LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Doktora

Bu koleksiyon için kalıcı URI

http://hdl.handle.net/11527/19402

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Yazar "Doğanay Dinler, Gizem" ile LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Doktora'a göz atma
  • Öge
    Clinical and molecular collaboration studies: i)comparison of CNV by NGS&MLPA in hereditary breast and ovarian cancer ii)effective covid-19 sampling and storage
    (Graduate School, 2024-04-26) Yıldız, Jale ; Doğanay Dinler, Gizem ; 521182106 ; Molecular Biology-Genetics and Biotechnology
    HBOC syndrome, as the focus of the study in chapter one, represents a significant concern in cancer genetics. HBOC is primarily associated with mutations in the BRCA1 and BRCA2 genes. In this context, the study aims to enhance the detection of CNVs, crucial genetic alterations often observed in malignancies, including HBOC. The research assesses the efficacy of NGS for CNV detection, comparing it against the established Multiplex Ligation-Dependent Probe Amplification (MLPA) method. In this study, 1276 cases were examined using targeted NGS panels. Of these, 691 cases were further validated through MLPA, encompassing 61 calls in 58 NGS-CNV positive and 630 NGS-CNV negative cases. The findings revealed a considerable disparity: 46% of NGS-CNV positive calls were consistent with MLPA results, while 54% displayed discrepancies. Two cases identified as single nucleotide variations (SNVs) by NGS were found to be CNVs by MLPA. Interestingly, 2-3% of the cases showed MLPA-confirmed CNV regions in the BRCA1/2 genes. Notably, while the NGS-CNV algorithm had a high rate of false positives, it did not yield false negatives. The instances where NGS indicated negative but MLPA showed positive was due to SNVs at MLPA probe binding sites. The study concludes that NGS-CNV analysis shows promising diagnostic capabilities in detecting CNVs, specifically for negative CNV cases. However, false positives and the necessity for confirmation through alternative methods highlight the need for an integrated approach in clinical diagnostics. This ensures more accurate and reliable detection of CNVs, which is crucial for understanding and treating HBOC. Chapter Two focuses on PCR tests, which have been widely adopted as an essential diagnostic tool for identifying SARS-CoV-2 infections during the COVID-19 pandemic. However, proper handling and storage of collected samples are essential to confirm the precision and reliability of test results. Understanding the impact of sample storage conditions on PCR assay performance is critical to maintaining the effectiveness of test protocols. Moreover, this thesis investigates the effectiveness of using saliva samples as an alternative to oro-nasopharyngeal swabs for the detection of SARS-CoV-2, the virus responsible for COVID-19, through reverse transcription-polymerase chain reaction (RT-PCR) testing.
  • Öge
    Targeting bag-1S/C-raf interaction for therapeutic intervention in cancer
    (Graduate School, 2022-06-05) Tatlı, Özge ; Doğanay Dinler, Gizem ; 521152114 ; Molecular Biology-Genetics and Biotechnology
    In this context, this study aims to map the interaction surface of the complex formed by Bag-1 and C-Raf, which was accomplished through the use of both molecular and structural techniques. For this, the three dimensional structure and domain architecture of the small isoform of Bag-1 were first examined by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS), and the regions on Bag-1S that can accommodate small molecule binding were probed to assess its "druggability". To this end, Bag-1S was first purified from cell lysate using Ni-NTA affinity purification through the incorporated hexahistidine tag, and subsequently, the tag was cleaved with TEV protease. A subsequent Ni-NTA purification was carried out in a flow-through mode to collect Bag-1S separate from His-tagged TEV enzyme and impurities that contained neighboring histidines. The purified Bag-1S showed an apparent 33-kDa band in gel electrophoresis. Sample purity was estimated at over 90% using ImageJ analysis of SDS-PAGE gel. To monitor the deuteration level of the Bag-1S isoform, HDX-MS experiments with five time-points that ranged from 12 s to 24 h were carried out and revealed the identification of ~150 peptides of the Bag-1S with a sequence coverage of 98%. Using HDX-MS data, peptide-specific deuterium incorporation rates were projected onto the modeled structure of Bag-1S and deuterium uptake was analyzed on the Bag-1S full-length structure. BAG domain exhibited a more solvent-protected and