Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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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 Role of Human Aprataxin Protein in P53-Related Cellular Processes in Breast Cells(01. Izmir Institute of Technology, 2021) Doğan, Hülya; Yalçın Özuysal, Özden; Yalçın Özuysal, Özden; Yalçın Özuysal, Özden; 01. Izmir Institute of Technology; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of ScienceAprataxin 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; Yalçın Özuysal, Özden; Yalçın Özuysal, Özden; Yalçın Özuysal, Özden; 01. Izmir Institute of Technology; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Sciencep53, 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.Master Thesis Determination of Therapeutic Effects of Multifunctional Antibody and Peptide Micelle-Based Nanocarriers on Breast Cancer Cells(01. Izmir Institute of Technology, 2021) Abdulhadi, Nusaibah Abdulsalam Abdulhad; Baran, Yusuf; Baran, Yusuf; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyBreast cancer is the most prevalent type of cancer and a major cause of death among women globally. Currently, many treatments are developed to reduce breast cancer death risks. Targeting therapy represents an advanced and successful approach. It provides targeting specific tumor sites by using specific ligands and modifying physicochemical characterization of nanocarriers to increase drug efficiency. In this study, we aim to determine and compare the therapeutic effects of doxorubicin (DOX)- loaded nanocarrier that was synthesized by using two properties a core cross-linked and pH sensitivity to increase drug stability and DOX releasing at the tumor site. The effects of DOX-loaded micelles (DM), HER2 targeting peptide (LTVSPWY)-conjugated-DOX-loaded micelles (DMP), and antibody (Herceptin) conjugated-DOX-loaded-micelles (DMA) on HER2 positive SKBR-3 cell line and HER2 negative MCF-10A normal epithelial breast cell line were determined by using cytotoxic, apoptotic, cytostatic, and genotoxic assays. According to the cytotoxic assay, the IC50 value of DM, DMA, and DMP were 0.71-, 0.49-, 0.34-µM, respectively. Additionally, the fluorescence image showed higher DOX uptake by SKBR-3 cells treated with DMP. According to the apoptotic assays, the mitochondrial membrane potential on SKBR-3 cells with treated DMP decreased as well as higher apoptosis and necrosis rate that was regulated by Bcl-2, Pro-Caspase-3, PARP1, Bax, Bak, and Bcl-xL. Besides, the application of DMP caused cell cycle arrest at the G2/M phase. Lastly, DNA damage was observed in response to DMP determined by comet assay. This study provides a novel and effective therapeutic option for breast cancer through using this nanocarrier system with targeting properties.Master Thesis Time Dependent Expression and Localization of Connexin 32: Implication in Epithelial To Mesenchymal Transition of Mammary Epithelial Mcf10a and Triple Negative Breast Cancer Mda Mb 231 Cells(01. Izmir Institute of Technology, 2020) Ünal, Yağmur Ceren; Meşe Özçivici, Gülistan; Meşe Özçivici, Gülistan; 01. Izmir Institute of Technology; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of ScienceBreast cancer is the most frequent and the second leading cause of cancer-related deaths among women worldwide. Epithelial to mesenchymal transition (EMT) is critical driving force in metastasis. Connexins as a basic subunit of gap junctions indicate critical roles in regulation of EMT. In addition to Cx26 and Cx43, Cx32 is associated with breast cancer and elevated levels of Cx32 has been reported in lymph node metastasis compared to primary breast cancer while the role of Cx32 in breast cancer is still elusive. Here we aimed to shed light on the effect of Cx32 on breast cancer. Our study suggested that Cx32 acquired mesenchymal morphology and decreased proliferation in MCF10A cells but not in MDA MB 231 cells. To further elucidate whether Cx32 indicate these changes through EMT, EMT markers were examined and subsequently it was revealed that Cx32 expression was strongly correlated with increased E-cadherin and Vimentin in MCF10A cells while decreased E-cadherin and Snail in MDA MB 231 cells. Importantly majority of Cx32 did not localize to the plasma membrane and indicated dynamic changes in a day dependent manner in both MCF10A and MDA MB 231 cells. Moreover, day dependent expression and localization of Cx32 revealed strong correlation with Zeb2 expression in MCF10A cells. In conclusion, Cx32 indicated differential effects in regulation of EMT between MCF10A and MDA MB 231 cells. It was the first time that the role of Cx32 on EMT was investigated in breast cancer and differential localization of Cx32 was identified.Master Thesis Investigating Oncogenic Role of Sema6d in Breast Cancer Cells(Izmir Institute of Technology, 2019) Günyüz, Zehra Elif; Yalçın Özuysal, Özden; Yalçın Özuysal, Özden; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyBreast cancer, the most commonly diagnosed