Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Blood-Brain Model Comprising Mouse Brain Tissue(Izmir Institute of Technology, 2023) Demi̇r, Ceylan; Okvur, Devri̇m Pesen; 01. Izmir Institute of TechnologyBeyin kan bariyeri (BBB), beyin hastalıklarının patolojilerinde ve ilaç dağıtımı gibi tedavi yaklaşımı çalışmalarında büyük önem taşımaktadır. Kan beyin bariyerinin yüksek verimli ve gerçekçi tasarımlara sahip mikroakışkanlarla modellenmesi, araştırmalarda hayvan kullanımının azaltılması, hassas tıbbın yaygınlaştırılması ve bilimsel araştırmaların ilerletilmesi için etkili bir seçenektir. Bu çalışmada yetişkin fare beyin dokusunu içerecek şekilde bir BBB modelinin geliştirilmesi amaçlandı. Bu amaçla yaklaşık olarak eşit büyüklükte ve besin alımına yetecek kadar küçük beyin dokusu elde edilmesini sağlayacak bir araç ve yöntem geliştirildi. BBB modeline uygun mikroakışkan model geliştirilmiş ve çalışmanın ilerlemesine göre özellikleri optimize edilmiştir. Doku canlılığına ilişkin parametreler optimize edildi ve doku, mikroakışkan içinde yaşadı. Farklı bileşenlerin dahil edilmesiyle doku içeren ve doku içermeyen ortamların mikrovasküler ağ oluşumları incelendi. BBB modelinin işlevsel durumu, optimize edilmiş vasküler ağ ve endotel hücre katmanının oluşturulmasından sonra geçirgenlik tahlili ile test edildi.Master Thesis Investigation of Metastatic Potential of Breast Cancer Cells in Lung-On(Izmir Institute of Technology, 2023) Turgut, Deni̇z Cemre; Pesen Okvur, Devri̇m; 01. Izmir Institute of TechnologyMeme kanseri kadınlar arasında en sık görülen kanser olup, dünya çapında kansere bağlı ölümlerin önde gelen ikinci nedenidir. Mortaliteye katkıda bulunan birincil faktör ise metastazdır. Üçlü negatif meme kanseri, akciğerlere metastaz yapma eğilimi gösterir. Bununla birlikte akciğerler, meme kanseri metastazlarının en yaygın hedeflerinden biri olarak tanımlanır. Bu yüzden, akciğere özgü metastatik hareketi yöneten kesin mekanizmaları incelemek, akciğer fizyolojisini ve yapısını gerçekten karşılayabilen bir 3 boyutlu akciğer modelini gerektirir. Bu çalışma, MDA-MB-231 meme kanseri hücrelerinin metastatik davranışını ve bunların, yeni bir biyopsi delme tekniği ile oluşturulan 3 boyutlu akciğer modeli içerisinde tümörle ilişkili makrofajların (TAM'ler) etkisini araştırmaktadır. Elde edilen delinmiş akciğer dokuları, akciğer çalışmaları için hibrit bir 3 boyutlu kültür modeli sunan mikrofizyolojik sistemlere entegre edilir. Bu araştırmanın temel bulguları, biyopsi yumruklarının doku bütünlüğünü koruma ve tutarlı bir boyut dağılımı sağlama konusundaki etkinliğini ortaya koymaktadır. Ayrıca çalışma, biyopsi uygulanmış akciğer dokularının 72 saate kadar canlılığını koruyabildiğini de ortaya koymaktadır. En önemlisi, MDA-MB-231 hücreleri, farklılaşmış U-937 hücrelerinin varlığında gözlenen daha da büyük bir göçle delinmiş akciğer dokularını istila etme konusunda önemli bir kapasite sergiler. Bu bulgular, meme kanseri hücrelerinin metastatik potansiyeli hakkında değerli bilgiler sağlar ve TAM'lerin, 3 boyutlu akciğer mikro ortamında kanser ilerlemesine olan yardımcı rolünün önemini vurgular. Bu yeni doku kültürü tekniği, kanser metastazı, hassas tıp ve tümör-mikroçevre etkileşimlerini incelemek için yeni bir araştırma tekniği ortaya koymaktadır.Master Thesis Examination of Stable Intronic Sequence Rna Profile Under Apoptotic Conditions(Izmir Institute of Technology, 2022) Akgül, Bünyamin; Akgül, Bünyamin; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyApoptosis is a process of programmed cell death. Cisplatin, a chemotherapeutic drug, activates intrinsic pathway of apoptosis while TNF-alpha, a death ligand, activates the extrinsic pathway of apoptosis. Noncoding RNAs involve in regulation of apoptotic pathways at post-transcriptional level. Stable intronic sequence RNAs (sisRNAs) are the novel class of non-coding RNAs which can be generated by splicing- dependent and independent mechanisms. sisRNAs transcribed from their intronic promoter may contain 5’ cap and polyA tail. Despite the reports of several studies about sisRNAs in Xenopus and Drosophila, a genome-wide profile of sisRNAs in human is lacking. Therefore, we aimed to identify sisRNAs profile that are transcribed from their intronic promoter under cisplatin- and TNF-alpha- mediated apoptosis conditions. In this thesis study, the deep sequencing of total RNA, polyA + and polyA eliminated fractions from cisplatin-, TNFalpha-, DMSO-treated cells were performed. Differentially expressed intronic transcripts were analysed by DE-kupl algorithm. The intronic transcripts both in total RNA and polyA + RNA fractions but not in polyA eliminated fractions were screened visually on Integrated Genome Viewer (IGV) and selected as sisRNA candidateS. 