stabilized structure compared to the UBL (ubiquitin-like) domain. While turn regions are more labile, the regions where the helical conditions exist remained unexchanged during the entire monitored time. Multiple interaction partners of the adapter protein Bag-1 engage specifically with the BAG domain. Interestingly, the interaction sites of these partners coincide with the regions that are most solvent-protected. The interaction site is supposed to be located in the solvent-protected region of the BAG domain, which is surrounded by charged and hydrophilic regions. This solvent-protected region in the BAG domain likely possesses an interaction region, revealing a potential "druggable" binding site. To further evaluate the binding stoichiometry of the Bag-1S with C-Raf, cross-linking assays were performed in the subsequent experiments. To accomplish this, C-Raf and Bag-1S proteins were affinity-purified, which was followed by the combination of purified proteins to form an in vitro complex. Covalent coupling of the formed complexes was then performed with a cross-linking agent, DSS (disuccinimidyl suberate). According to the results obtained after immunoblotting of cross-linked samples, Bag-1S and C-Raf formed a 2:2 stoichiometric complex, suggesting that Bag-1S might contribute to C-Raf activation by triggering its dimerization. After the Bag-1S/C-Raf interaction was affirmed and stoichiometrically tested, on-membrane in vitro binding experiments were conducted to selectively identify the interface of the complex. The purified C-Raf was immobilized on a PVDF membrane and incubated with purified Bag-1S in vitro. Bag-1S-bound peptides were recovered and analyzed by LC-MS/MS after the formed complex was subjected to limited tryptic digestion on the membrane. A 20-amino acid length peptide was identified as a plausible C-Raf interacting peptide in the BAG domain of Bag-1S. Further, an in silico docking study was also conducted using the protein structure of the kinase domain of C-Raf (PDB ID 5OMV) and the modeled full length protein structure of Bag-1. In some of the poses with the lowest docking energy score, K137, T140, Q144, K149, and L156 residues of Bag-1S were found to occupy the Bag-1/C-Raf binding site. This region coincides with the plausible "druggable" interaction site identified in HDX-MS and on-membrane in vitro binding experiments. Site-directed mutagenesis experiments were then carried out to confirm the identified binding interface and to evaluate if mutations in the determined peptide sequence affect the binding of Bag-1S/C-Raf or not. Upon mutagenesis, K149A and L156R substitutions significantly decreased the endogenous levels of p-C-Raf (S338) and p-MEK1/2 (217/221) in MCF-7 cells. Consistently, TAP-pull down experiments demonstrated that these substitutions impaired the interaction of Bag-1S with C-Raf, without affecting its HSP70 contact. They also led to a significant decrease in the survival of MCF-7 cells compared to wild-type Bag-1S. In addition, while these mutations did not affect the interaction of Bag-1S with its known direct interaction partners, Bcl-2 and HSP70, they resulted in the disruption of its interaction with the complexes involved in other regulatory cell survival pathways, including B-Raf, Beclin 1, and Akt. Subsequent in vitro binding experiments did not reveal a binary interaction of Bag-1S with either Beclin 1 or B-Raf, at least under our experimental conditions. Therefore, it has been hypothesized that the formation of a Bag-1/Beclin 1 or Bag-1/B-Raf complex might require the presence of C-Raf as a mediator. Further, Bag-1S interacting C-Raf region was identified by on-membrane in vitro binding experiment coupled with LC-MS/MS. Four different peptides derived from native Bag-1 and C-Raf sequences corresponding to the plausible interaction segments of the complex were designed and then synthesized by using solid-phase synthesis. The ability of the peptides to hamper the formation of a Bag-1S/C-Raf complex was tested in vitro. Of these peptides, Pep 3 that targets C-Raf binder region of Bag-1S significantly altered Bag-1S/C-Raf interaction. Pep 3 not only impeded the binary interaction of C-Raf with Bag-1S but also disrupted BAG-associated complexes of Bag-1 in TAP pull-down experiments. Inhibition of multiple Bag-1S interactions afforded by Pep 3 bolsters its potential to impair the prolonged survival of cancer cells. We therefore not only affirmed that this region on C-Raf is responsible for Bag-1 binding, but also discovered a novel peptide inhibitor targeting Bag-1S, which has the potential to be improved for cancer therapy.
