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Gliom oluşumunun erken aşamalarında IDH1 mutasyonlarının epigenomda yol açtığı değişimlerin karakterizasyonu
Isocitrate dehydrogenase (IDH1/2) mutations, which have been shown to develop before other mutations known to play a role in glioma development, are believed to trigger gliomagenesis. Studies to date have mostly used heterogeneous tumor samples or cells with oncogenic mutations when investigating the early stages of glioma development. This limits our knowledge of how gliomagenesis is triggered by IDH1/2 mutations. Moreover, examination of epigenomic changes caused by IDH1/2 mutations has not gone beyond classical histone methylation analyzes based on methylation arrays, RNA-seq and expression microarrays, and ChIP-seq. The aim of this study was to understand the permanent changes caused by the IDH1-R132H mutation, which is the earliest seen in gliomagenesis, in the epigenome of neural progenitor cells (NPCs) cultured in a 3D environment. For this, firstly, conditioned medium with Immortalized Human Astrocytes (IHAs) expressing doxycycline-inducible IDH1 (Mutant and wild type) was obtained. These media samples were then delivered for specified periods of time to NPCs differentiated from human pluripotent stem cells (hiPSC) in an alginate-based 3D matrix. RT-qPCR and RNA-seq analyzes were performed by isolating RNA from cells taken at three time points (day 0, day 14, day 17) from NPCs exposed to different media. Key genes reported to be altered in IDH1 mutant gliomas were checked by RT-qPCR. Then, epigenetic marks in histones and DNA were analyzed by ChIP-qPCR and MeDIP-qPCR. In selecting the loci for these analyses, we particularly prioritized the aim of furthering the mechanistic information regarding the IDH1-MYC relationship in the risk of glioma located at 8q24, which we brought to the literature for the first time in our group's previous studies, and directly demonstrating it in an in vitro model. Our findings indicate that one of the direct targets of the oncogenic changes that occur in the IDH1-MYC axis, which we think are initiators of gliomagenesis, is the ID1 (inhibitor of differentiation) gene, and that as a result of the induction of this gene, the cells take on a less differentiated phenotype.
Omics veri entegrasyon yaklaşımlarını kullanarak yüksek dereceli gliomaya (HGG) yönelik yeni hedefli tedavilerin geliştirilmesi
Gliomas are the brain tumors that develop in glial cells and present severe challenges based on intertumoral heterogeneity associated with different subtypes, further leading to poor prognosis and outcomes for patients. This study was conducted to utilize the transcriptomics and DNA methylation datasets available to researchers to arrive at conclusions that can be utilized for either screening novel targets or use already established drugs that can target the specific gene signatures associated with low grade gliomas (LGGs), which develop into high grade gliomas (HGGs) or Glioblastoma multiforme (GBM). We identified co-expression modules and their associated pathways for specific subtypes of LGG IDH mut pTERT-, IDH mut pTERT+, IDH wt pTERT-, and IDH wt pTERT+. We constructed co-expression modules based on these subtypes and found common and different enriched pathways as synapse pathways and immune-related pathways, respectively. We further explored the differentially expressed genes (DEGs) and found a gene signature of upregulated GNG12 and downregulated PLCB1, GRIA2, GABRA3, and GNAL after mapping DEGs on our co-expression modules of interest. This gene signature was included in our drug-gene interaction analysis, leading us to 4 drugs (Vemurafenib, Vanadium Pentoxide, Imatinib, and Cisplatin,) that can target 4 out of 5 genes. Therefore, we recommend exploring the synergistic effects of the combination of these drugs against low and high grade gliomas. We also integrated transcriptomics and DNA methylation data to develop networks including epigenetic factors that can be targeted in a subtype specific manner. Our analysis revealed that PRMT5 can be used as a target irrespective of the LGG subtype and WEE1 is a specific target for IDH wt regardless of pTERT status. The specific chemical inhibitors of these targets being available could facilitate translation of our findings into preclinical settings. Keywords: Glioblastoma, Lower Grade Glioma, Transcriptomics, Co-expression Analysis, DNA Methylation, Bioinformatics Pipeline, Drug-Gene Interaction