48 sisRNA candidates were detected in cisplatin-treated data while 33 sisRNA candidates were detected in TNF-alpha- treated data. 5’ and 3’ RACE PCRs were performed for determination of transcriptional units of sisRNA candidates. Overexpression of sisRDOCK7-IT1 caused 8.09% increase in total apoptosis of HeLa cells in 48 hours. sisRDOCK7-IT1 triggers the activation of apoptosis but the mechanism of its induction of apoptosis is still unknown.Master Thesis Investigation of Long Non-Coding Rna and Chromatin Interactions in Hela Cells(Izmir Institute of Technology, 2022) Akgül, Bünyamin; Akgül, Bünyamin; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyThe DNA in the cells is surrounding histone proteins to form nucleosomes. The structure is packed further into chromatin. The chromatin structure is dynamic and flexible. It is regulated by many factors including long non-coding RNAs (lncRNAs). LncRNAs are a class of non-coding RNAs, transcripts that do not encode protein. They are longer than 200 nucleotides and might contain a polyA tail and a 5’ cap. Thus, they are localized in the nucleus. lncRNAs interact with chromatin in two ways, indirect and direct. Direct interaction occurs via two mechanisms: R-loop and triplex formation. These interactions affect the folding of chromatin inducing gene expression under various cellular conditions. LncRNAs interacting with chromatin regulating genes are found in HEK cells. Thus, it is hypothesized that lncRNA – chromatin interactions may differ in cancerous cells as well. In this study, the iMARGI method is optimized to be used in adenocarcinoma HeLa cells. The chromatin digestion and incubation conditions are adjusted to give optimal results for HeLa cells. iMARGI is a recently developed method employed to investigate such interactions in a genome-wide manner. iMARGI allows the isolation of all lncRNAs interacting with the whole genome. The interacting RNA – DNA molecules are pulled down with streptavidin conjugated beads after linker ligation. The chimeric molecules are amplified with PCR forming lncRNA – chromatin libraries of HeLa cells. In the future, new libraries can be formed after inducing apoptosis in HeLa cells. Identification of lncRNAs involved in chromatin remodeling in apoptotic conditions can facilitate new therapeutic methods for fighting tumor initiation and development.Master Thesis Investigation of the Interaction Between Dr5-As Long Noncoding Rna and Caprin1 Protein(Izmir Institute of Technology, 2022) Akgül, Bünyamin; Akgül, Bünyamin; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyCell proliferation is the crucial process for many physiological incidents such as tissue and organ development, wound healing, and immune system reactions. It is achieved by the growth and division of cells in a multicellular organism. Investigation of molecules involved in the regulation of cell cycle mechanism provides insight into reasons and treatments of the diseases such as cancer. In recent years, information that acquired from deep sequencing reveals that several proteins and non-coding RNAs have crucial role in the regulation of cell cycle and proliferation. Death receptor 5 antisense (DR5-AS) is a novel long non-coding RNA (lncRNA) transcript that is cisplatin inducible and is involved in modulation of cell proliferation and cell cycle in HeLa cells. When DR5-AS lncRNA was knocked down, the morphology of HeLa cells became spherical without inducing apoptosis. Although this lncRNA reduces cell proliferation via a cell cycle arrest at S and G2/M phases, mechanism behind this cell cycle arrest is not known. lncRNAs work in complexes with RNA, DNA, and protein interactions in the cell. There are several experimental and bioinformatical approaches to investigate RNA: protein interactions such as PAR-CLIP. In this approach, proximal protein and RNAs are covalently bonded with UV radiation. Then this complex is immunoprecipitated with specific antibodies. According to PAR-CLIP data of DR5-AS lncRNA, CAPRIN1 is a cell cycle associated protein that has the highest interaction score. The results suggest that CAPRIN1 and DR5-AS work reversely in cell proliferation although under the cisplatin treatment, CAPRIN1 enhances the expression of DR5-AS lncRNA. All these observations were confirmed by many quantitative experiments. Conclusively, this study provides a clue about how DR5-AS lncRNA might regulate cell cycle and proliferation through CAPRIN1 protein.Master Thesis Investigation of the Interactions Between Cancer Cells and the Microenvironment at the Cellular Level(Izmir Institute of Technology, 2022) Pesen Okvur, Devrim; Pesen Okvur, Devrim; Pesen Okvur, Devrim; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyBreast cancer is the most frequently diagnosed