cancer type and the leading cause of cancer-associated deaths, is the major health issue among women worldwide. In many cancer types, the expression of the semaphorins and their receptors such as plexins and neuropilins are dysregulated. SEMA6D is a member of class-6 family transmembrane semaphorin proteins and acts through Plexin-A1 receptor. It was previously shown that overexpression of SEMA6D in breast cancer cell line MCF-7 leads to a reduction in proliferation and an increase in migration. On the other hand, in the MDA-MB-231 breast cancer cell line, overexpression of SEMA6D had no significant effect on proliferation but enhanced migration. In this study, we aimed to analyze the effects of SEMA6D overexpression in normal breast cell line MCF10A and investigate the invasive behavior and transformation capacity of SEMA6D overexpressing breast cancer cell lines. We demonstrated that overexpression of SEMA6D leads to elevated proliferation, viability and migration in MCF10A cells, whereas it did not trigger their anchorage-independent growth. On the other hand, MDA-MB-231 and MCF7 cells stably expressing SEMA6D showed reduced colony formation in the soft-agar assay. Furthermore, the invasiveness of MDA-MB-231 cells was elevated with SEMA6D overexpression, whereas SEMA6D overexpression did not stimulate the invasiveness of MCF-7 cells through matrigel microenvironment, whereas slightly trigger invasion through bone microenvironment. In conclusion, SEMA6D overexpression has cell-specific effects on breast cancer. The exact role of SEMA6D in breast cancer development remains undefined and must be further investigated.Master Thesis Determination of Therapeutic Effects of Multifunctional Micelle-Based Nanocarriers on Breast Cancer Cells(Izmir Institute of Technology, 2019) Ulu, Gizem Tuğçe; Baran, Yusuf; Ulu, Gizem Tuğçe; Baran, Yusuf; 01.01. Units Affiliated to the Rectorate; 04.03. Department of Molecular Biology and Genetics; 01. Izmir Institute of Technology; 04. Faculty of ScienceBreast cancer is the most common and frequent cause of death among women composed to all types of cancer. Current treatment protocols do not provide complete cure or selective drug delivery while targeted therapy can provide an important avenue for successful treatment of breast cancer. In this study, therapeutic effects of drug-conjugated nanocarrier system with enhanced stability and double moiety pH-sensitivity on breast cancer (SKBR-3- HER-2- positive), normal breast epithelial (MCF-10A, HER-2-negative) and chronic myeloid leukemia (K562, HER-2-negative) cells were determined. With this approach, SKBR-3 cells were targeted by single nanocarriers having selectivity with unused peptide ligand (HER-2), stability with cross-linking of core moiety, and cleavage by two sites of pHeffect and drug release properties. After physicochemical characterization of micellebased nanocarriers, cytotoxic, apoptotic and cytostatic effects of doxorubicin conjugated micelles were determined. Doxorubicin conjugated micelles with HER-2 peptide (DOX-HER-2-NCs) had more cytotoxic effects on HER-2 positive cells. Additionally, intracellular amounts of doxorubicin is higher in SKBR-3 cells with applied DOX-HER-2-NCs as determined by fluorescence imaging. The apoptosis rate was increased on SKBR-3 at 50% cell growth inhibition (IC50) as determined by Annexin-V/Propidium iodide double staining. However, there was not any significant change in loss of mitochondrial membrane potential. Additionally, DOX-HER-2-NCs resulted in cell cycle arrest at G2/M-phase in response to IC50 value. Besides, protein level of Bcl-2 did not change while protein level of Bax and Caspase-3 were increased as determined by Western Blotting. This project provides novel and more effective treatment of breast cancer by using multifunctional properties of nanocarriers.Master Thesis Cyr61-Notch Interaction During Epithelial-To Transition, Migration and Invasion in Breast Cancer Cells(Izmir Institute of Technology, 2017) İlhan, Mustafa; Yalçın Özuysal, Özden; Yalçın Özuysal, Özden; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyNotch signaling is one of the major pathways involved in development and tumorigenesis. Activated Notch is correlated with increased migration, invasion and epithelial-to-mesenchymal-transition (EMT) in breast cancer. However, mechanism of Notch-mediated cancer progression is poorly understood. CYR61 is a secreted protein and its upregulation is also related to increased breast tumorigenesis and EMT. Microarray analyses revealed that CYR61 was differentially expressed in response to Notch activation in breast epithelial cells. We hypothesized that CYR61 is a downstream mediator of Notch during EMT, migration and invasion. To test whether Notch requires CYR61 during EMT, migration and invasion, two complementary approaches were followed: (i) CYR61 expression was silenced by using shRNA in MCF10A epithelial breast cell line in the presence of Notch activation, (ii) CYR61 was over-expressed