Nörogelişimsel hastalıklarla ilişkilendirilen aday genlerin işlevsel validasyonu
This study aims to elucidate genetic diseases through the validation and characterization of novel mutations identified in a group of children born of consanguineous marriages. Three such pediatric patients were selected for further investigation. They were all clinically suspected of genetic diseases but were unable to be diagnosed with known genetic mutations. Clinical findings included microcephaly, motor-mental retardation, neuro-developmental disorders, and epilepsy. Whole exome sequencing identified previously unreported candidate mutations: nucleotide deletions in WDR62 and ATG9B genes resulting in frameshift and premature termination of translation, and a missense mutation in VARS1 gene resulting in single amino acid alteration. In the literature, WDR62 mutations were extensively reported as causative factor for microcephaly. Therefore, the WDR62 mutation was only validated by Sanger sequencing. The effects of the VARS1 mutation were studied by structural modeling and shown to have destabilizing effects on the VARS1 protein. The focus of this thesis was the ATG9B mutation. ATG9B has not been associated with any genetic disease and its function is poorly characterized in literature. The eleven-nucleotide deletion identified in the ATG9B gene causes a frameshift resulting in protein truncation. We characterized WT and mutant ATG9B by ectopic expression in cells and developed several mouse models for in vivo studies. We showed that truncated protein is not stable when expressed in cells. Using in vivo models, we demonstrated that Atg9b is expressed in the placenta and testis, but not essential for viability or fertility. We didn't observe any major histological differences between WT and mutant placenta. On the other hand, behavioral assays revealed reduced fear memory in the homozygous knockin animals. We additionally developed knockout and FLAG knockin mouse models of ATG9B, and generated antisera to study its functions in vivo and in vitro.
Farklı taksonomik gruplarda kanserin moleküler mekanizmalarının hesaplamalı biyoloji uygulamalarıyla araştırılması
Investigating the fundamental mechanisms of tumorigenesis is considered crucial in developing alternative therapies and new diagnostic strategies. These mechanisms, which were investigated in this study, could provide an indispensable guide to identifying alternative treatment methods in cancer research. Data mining methods allow the investigation of large-scale biological data. To this end, cross-disciplinary in silico systems biology methods, by combining comparative genomics, differentially expressed gene profiles, biological networks and biological text mining, were employed to investigate the molecular mechanisms of carcinogenesis in diverse taxonomic groups. For this purpose, the following steps were carried out: (1) Identification of novel genes/proteins involved in different aspects of cancer through mining publicly accessible primary genomic databases, (2) Conduct of comprehensive in silico phylogenetic analyses of genes/proteins, covering the major taxonomic divisions, (3) Identification of patterns of differentially expressed genes in different types of cancers through processing high-throughput data derived from public repositories, (4) Construction of functional networks and subsequent identification of gene/protein modules by employing biological network inference techniques, (5) Natural language processing using artificial intelligence techniques for the detection of semantic relations. The findings of this study could have potential application in the clinical setting. In this regard, the corresponding genes can be exploited as diagnostic or prognostic tumor markers, and they could also be utilized in cancer therapeutic decision-making or in future experimental studies for the rational design of targeted cancer drugs.