  • Öge
    Variant detection in inflammatory diseases
    (Graduate School, 2024-04-30) Alkurt, Gizem ; Doğanay Dinler, Gizem ; 521162104 ; Molecular Biology-Genetics and Biotechnology
    Cancer remains a public health concern, with a significant number of reported cases worldwide in 2020. 9.3 million cases were diagnosed in men and around 8.8 million cases were diagnosed in women according to 2022 data. From a healthcare perspective, cancer is an influenced by factors such as genetics, environment and inflammation. Cancer is widely recognised as an inflammatory disease, as it is closely linked to an interaction of processes within the body. Inflammation plays a role in the initiation, progression and promotion of cancer. It involves the activation of cells, the release of cytokines and the creation of an inflammatory environment. All of these contribute significantly to stages of cancer development. Therefore it can be deduced that cancer is not caused by mutations, but is also strongly influenced by inflammations. Beyond the events, genetic predisposition adds another layer of complexity that affects not only how the disease manifests, but also how it is treated. The development of cancer is the result of an interplay between hereditary mutations. Tumors associated with cancer syndromes provide insights into genetic conditions. One such syndrome is Lynch syndrome, an inherited disorder that increases the risk of colorectal and endometrial cancers, among others. Mutations in genes such as BRCA1 and BRCA2 are associated with breast and ovarian cancer syndromes, also significantly increase the likelihood of developing these cancers. Our research has explored the link between genetics and cancer by focusing on genes beyond BRCA1 and BRCA2. We examined genes such as ATM, PTEN and CHEK2 that play a role in cancers. For example, ATM mutations increase the risk of breast cancer. PTEN mutations are associated with Cowden syndrome, while CHEK2 mutations increase the risk for both breast and colorectal cancer. We have also investigated Lynch syndrome associated mutations in genes involved in mismatch repair (MLH1, MSH2, MSH6 and PMS2). These mutations are known to make people more susceptible to cancer. Recent studies have highlighted the importance of inherited mutations in determining cancer susceptibility. Therefore, in the first part of this thesis, our aim was to analyze variations in 25 genes associated with cancer susceptibility in a population through a large collaborative effort. The study included 732 breast cancer patients, 189 colorectal cancer patients and 490 elderly cancer-free controls who were free of cancer. The analysis revealed 119 likely pathogenic variations in a total of 149 individuals. Interestingly, around 22.7% of these variations were newly discovered, indicating genetic risks. In addition, 16.0% of the variations were found recurrently among individuals suggesting shared genetic predispositions. In high-risk breast cancer patients, harmful variations in BRCA1 and BRCA2 genes accounted for 61.3% of all identified mutations. Other high-risk breast cancer genes like PALB2, TP53, STK11 and CDH1 also contributed to the mutation spectrum. We also found mutations in genes with penetrance such as CHEK2 and ATM. In cancer cases, Lynch syndrome-related genes accounted for 62.5% of all disease-causing mutations observed. In addition, MUTYH pathogenic variants were present in about 4.4%of high-risk colorectal cancer patients. Interestingly, 2.9% of the control group had a genetic predisposition to cancer based on their family background, but none of them actually had any mutations. It's worth noting that individuals with these mutations also had no family history of cancer. This is possible because of the interaction of proteins that ensure cell survival with these genes and provides preliminary data for a viable project in the future. In the second part of this thesis, we analysed the expression levels of microRNAs in wild type and BAG-1 knockout breast cancer cells. The BAG-1 protein is known for its ability to prevent cell death. It interacts with various other molecules in the cell. These interactions play a role in determining whether cancer cells survive or die. Studies have shown that changes in the levels of BAG-1 protein are associated with types of cancer. In addition, BAG-1 has shown promise as a marker for breast cancer, which is the most common type of cancer in women. Higher levels of BAG-1 are generally associated with increased growth, development and aggressiveness of breast cancer. The presence of miRNAs plays a crucial role in regulating gene expression. To identify these miRNAs, we performed high-throughput sequencing and also determined the target genes and molecular signaling pathways associated with these expressed miRNAs. Of the 25 miRNAs sequenced, we found that 11 were significantly upregulated and14 were downregulated. We experimentally validated 14 out of the 25 miRNAs, among which hsa-miR-429 stood out as a miRNA in BAG-1 KO MCF-7 cells due to its downregulation observed through bioinformatics analysis and other assays. Shifting focus to the global health crisis, the final phase of the thesis examined antibody levels in healthcare workers in the context of the COVID-19 pandemic. The COVID-19 pandemic has highlighted the relationship between infections and the body's immune response particularly inflammation. When someone contracts COVID-19 from the SARS-CoV-2 virus, they may experience levels of inflammation. This natural defense mechanism is the bodys' way of fighting off the virus. Understanding and effectively managing this response is critical to treating COVID-19. Researchers and health professionals are focusing on strategies to regulate how the immune system responds to the virus. The aim is to prevent inflammation that could lead to illness. During the pandemic, we conducted a study of antibody levels in healthcare workers related to COVID-19. As infections continue to increase, it is important to research aspects of the disease. One aspect is to access the extent to which healthcare workers have been exposed to SARS-CoV-2 by testing for immunoglobulin G (IgG) antibodies. This study took place in three hospitals to determine whether healthcare workers (HCWs) had contracted SARS-CoV-2. The main objective was to determine the percentage of HCWs who tested positive for antibodies indicating their exposure to the virus. To measure this we used an microparticle immunoassay that detects antibodies. By analysing the prevalence of IgG antibodies, this research provides insights into HCWs' exposure and immune response to SARS-CoV-2. This information is critical for making decisions on vaccination programs, the need for personal protective equipment (PPE) and implementation of infection control measures in healthcare settings such as hospitals.