Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Development of Visual Analysis Interfaces for Large Biological Data and Characterization of Immunomodulatory Noncoding Rna Networks Cancer(01. Izmir Institute of Technology, 2023) Kuş, Muhammet Emre; Ekiz, Hüseyin Atakan; Ekiz, Hüseyin AtakanThese days we are collecting data in higher and higher dimensions, processing it, and developing tools that have strong descriptive and predictive powers. Especially in the field of cancer, the processing of data collected from patients has substantial potential in terms of discovering new biomarkers, developing personalized treatment methods, and better prognosticators. However, there are significant difficulties in utilizing and analyzing high-dimensional data. A good level of coding skills is required to bring the data together and apply different analysis methods. With the visual interfaces created in this study, we offer the opportunity to examine and analyze the high-dimensional data of thousands of cancer patients, which are open to the public through The Cancer Genome Atlas initiative, especially for bench scientists who has no prior coding expertise. The Cancer Genome Explorer, shortly TCGEx, is a robust bioinformatic tool that we developed to facilitate high-throughput cancer data analysis through several sophisticated algorithms. With special features like subset-specific analysis and comparative analysis by using multiple cancer data, TCGEx can contribute to the literature by accelerating the studies, especially in hypothesis-driven research. This study also describes a use-case scenario that demonstrates how hypothesis-driven research can be performed using TCGExplorer for melanoma. In melanoma, elucidating the interactions between the tumor and the immune system at the miRNA level is crucial for developing new therapeutics. In this study, we characterize the properties of potential therapeutic targets that act on tumor and immune cells, which we have identified using various statistical analysis methods including machine learning, dimensionality reduction, and survival modeling using the TCGEx portal.Master Thesis Optimization of a Liver Organ on Chip System for the Investigation of Breast Cancer Cell Invasion(01. Izmir Institute of Technology, 2023) Tosunoğlu, Perge Bilgesu; Yalçın Özuysal, ÖzdenThere are many challenges in creating an accurate and physiologically relevant three-dimensional (3D) model of the liver such as finding the suitable liver extracellular matrix (ECM) components and cell types for the development of cell-laden liver-on-a-chip systems. In recent years, precision tissue slice-based methods are used to reduce the problems caused by the cell-laden liver-on-a-chip system complexity. However, these methods require sophisticated tools which are not easily accessible. This study focuses on the development of a 3D liver model using simple and cost-effective methods. To achieve this, we aimed to optimize liver tissue size, maintenance and culture medium, scaffold gel, and viability assay. The needle method was found to be the easiest, most efficient, and cheap method for tissue processing as the equipment used was easily accessible and evaded enzymatic steps. Moreover, consistent samples that were all similar and in the desired size were easy to obtain. In addition to these, functionality and viability were analysed for 72 hours to assess the physiological state of the liver after tissue processing. Based on these findings a novel liver-on-a-chip system was successfully developed and as the next step, the invasion of the MDA-MB-231 breast cancer cell line towards the liver was investigated with and without the presence of C-X-C chemokine receptor type 4 (CXCR4) antagonists AMD3100 and AMD3465. In conclusion, this study demonstrated the development of a novel 3D model for the liver and provided a platform for studying breast cancer invasion with its potential implications for cancer therapy research.Master Thesis Elucidation of Lysosomal Cathepsin a in the Regulation of Autophagy(01. Izmir Institute of Technology, 2023) Yanbul, Selman; Seyrantepe, VolkanLysosomal Cathepsin A (CathA) is a multifunctional enzyme with independent catalytic and protective functions. It has a serine carboxypeptidase activity in acidic pH conditions for the degradation of short bioactive peptides that are vasoactive peptides including endothelin-1, angiotensin-I, bradykinin and neuropeptides including oxytocin and substance P. Lysosomal CathA enzyme also forms a lysosomal multienzyme complex (LMC) with α-neuraminidase (Neu1) and ß-Galactosidase (ß-Gal) enzymes to protect them from hydrolytic degradation in lysosomes and due to its protective function. Genetic defects in the lysosomal CathA enzyme causes a rare lysosomal storage disorder, Galactosialidosis (OMIM #256540), with secondary deficiencies of Neu1 and ß-Gal enzymes. Catalytically inactive Cathepsin A knock-in mouse model, CathAS190A has point mutation in the active catalytic site which serine was replace with alanine amino acid. Accumulation of short bioactive peptides has been reported in previous studies in different tissues of the CathAS190A mouse model. In this thesis study, investigation the role of the lysosomal CathA enzyme in the regulation of autophagic flux in neuroglia and fibroblast cell lines derived from CathAS190A mice was aimed. For this aim; RT-PCR, Western Blot and Immunocytochemical analyses were for performed for autophagy markers. Thesis study results have exhibited that catalytically deficient CathA causes the impairment in autophagic machinery with secondary accumulation of autophagic substrates and alterations in the expression of the autophagy marker genes. Accumulation of the short bioactive peptides due to the catalytically inactive CathA enzyme may be related to to impaired autophagic flux. Autophagy-inducing Rapamycin and Starvation treatment conditions may restore the impaired autophagic flux due to catalytically inactive CathA enzyme with the clearance of accumulation of secondary autophagic substrates.Master Thesis Examination of Therapeutic