Investigation of the role of wnt/β-catenin signaling in development of Alzheimer's disease in a zebrafish model of amyloid-β toxicity
The Wnt/β-catenin signal transduction pathway is evolutionarily conserved and plays a key role in the regulation of development in the embryonic period, synapse formation in adulthood, apoptosis, and adipogenesis. Due to its pleiotropic and basic function, an abnormal activation or disruption in this signaling pathway may be associated with many diseases such as Alzheimer's, cancer, diabetes, and Parkinson's. Alzheimer's disease (AD) is a neurological disease characterized by dementia and loss of synaptic functions and associated with aging. Alzheimer's disease is characterized by three distinctive pathologies: (1) accumulation of extracellular beta-amyloid plaques, (2) accumulation of intracellular Tau clusters, and (3) diffuse cell death seen in advanced Alzheimer's disease. However, synapse losses that can be seen in the early course of the disease represent the most common pathological link of the disease and are the most important cause of cognitive losses. Current animal models suggest that this synapse degeneration and loss is triggered by accumulating Aβ oligomers. Intercellular accumulation of this oligomer, which is very important for synaptic stabilization and function, causes deregulation of Wnt signaling pathways. This deregulation of the Wnt signaling pathway triggers synapse degeneration and leads to damping in synapse functions. This disruption in the signaling pathway is a trigger for the further progression of AD. In the scope of the thesis, Alzheimer's disease, which can be triggered by the Aβ42 peptide, was modeled using adult transgenic (6XTCF) zebrafish. Zebrafish have genes orthologous to some human genes thought to play an important role in Alzheimer's disease. Throughout the thesis, Aβ42 was injected into the brains of adult zebrafish using the CVMI technique, and a model of Alzheimer's disease was created. The regenerative response of zebrafish to toxicity was analyzed by qPCR and confocal microscopy. The change in AD pathogenesis was investigated in zebrafish treated with IWR-1 drug on days of high regeneration. As a result, more Aβ accumulation and cell loss were observed in zebrafish treated with IWR-1. Key Words: WNT/β-CATENIN, Zebrafish, Alzheimer's Disease
Kuzeybatı ve Orta Anadolu'daki yarasaların dışkı örneklerinde virom içeriğinin belirlenmesi
Bats carry various virus families that infect vertebrates, invertebrates, plant, fungi, or bacteria. Viruses may be shed via several routes and transmitted to humans. In current literature, there is insufficient information about bat-related viruses in Türkiye. In this study, feces of bats were collected from three different locations in the Northwest and Central Anatolia of Türkiye and investigated via metagenomics approach to profile viruses shed in feces. Then, virome content of bats were identified using three different bioinformatics approaches in the analysis of sequencing data. According to our results, different virus families could be detected by different bioinformatics approach and the virome content of bat feces was affected by bat species in terms of dietary choice, microbiome, and interaction with other animals. In Cilingoz Cave, virus families that infect bacteria, vertebrates, invertebrates, plant, and algae were detected. In Sefer Yitigi Cave, virus families that infect, bacteria, invertebrates, plant, and fungi were detected. In Sarihidir Cave, virus families that infect bacteria, vertebrates, invertebrates, archaea, and fungi were detected. While the highest viral diversity was detected in Sefer Yitigi Cave by BLAST before and after de novo assembly, the highest viral diversity was detected in Sarihidir Tunnel by Kraken 2. Overall, bacteriophages were the most dominant virus families by each bioinformatics approach for all regions. The sequence reads that hit virus families infecting vertebrates were investigated further. It was found that none of them carry risk for zoonosis. Such studies are crucial for monitoring emerging viruses.
İnsan indüklenmiş pluripotent kök hücre kaynaklı lakrimal bez organoidlerinin metabolomik analizi
The lacrimal gland (LG), commonly known as the tear gland, regulates the ocular surface's homeostasis by secreting proteins and electrolytes that form the tear film's aqueous layer. It produces lacrimal fluid, which lubricates and protects the eye as the major exocrine gland of the eye. Loss of function brought on by LG damage has a negative impact on eye health and visual quality. Studies on human LG formation and development are few, despite the fact that a large percentage of the human population suffers from eye diseases caused by functional disorders in the LG, such as dry eye disease. The LG's structure and morphology at the molecular level are not well known in the literature. In this study, human induced pluripotent stem cells (hiPSCs) were differentiated into cells with characteristics of LG in vitro by recapitulating functional and mature LG morphology at different time points. Differentiation was carried out according to the protocol developed by our lab in previous studies. Additionally, LG's reaction was monitored upon forskolin stimulation to understand secretion functionality better. In this context, different maturation levels and secretion functionality of LG's upon forskolin stimulation were examined by untargeted gas chromatography-mass spectrometry (GC-MS) metabolomics. The results suggest that the metabolome of LG organoids changed significantly at different maturation levels and upon stimulation with different small molecules. In addition, different –omic analyzes can be performed on the produced LG organoids and can be integrated with other -omic analyzes to investigate LG organoids at the multi-omics level.
