thus suitable for many light microscopy techniques. Exposure times were significantly reduced using the line exposure mode with micrometer scale step sizes. Micrometer scale spacings of 2, 4, 8 microns and gradients between FN nanodots modulated cell adhesion for both breast cancer and normal mammary epithelial cells, through modification of cell area, cell symmetry, actin organization, focal adhesion number, size and circularity under both static and flow conditions. Overall, cell behavior was shown to shift at the apparent threshold of 4 μm spacing. Results showed that there were significant differences in terms of cell adhesion between breast cancer and normal mammary epithelial cells: Breast cancer cells exhibited a more dynamic and flexible adhesion profile than normal mammary epithelial cells.Master Thesis Design and Fabrication of Microfluidic Device That Allows Investigation of Distance Dependent Interactions of Two Different Cell Types(Izmir Institute of Technology, 2014) Sağlam, Murat; Pesen Okvur, Devrim; Özyüzer, LütfiThe main studies of in this thesis, the mold and a microfluidic device are achieved by using SU-8 photoresist and PDMS polymer. Firstly, molds are obtained which are thickness ranging from 30 to 400 μm by using SU-8 photoresist with UV lithography technique and this molding will use for shaping polydimethylsiloxane (PDMS) polymer. Finally, PDMS molds combined with the glass surface to create a three dimensional reservoirs. Microfluidic device that allows investigation of distance dependent interactions, two factors are positioned at certain distances from each other and the microfluidic device is allowed to investigation of distance dependent interaction of two factors. There is an alternating width channel between two channels which have each of two factors. These three channels are separated from each other by colonnades, not by walls, therefore physical, chemical and biological interactions are possible between the factors. Necessary physical, chemical, and biological conditioning can be provided by the reservoirs which are neighbor of channels including factors. Microfluidic chip has a lot of advantages that are small liquid volumes (pL-μL), precise spatial & temporal control, successfully mimic the physiological context, highthroughput analysis, low fabrication costs; portable and safer therefore it facilitates us to refine our methods of analysis and development in cell biology investigations and determining the content of chemical samples.Master Thesis Preparation and Characterization of Tyrosinase Immobilized Gelatin Films(Izmir Institute of Technology, 2015) Tetik, İlkin; Çakıcıoğlu Özkan, Seher Fehime; Şanlı Mohamed, GülşahIn this work, tyrosinase enyzme was immobilized on gelatin films by using entrapment and adsorption. In entrapment method, glutaraldehyde was used to crosslink the gelatin matrix. Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), contact angle were used for characterization of the films; UV spectrophotometer was used for measurement of the catalytic activity of the tyrosinase immobilized films in catechol oxidation reaction. The pH and temperature, enyzme and substrate concentration effects on the activity of enzyme were investigated. Immobilization of enyzme to gelatin support increases the pH, temperature and operational stability due to the appropriate flexibility and structural rigidity. Activity of immobilized enyzme has decreased compared to free enyzme activity due to the steric hinderance effect and mass transfer limitations. The reusability of the enzyme entrapped gelatin film was also investigated; there was almost 60% decrease in activity of the film after fifth usage. During activity measurement, the color of the gelatin films becomes brown showing that quinone produced with enyzmatic reaction was bound on the film.Master Thesis Method That Positions Cell-Laden or Cell-Free Matrices at Defined Positions From Each Other Inside a Single Microfluidic Channel(Izmir Institute of Technology, 2014) Tarım, Emre; Pesen Okvur, Devrim; Özyüzer, LütfiIn recent years, the use of microfluidic has increased in the field of many biological studies. Microfluidic technology has a large area which is a joint product of biology and industry covering all branches of science. The small size of the microfluidic chip offers many advantages in the use of microfluidic. During the analysis, the microfluidic chip offers many advantages such as, use of less material, less waste generation, temporal control, opportunity of analysis under the microscope and high throughput analysis. In addition to these, while microfluidic chip is providing a safe environment for users, via mimicking the physiological environment, it also provides a suitable environment in order to make cell, tissue and organs based assays. Microfluidic devices especially use in cancer studies, chemical analysis, tissue engineering, drug screening, immunology and stem cell differentiation. In this study, we aimed to develop methods depending on the distance to position the MDA-MB 231 breast cancer cells in the microfluidic channels. Firstly, the microfluidic channels were obtained by using the soft lithography and experiments with breast cancer cells were performed using these channels. Breast cancer cells containing matrix was loaded into microfluidic chips and precipitated onto blank matrix by using centrifuge. The aim of repeating this process was to position the breast cancer cells at different distanced locations.