Potential of Luteolin on Acute Lymphoblastic Leukemia Cells and Changes in Macromolecules(01. Izmir Institute of Technology, 2023) Çetinkaya, Melisa; Baran, YusufAcute lymphoblastic leukemia (ALL) is a genetic disease that arises from the various recurrent genetic alterations blocking the differentiation of the precursor B-and T-cells, resulting in the aberrant proliferation and survival of immature lymphoblasts within the peripheral blood and bone marrow. T-ALL is an aggressive type of ALL, and the current treatment strategies, including the high-intensity combination chemotherapy, result in different side effects which are difficult to accept or ultimately lead to the death of patients as substantial toxicity of those chemotherapeutics is known to healthy cells alongside with the cancer cells. Therefore, there is a need to identify nontoxic, costeffective, potent, and readily available treatment options for T-ALL patients. One alternative option is the flavonoids in cancer therapeutics, which are secondary metabolites of plants mainly responsible for plants' colors and flower aromas. Luteolin is an extensively researched member of the flavonoids with anticancer properties shown in various cancer types, except for the T-ALL. This study demonstrated Luteolin's time- and dose-dependent antiproliferative, cytostatic, and apoptotic effects on T-ALL cells for the first time in the literature. Also, the macromolecular changes caused in response to Luteolin treatment in T-ALL cells were examined for the first time. As a consequence, it was found that Luteolin had antiproliferative, apoptotic, and cytostatic effects on T-ALL cells, suggesting its therapeutic potential and was demonstrated to cause an increase in the lipid-to-protein ratio and the hydrocarbon chain length of the lipid acyl chains in a dose-dependent manner on T-ALL cells.Master Thesis Examination of Stable Intronic Sequence Rna Profile Under Apoptotic Conditions(Izmir Institute of Technology, 2022) Kara, Merve; Akgül, BünyaminApoptosis 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) Atbinek, Melis; Akgül, BünyaminThe 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) Kaçar, Vahide İlayda; Akgül, BünyaminCell 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) Yöndem, Eyüp; Pesen Okvur, Devrim; Pesen Okvur, DevrimBreast 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 Role of Human Aprataxin Protein in P53-Related Cellular Processes in Breast Cells(01. Izmir Institute of Technology, 2021) Doğan, Hülya; Doğan, Hülya; Yalçın Özuysal, Özden; Yalçın Özuysal, ÖzdenAprataxin encoded by APTX, which is the human homolog of yeast HNT3, reverses adenylation damages emerged from abortive DNA ligation during ribonucleotide and base excision repair. Thus, it corrects AMP-modified nucleic acid termini and protects genome integrity as a DNA ligase "proofreader". Role of HNT3, which is a candidate p53-related gene, against DNA oxidative and alkylating damage indicates its antioxidant importance. Besides, previous studies demonstrated that absence of Aprataxin gives rise to ROS generation and oxidative stress in addition to mitochondrial dysfunction. Also, role of Aprataxin in drug and radiotherapy sensitivity was shown in many cancer. Since conformation of cysteine residues in p53 DNA-binding domain can be modified by oxidizing environment, functionality can be influenced by defective APTX. Although p53-Aprataxin interaction has been shown by co-immunoprecipitation, effects of APTX on p53 pathway were not studied. Aim of this study is to investigate Aprataxin-driven changes in p53-regulated processes in p53 wild-type cells through. According to results, Aprataxin overexpression leads to cell cycle arrest in low stress levels. However, it triggers cell death against induced stress in MCF10A cells. Moreover, apoptotic assay on MCF10A APTX Crispr cells indicated elevated level of basal cell death. Also, expression analysis of p53 targets in APTX knockdown MCF7 cells revealed that extrinsic apoptosis pathway might be induced. Consequently, these results help us to gain insight into how Aprataxin affects activity of p53 pathway. Further investigation providing stress accumulation based assays and protein level analysis is needed to figure out whether resulting changes are p53-dependent or not.Master Thesis Investigating the Effect of Human Sacm1l Gene in the P53 Wild Type Breast Epithelial Mcf10a and Breast Cancer Mcf7 Cells(01. Izmir Institute of Technology, 2021) Efe, Eda; Efe, Eda; Yalçın Özuysal, Özden; Yalçın Özuysal, Özdenp53, tumor suppressor protein, plays role in the regulation of many cellular processes. Thus, p53 activity is controlled by a series of mechanisms, one of which is a redox reaction. However, redox regulation of p53 is not well defined in the literature. As a candidate of antioxidant, Sac1 gene mutation resulted in decreased levels of human p53 protein in transformed yeast, but the human homolog of Sac1 (SACM1L) has not been studied yet. SACM1L is known to function as a phosphoinositide phosphatase, hydrolyzes PI4P in the Golgi and ER. Previous studies demonstrated SACM1L depletion in HeLa cells led to decreased viability and arrest at the G2/M phase. However, no data were found on the association between the SACM1L and either directly p53 or p53 mediated cellular processes. We aimed to investigate the role of SACM1L in p53 controlled cellular processes like cell cycle and apoptosis in p53 wild type (wt) breast epithelial cells MCF10A and breast cancer cells MCF7 in the presence or absence of SACM1L gene. We demonstrated that SACM1L knockout MCF7 cells were arrested in the G1 phase, and number of proliferating cells was reduced, whereas overexpression of SACM1L did not change the proliferation, and cell cycle. Further, the rate of apoptosis was increased in SACM1L overexpressing and knockout MCF10A and MCF7 cells, supported by the findings of transcriptional analysis for p53 target genes. In conclusion, the greatest effect of SACM1L was observed in the apoptosis, but the underlying mechanisms are still unclear and must be further studied.