cancer type and the first leading cause of cancer-related deaths in women. Breast tumor mass is not only harboring cancer cells but also several types of stromal cells, including fibroblasts. While all of these stromal cells may have a calamitous effect on cancer progression, fibroblasts which make up nearly 80% of tumor mass present unique characteristics such as extensive extracellular matrix (ECM) production. In the context of tumors, the activated cells are referred to as cancer-associated fibroblasts (CAF), expressing several markers such as αSMA, FSP1, FAP, vimentin, and PDGFRβ. However, an in-depth understanding of the transdifferentiation of fibroblasts to CAFs is lacking. ECM components may change when cells become cancerous, which can alter cell behavior, facilitating proliferation, differentiation, and migration. Decellularized ECM(dECM) has recently been considered one of the tools to study in-vitro cell-ECM interaction. In this work, we utilized cancer cell-derived ECM(ccECM) to investigate its effect on the differentiation of the fibroblast to CAFs by compering decellularization methods called the extraction buffer and the freeze-thaw cycle. Our study suggested that ccECM from MDA-MB-231 impacted the fibroblasts' behavior from proliferation to differentiation via its ECM components, including fibronectin and laminin. The fibroblasts cultured on ccECM showed increased CAFs markers indicated above. Overall, ccECM could be one of the intermediate steps in fibroblast differentiation, but in the future, the factors present in ccECM should be scrutinized to understand the mechanisms behind this effect.Master Thesis Genetic Modification of Glycoalkaloid Content in Eggplant by Agrobacterium-Mediated Transformation Technique(Izmir Institute of Technology, 2022) Frary, Anne; Frary, Anne; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyEggplant is one of the first plantsthat was cultivated in the Old World. It is economically and agriculturally important for many countries such as Turkey. In addition, eggplant has been used medicinally since ancient times. The medicinal effects of eggplant come from its secondary metabolites. One of the most useful secondary metabolites is steroidal glycoalkaloids (SGA). SGAs have been used in cancer treatment research. The biosynthesis of SGAs in plants is controlled by enzymes and transcription factors. GAME 9 is a transcription factor that has been investigated in tomato and potato and has been shown to be an important element in the production pathway. In this research, a copy of eggplant’s own GAME 9 transcriptional factor was transformed to the eggplant genome with Agrobacterium-mediated transformation. To this end, the GAME 9 gene was amplified from the eggplant genome and inserted into the pSoup/pGreen0029 vector system. This vector system was inserted into Agrobacterium. Eggplant seedling explants were incubated with transgenic Agrobacterium. The transformation of regenerated plants was confirmed with PCR and efficiency was found to be 1.3%. RNA expression levels were checked by RT-QPCR for 12 confirmed transgenic plants. On the metabolite level, the leaves of transgenic plants were assayed for SGA content using Dragendorff’s reagent method. The transgenic plants’ mRNA and metabolite levels showed plant to plant variation due to the random nature of transgene insertion. Of the 12 transgenic eggplant, nine plants had significantly increased production of both mRNA (at least 2-fold difference) and metabolite levels. In addition, two plants did not show any difference in mRNA level but their SGA amount was significantly increased. The remaining plant had a 0.16-fold decrease in mRNA level but a significantly greater level of SGA than control plants. In the future, these transgenic plants can help illuminate the SGA production pathway of eggplant and be used for the production of economically and medicinally valuable SGAs.Master Thesis Investigation of the Biological Role of Mouse Acylneuraminyl Hydrolase Enzymes in the Regulation of Neuroinflammation(Izmir Institute of Technology, 2022) Seyrantepe, Volkan; Seyrantepe, Volkan; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologySialidosis is a lysosomal storage disorder, and it is inherited by autosomal recessive mutations in the Neuraminidase 1 (NEU1) gene. Neuraminidases or sialidases are catalytic enzymes that carry out the desialylation of glycoconjugates. Deficiencies of neuraminidases lead to the accumulation of sialoglycoconjugates in membranes of cells. Neuroinflammation and neurodegeneration are present in some lysosomal storage diseases such as Tay-Sachs. However, in the sialidosis mouse model, neuroinflammation was never studied. In this study, we investigated the effect of neuraminidase 