Mikroglial hücrelerde dietil maleat'in NLRP3 inflamazom aktivasyonu üzerine etkilerinin araştirilmasi
Microglial cells are tissue-resident macrophages of the Central Nervous System (CNS) (Dubbelaar, Kracht et al. 2018). In case of an injury in the brain, microglia act through their phagocytic function and eliminate invading microbes and other substances that harm the CNS. Also, they are responsible for secretion of cytokines assisting the immune response within the CNS (Dubbelaar, Kracht et al. 2018). Inflammasome activation initiates inflammatory responses leading to secretion of IL-1β and IL-18 cytokines and activation of caspase-1, and may also lead to pyroptotic cell death (Yang, Wang et al. 2019). For this reason, inflammasome activation should be prevented under such conditions. Chemicals with anti-inflammatory effects are appropriate candidates to inhibit inflammasome activation. Diethyl Maleate (DEM) is a Nrf2 activator and correlatively, DEM has been estimated to inhibit secretion of inflammatory cytokines and possible cell death. It is hypothesized that DEM represses LPS+ATP induced NLRP3 inflammasome activation caused cell death of N9 cells. In this thesis, effects of DEM on NLRP3 inflammasome activation in N9 microglia was investigated. NLRP3 inflammasome activation via LPS+ATP was performed. N9 cells were treated via DEM in order to determine the effect on inflammasome activation. The cytotoxicity of DEM in regarding conditions was determined via LDH (Lactate Dehydrogenase) and PI visualization. Afterwards, inflammasome markers were examined at mRNA level via PCR, at protein level via Western Blotting and ELISA. It was determined that DEM pretreatment represses inflammatory cytokine secretion and cell death arising from NLRP3 inflammasome activation. Keywords: Diethyl Maleate, microglia, N9, inflammasome, NLRP3, pyroptosis, LPS, ATP
Alzheimer hastalığının tanısında ve yayılımında nöron kökenli ekzozomlar
Alzheimer's Disease (AD) is the most widespread neurodegenerative disease seen in elderly population over the world. Since the lack of definitive diagnosis, treatment, and also clear understanding of its pathogenesis; AD is very interesting and challenging topic to search. Exosomes are the smallest class of extracellular vesicles that are nano-sized and secreted from every cell type including central nervous system cells. Exosomes contain nucleic acids, proteins, pathogens, etc. as cargo molecules and they carry them to recipient cells in their environment. Since they have exosome-specific proteins in their membranes, exosomes can be separated from body fluids and disease-specific molecular alterations in exosomes can be determined. In this thesis, neuron-derived exosomes (NDE) were isolated from plasma samples of AD patients and healthy individuals. Dysregulated miRNAs in those exosomes were determined by small-RNA sequencing. 10 miRNAs were determined as dysregulated and validation of the most dysregulated miRNA, hsa-let-7e-5p, was performed by qRT-PCR to demonstrate its biomarker potential in NDEs of AD patients. Also, human microglial cells were treated with NDEs of patients which contain high level of upregulated hsa-let-7e-5p and miR-96-5p in order to examine their role in cell-to-cell interaction by affecting cytokine levels at protein level and gene expression of IL-1β, IL-6, TNF-α were determined via ELISA and qRT-PCR methods. Results showed that certain miRNAs were altered in NDEs of AD patients and validation of them may confirm their biomarker potential. Also, exosomal miRNAs may be involved in cell-to-cell interactions.