in MDA-MB-231 cancer breast cell line in the absence of Notch activity. Then, expression of EMT markers was analyzed in mRNA and protein levels via RT-qPCR and immuno-blotting, respectively. Migration and invasion ability of cells were investigated by wound healing assay and a lab-on-a-chip-system, respectively. Here, it was shown that CYR61 inhibition decreased Notch-induced EMT, migration and invasion of MCF10A and CYR61 overexpression in the absence of Notch activity partially rescued Notch-mediated invasion in MDA-MB-231 cell lines. Our findings suggest that CYR61 may act in downstream of Notch and is regulated by Notch. When we consider importance of CYR61 in Notch-induced EMT and cancer progression, targeting CYR61 may hold promise to develop novel strategies for treatment of breast cancer in early stages.Master Thesis Role of Sema6d in Proliferation, Epithelial-Mesenchymal Transition and Migration of Breast Cancer Cell Lines(Izmir Institute of Technology, 2017) Şahi, Ece; Yalçın Özuysal, Özden; Yalçın Özuysal, Özden; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyBreast cancer is one of the most common cancer types around world and the second leading cause of cancer related deaths among women. Not the primary tumor but distant metastases are mainly the reason of deaths. For metastasis, the cells may go through epithelial-mesenchymal transition (EMT), and acquire migration and invasion abilities. SEMA6D is a transmembrane protein that belongs to a large semaphorin family. SEMA6D is involved in the migration of embryonic cardiac cells. Recently it was validated as an oncogene in osteosarcoma. Also, its oncogenic roles were investigated in gastric cancer and mesothelioma. According to in silico analysis of the Cancer Genome Atlas (TCGA), high SEMA6D expression level is associated with better survival of triple negative breast cancer patients. However, there is not any published study which investigates roles of SEMA6D in breast cancer yet, other than bioinformatic analysis. Therefore, we aimed to understand role of SEMA6D in proliferation, EMT and migration of breast cancer cells. We observed that overexpression of SEMA6D reduces proliferation but enhances migration in non-invasive breast cancer cell line MCF7. Thereby, SEMA6D may increase metastatic ability of MCF7 cells. Its metastatic ability was also supported by changes in EMT markers. On the other hand, proliferation of metastatic breast cancer cell line MDAMB231 was not significantly changed by overexpression of SEMA6D and migration ability was slightly reduced but mesenchymal markers tended to increase in SEMA6D overexpressing MDAMB231 cells. As a conclusion, SEMA6D tends to enhance proliferation, migration through EMT in MCF7 cell line whereas overexpression of SEMA6D did not demonstrate significant effect on metastatic MDA-MB-231 cell line. Therefore, we should separately evaluate role of SEMA6D in different breast cancer cell lines and further studies are required to understand role of SEMA6D in breast cancer.Master Thesis Comparison of Connnexin32 Expression and Function Between Mcf10a Normal Breast and Mda-Mb Breast Cancer Cell Lines(Izmir Institute of Technology, 2017) Meşe Özçivici, Gülistan; Meşe Özçivici, Gülistan; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyBreast cancer is one of the most prominent cancer-related deaths among females. Among many molecules, connexins have role in breast cancer. Gap junctions, formed from connexins (Cx), facilitate intercellular communication between adjacent cells. Different connexins were expressed during different stages of breast cancer. Cx32 was found both in normal pre-menopausal and tumor breast tissue samples. In lymph node metastases, elevated Cx32 level was observed compared to primary cancer. However, the role of Cx32 in breast cancer is not known but its elevation in lymph node metastasis may indicate its diverse functions in breast cancer. To verify this, in MCF10A and MDA-MB-231 cell lines, Cx32 was overexpressed. The protein localization was compared with immunostaining. In MDAMB- 231 cells, Cx32 localized in nucleus and cytoplasm, although in MDA-MB-231 Cx32-EGFP cells, Cx32 localized mostly in the cytoplasm. In MCF10A cells, Cx32 localized in nucleus, whereas Cx32 formed gap junctional plaques between MCF10A Cx32-EGFP cells. By Cx32 overexpression, gap junction coupling increased in MCF10A cells significantly, although it did not change in MDA-MB-231 cells. In both cells, hemichannel activity was not altered with Cx32 overexpression. The effects of Cx32 overexpression on cell viability demonstrated a significant decrease in MCF10A cells and an increasing trend in MDA-MB-231 cells. Furthermore, the percentage of G1 phase decreased, G2 and S phases increased in MDA-MB-231. However, Cx32 overexpression did not alter cell cycle profile of MCF10A significantly. Determination of the differential role of Cx32 in different stages of breast cancer may help to understand its diagnostic and/or therapeutic potential.