1, neuraminidase 3, and their combined deficiency on neuroinflammation by using Neu1-/-, Neu3-/- knockout, and Neu1-/- Neu3-/- double knockout mouse models. Neu1-/- Neu3-/- knockout mouse model was smaller in comparison to its littermates and showed muscle weakness, tremoring, and 2-3 weeks of a lifetime. Some of the Neu1-/- Neu3-/- mice died prematurely. To unravel the pathology immunohistochemical, biological, and chromatographic techniques were used. The expression of inflammatory cytokines was altered in the Neu1-/-, Neu3-/-, and Neu1-/- Neu3-/- mice with respect to the brain section. Neu1-/- Neu3-/- mice showed generally the highest expression of cytokines in the cerebellum compared to the cortex. Neuronal loss was observed in the Neu1-/- Neu3-/- mice in the cortex, thalamus, and cerebellum. The most remarkable change was in the ganglioside expression pattern in the Neu1-/- Neu3-/- mice cortex. GD3 expression was present in the cortex of Neu1-/- Neu3-/- mice where expression of this ganglioside is related to neuroinflammation, neurodegenerative stimuli, autophagosome remodeling and programmed cell death.Master Thesis Induction of Hairy Root Culture in Eggplant With Rhizobium Rhizogenes(Izmir Institute of Technology, 2021) Öztürk, Tuba; Doğanlar, Sami; Öztürk, Tuba; Doğanlar, Sami; 04.03. Department of Molecular Biology and Genetics; 01. Izmir Institute of Technology; 04. Faculty of ScienceIn this study, It has been used two agropine strains (ATCC 15834 and ATCC 43057) of R. rhizogenes to compare hairy root induction in hypocotyl and cotyledon explants of eggplant. Transformation frequency was found to be the highest in hypocotyl explants (49.4%) infected with ATCC 15834. Thirty nine hairy root clones were selected based on their hairy root phenotype. The transgenic status of 15 of these clones was tested by PCR using rol gene primers. Hairy roots infected with ATCC 15834 were grown for 8 weeks in MS liquid medium. At the end of this growth period, the growth ratio of the hairy roots was measured. The root biomass was 6.52 g/DW for hypocotyl and 9.83 g/DW for cotyledon-derived clones. Biochemical analyses of clones were performed. Total phenolic contents were evaluated for hypocotyl and cotyledon-derived clones yielding 8.56 mg/g DW and 8.81 mg/g DW, respectively. The average total flavonoid content was 2.12 mg/g DW in hypocotyl-derived clones, and 1.90 mg/g DW in cotyledon-derived clones. On average, these amounts of both phenolic and flavonoid contents were significantly higher than those obtained for untransformed eggplant roots. Overall the results indicated that eggplant hairy roots are a useful source of secondary metabolites. In the future, the production of metabolites of interest such as steroidal glycoalkaloids or phenolics can be improved using elicitors in eggplant.Master Thesis Investigation of the Effect of Dr5-As Long Non-Coding Rna on Cell Proliferation(Izmir Institute of Technology, 2020) Akgül, Bünyamin; Akgül, Bünyamin; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyCell proliferation is the process of increasing cell number in a multicellular organism. In literature, there are numerous proteins and non-coding RNAs reported as regulators of cell proliferation, yet, many of others are waiting to be explored. Unravelling the mechanism behind the regulation of cell proliferation is crucial to develop new strategies for fighting numerous diseases such as cancer, immune diseases, or neurodegenerative diseases. Long non-coding RNAs (lncRNAs) are known to regulate various cellular processes. To determine which ones are related to cell proliferation and apoptosis in HeLa cells, a transcriptomics study was performed under cisplatin, doxorubicin, TNF-? and Anti-Fas treatments. DR5-AS is a novel lncRNA transcript selected from this transcriptomics study as a promising regulatory lncRNA candidate due to its overlap with DR5 protein-coding gene which is known to regulate apoptosis and proliferation. Several phenotypic characterization methods were performed to understand the function of DR5-AS lncRNA. These studies showed that DR5-AS knockdown causes a significant decrease in cell proliferation, an alteration in the normal HeLa cell morphology, a shift through S and G2/M phases in cell cycle profile, and significant accumulation of cells in the metaphase phase. A second transcriptomics study was performed with DR5-AS knockdown HeLa cells to uncover which pathways are responsible for these changes. The results suggest that DR5-AS lncRNA regulates expression of numerous key proteins in cell cycle regulation. This observation was confirmed by several qPCR experiments. In conclusion, this study provides the first evidence that DR5-AS lncRNA modulates cell cycle and proliferation in HeLa cells.