Yumurtalık tümör mikroçevresindeki hücrelerde tümör oluşumu ve gelişiminin eksozom aracılığıyla uyarılması
Ovarian cancer, seventh most extensive reason of cancer demise in women, has a low incidence of 3% among gynecological diseases. It is substantial to explain the carcinogenesis mechanism of ovarian cancer because of difficulties in diagnosis at early phase, prognosis at late stages, and low survival rate after surgery and chemotherapy. The purpose of this research is to demonstrate the induction of tumorigenesis in tumor microenvironment cells after uptake of oncogenic signal carrying ovarian cancer-related exosomes. Exosomes were isolated from cisplatin-resistant A2780 cells by ultrafiltration and differential centrifugation. Primary ovarian cancer (A2780), ovarian surface epithelial (OSE) and mesothelial (MeT-5A) cells were used as recipients. Isolated exosomes were characterized by western blotting and particle size analysis. Internalization of PKH26-labeled exosomes into the recipient cells was examined by confocal microscopy. Uptake mechanisms of exosomes were detected by using inhibitors via flow cytometry. The effects on the differentiation of EMT, ROS production, invasion, migration and proliferation rates were examined following exosome uptake. Isolated exosomes ranging from 30-150 nm in diameter, contained exosome marker proteins Alix and Tsg101, except ER marker; calnexin. PKH26 labeled-exosomes were internalized by recipient cells in 6 hours by different endocytic mechanisms. ROS production, invasion and migration rates increased in A2780 and OSE cells except in MeT-5A cells. EMT and proliferation were induced following exosome uptake. In conclusion, the carcinogenesis mechanism of recipient cells in the primary and secondary tumor microenvironment of ovarian cancer was induced after the uptake of ovarian cancer-released exosomes.
Gediz deltasındaki kuş kaynaklı influenza a virüslerinin moleküler karakterizasyonu ve izolasyonu
Due to its location, Turkey is visited by thousands of birds from different origins annually. Coexistence of these birds in wetlands contributes to Influenza A virus (IAV) evolution. Gediz Delta, provides a great opportunity for avian IAVs to evolve and adapt to mammals, as it hosts many different aquatic bird and mammalian species together. Thus, this thesis study aims to determine the prevalence of avian origin low pathogenic IAVs in Gediz Delta and to investigate the molecular markers in their genomes that may provide ability for interspecies transmission and to cause an outbreak. Additionally, phylogenetic analyses were performed to identify the origins of antigenic segments. To achieve this aim, 500 fecal samples of wild aquatic birds were collected in Izmir Bird Paradise/Gediz Delta. The samples were examined to detect IAV prevalence in the Delta via molecular methods. IAV-positive samples were inoculated into embryonated chicken eggs for virus isolation. Whole genome analyses were performed to monitor virus evolution. According to the results of this study, the prevalence of IAVs in Izmir Bird Paradise/Gediz Delta was 31.2% based on matrix gene amplification. In this thesis study, four viruses were isolated and the subtype of one virus was determined as H6N2. Whole genome analyses of H6N2 virus revealed 30 molecular markers which confers potential for zoonosis and adaptation to poultry. Phylogenetic analyses indicate that antigenic genes were originated from Asia. The results of this study indicate IAVs circulating in the Delta carry molecular markers which pose a threat for public and veterinary health.
FAM134B'nin biyolojik işlevleri ve kanserle ilişkisi
Hepatocellular Carcinoma (HCC) is a major health problem worldwide and cause thousands of deaths each year. HCC development is mostly stemmed from the cirrhotic liver where hepatocytes escape cellular senescence to gain immortality. Our group has previously defined the expressional decrease of FAM134B as a hallmark for immortalization. Later, FAM134B was shown to be a receptor that mediates degradation of Endoplasmic Reticulum fragments through the autophagosome. However, how these properties are associated with development of cancer remains largely unknown. Therefore, we aimed to investigate the functional characterization of FAM134B in the context of physiology and cancer. Using in vitro and in vivo models, expressional changes of FAM134B variants were tested in response to various cellular stresses such as fasting, ER-stress, and inhibition of proteasome machinery. In the context of cancer, the expression of FAM134B variants was screened using databases in different cancer types. The effect of FAM134B variants on HCC tumor formation was evaluated by in vivo xenograft model. The findings have shown that there are at least two transcriptional isoforms in human, and bovine, and at least six isoforms in mouse. Starvation stimulated short FAM134B-2 expression in the skeletal and cardiac muscles as well as in the liver of Fam134b+/+ mice. Fasted Fam134b-/- mice yielded significant changes in body weight, serum biochemistry and liver transcriptome. Ectopic expression of FAM134B variants altered ER-stress response in vitro and suppressed xenograft tumor growth in vivo. Finally, short FAM134B variant was found to be significantly changed variant during the development of different tumors, including HCC. In conclusion, the short and long FAM134B isoforms were shown mediate different functions in physiology and cancer, play an important role in energy metabolism; and lastly, the short FAM134B-2 variant is the most altered in various cancers.
Türkiye'de akraba evliliğine bağlı nörogenetik hastalıkların temelinde yatan mitokondriyal etiyolojinin sıklığı
Neurogenetic diseases are conditions that affect the brain, spinal cord, nerves and muscles. Neurogenetic diseases can be categorized as single gene disorders with autosomal, X-linked or mitochondrial inheritance, polygenic disorders, multifactorial disorders and disorders caused by inherited or de novo chromosomal abnormalities. Due to complicated clinical features and genetic heterogeneity of them and limitations in traditional diagnostic approaches, neurogenetic diseases are difficult to diagnose. Advances in the next-generation sequencing (NGS) technologies facilitate the molecular diagnosis and the discovery of novel disease genes and variants. Today, high-throughput, cost and time-effective whole exome sequencing (WES) is mostly preferable strategy used in diagnostic process of genetic diseases including neurogenetic diseases. In the frame of this project, 191 consanguineous families with childhood neurogenetic disorders were recruited and deeply phenotyped. To date, 138 of them underwent WES and causative genetic defect for 79 of them were identified with diagnostic yield of 57%. Among identified genes, 28 of them are known variants and 32 of them are novel candidate variants in known disease genes which are already associated with neurogenetic diseases and 15 of them are novel candidate genes. Importantly, mitochondrial function associated genes were higher than expected. By obtaining a diagnostic yield of 57% in such a large cohort, we showed the diagnostic utility of WES in neurogenetic diseases, identified novel candidate disease genes and variants by explaining genotype-phenotype relations in detail and provided genetic counseling and disease-specific therapies for some of the families. Keywords: NGS, WES, neurogenetic diseases, mitochondrial, consanguineous families
Mitotik kinaz fonksiyonlarinin kimyasal genetik yaklaşimlarla i̇ncelenmesi
Mitosis is the shortest phase of the cell cycle yet it involves dramatic changes in the cellular architecture and processes. Mitotic entry and progression are tightly regulated by the dynamic actions of signaling pathway networks and autoamplification loops. Mastl kinase is responsible for preservation of the hyperphosphorylated state of the mitotic substrates by indirectly inhibiting the major antagonist protein phosphatase, PP2A. Loss of function studies of Mastl kinase have shown defects of chromosomal segregation and subsequent failure of cytokinesis, and underphosphorylation of the mitotic substrates. To better understand and further characterize the mitotic functions of Mastl kinase, we aimed to engineer its analogue-sensitive mutant variants. We initially worked on editing the endogenous loci in human cell lines using CRISPR-Cas9 system. After failed attempts of editing the endogenous loci, we decided to use the conditional knockout mouse embryonic fibroblasts. Using retroviral vectors, we ectopically expressed the mutant variants of mouse Mastl and deleted the endogenous loci by inducing knockout in these stable cell lines respectively. Our eventual aim was to create an enzyme that will bind ATP and have normal kinase activity, and yet, be sensitive to an ATP analogue inhibitor. As a result, we observed that the stable cell lines are not viable when knockout was induced. We suspect the possible negative impacts of the mutations on enzymatic activity and insufficient ectopic expression levels. In order to compensate for this possible partial enzymatic inactivation, we are currently switching to lentiviral expression systems to achieve higher expression levels.