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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.
Kanser tedavisi için protein mühendisliği yaklaşımları ile geliştirilmiş anti-vegf antikorlarının araştırılması
Monoclonal antibodies (IgGs) are powerful therapeutics for many diseases, including cancer, due to their high specificity and other favorable properties. However, their large size limits their penetration into large masses of biological tissue, such as solid tumors. The single-chain variable fragments (scFvs), on the other hand, have better tissue penetration due to their smaller size. Yet, due to the absence of constant domains, scFvs are not as thermally stable as their parental IgGs or even Fab (fragment antigen-binding) fragments, which poses a challenge for therapeutic applications. Therefore, in this thesis, we aimed to develop a general method for increasing the thermal stability of scFvs. Anti-angiogenic therapy has a critical importance in tumor-mediated angiogenesis, which is a hallmark of solid tumors. Bevacizumab (IgG) was the first therapeutic developed for anti-angiogenic therapy, and it is still used in the clinic for a variety of solid tumor types, mostly in combination with chemotherapy. In this thesis, we developed anti-VEGF scFvs derived from bevacizumab and they demonstrated better anti-angiogenic efficacy compared to bevacizumab on a transgenic zebrafish model. Next, by utilizing rational design approaches including computational methods, and directed evolution techniques including yeast surface display we obtained thermally more stable scFv variants, the highest of which has around 10 ºC higher transition mid-point (T1/2) compared to wild type scFv. This method described in this thesis can be used to improve the thermal stability of any kind of antibody or antibody fragment.
Maküler kornea distrofisinin mikroakışkan platform içerisinde in vitro hastalık modellenmesi
Cornea is an avascular structure that provides refraction, transparency, and protects the eye's anterior part. Collagen generates a unique tissue structure that promotes physical qualities and strengthens the rigidity of the outer eye. Corneal dystrophies are rare hereditary diseases that produce corneal defects. Macular corneal dystrophy (MCD) is an autosomal recessively inherited condition in which mutations in the CHST6 gene, which codes for the enzyme N-acetylglucosamine-6-sulfotransferase, induce aberrant proteoglycan production. Corneal opacity is caused by the accumulation of glycosaminoglycans among stroma keratocytes, resulting in visual impairment. MMP1 and MMP13 from the matrix metalloproteinase (MMP) family, which are involved in corneal remodeling, have been found to be reduced in the extracellular matrix in MCD disease. Current clinical methods are mostly focused on corneal transplants, which do not eliminate the risk of recurrence. Here, we create a dynamic microfluidic platform to test an alternate treatment technique that includes MMP enzyme therapy in an in vitro model of MCD. Primary human corneal cells were extracted and planted on a hydrogel made up of mainly collagen which mimicked the biochemical and mechanical properties of the cornea. The cells were cultivated in a microfluidic platform in continuous laminar flow under dynamic conditions. We demonstrate that imitating sulfation inhibition using sodium chlorate, a pharmacological inhibitor of 3′-phosphoadenosine-5′-phosphosulfate (PAPs), results in long-term cell survival and MCD (-) related molecular responses at the gene level. The bioengineered example disease model will be a good platform for learning more about MCD and creating new treatments in clinics. Key Words: Microfluidics, Disease Modelling, Rare Disease, Macular Corneal Dystrophy
Karaciğer kanserinin modellenmesinde indüklenebilir pluripotent kök hücre (IPSCs) kaynaklı hepatik organoidler
Liver cancer is a heterogeneous disease with very limited treatment options, which ranks 4th in the world in terms of death. Hepatocellular Carcinoma (HCC), the most common type, is cancer that usually develops on the basis of cirrhosis and is characterized by late diagnosis and drug resistance. The most important limitation in understanding the early molecular pathophysiological changes and identifying new treatment targets in HCC is lack of the in vitro and in vivo models mimicking human tumorigenesis. In this context, in this study, the interactions of the microenvironment with stemness and senescence morphology in hepatocytes produced from IPSCs were investigated (Karabicici, 2021) and it was aimed to develop a long-term and large-scale 3D cell culture model that models the formation and development of pre-malignant stages of liver cancer such as fibrosis and cirrhosis. Here we utilize CRISPR/Cas9 technology for targeted gene modification of three of the most commonly mutated liver cancer genes (p53, APC, and NF1) in Induced Pluripotent Stem Cell derived healthy organoids (eHEPO). Although the CRISPR/Cas9 strategy to make these driver genes knockout has been worked successfully in HEK293 cells, we only continued with p53 knockout on eHEPOs. For this, we firstly transfected px458 plasmids containing p53 specific gRNA to the eHEPOs via optimized Nucleofector conditions. After enrichment of the GFP+ cells by FACS, we manually selected p53 KO-eHEPO clones under Nutlin-3a treatment. To mimic pro-carcinogenic TME activated LX2 and M2 polarized cell secretoms were added to growing medium of p53 KO-eHEPOs as well as WT controls. P53-KO highly proliferative (Ki67) and all displayed features of pro-carcinogenic pathological marks of pre-malign stages including increased nuclear–cytoplasmic ratio, pleiomorphic and hyperchromatic nuclei, pseudo glandular and tubular rosettes and inflammatory-like cell morphology. In addition, mason trichome staining area and fibrotic gene expressions have been shown to increase significantly. As a result, it has been shown that a fibrotic organoid model can be created that will make significant contributions to the understanding of tumor formation mechanisms in a carcinogenic microenvironment by using p53 KO-eHEPOs, which is one of the driver genes at the onset of HCC.
Bağlanma afinite tahmin algoritmalarının spike: ACE2 derinmutagenez bağlanma verileri üzerinde değerlendirilmesi
COVID-19 is caused by the SARS-CoV-2 virus. The SARS-CoV-2 virus binds to the host receptor protein ACE2 via its spike protein to initiate viral entrance and viral infection. Because of their critical roles, these proteins have been widely investigated by experimental and computational approaches. Relatedly, ACE2 and spike variant binding rates were obtained by the deep mutational scanning experiments. The deep mutational scanning method provides a high throughput binding dataset and thereby facilitates benchmarking of computational tools. By taking advantage of the deep mutagenesis datasets of ACE2 and spike, we aimed to investigate the performances in predicting the impact of interfacial mutations of FoldX, EvoEF1, MutaBind2, and SSIPe as main predictors in addition to HADDOCK and UEP as naïve predictors. In addition to their overall performance analysis, we performed metric-based analysis using volume, hydrophobicity, flexibility changes upon mutations, and change of side chain properties of mutations. As a result, we found that FoldX has the highest performance with a 64% success rate. Additionally, we highlighted that none of the predictors performed well on the binding increasing mutations. As a result, we revealed that (i) the top-ranking predictor, FoldX, predicts most of the hydrophobicity increasing mutations as affinity depleting, (ii) HADDOCK tends to tag mutations with increasing volume as affinity enhancing, (iii) Conservation-based tools, MutaBind2 and SSIPe predict most of the mutations as affinity depleting. All in all, we concluded that classical binding affinity predictors are not yet sufficient to predict binding affinity changes across the interface of the host-pathogen protein system SARS-CoV-2 spike -ACE2.
Nörotrofin reseptörü ile ilişkili ölüm bölgesi proteininin WNT/ß- katenin sinyal iletiminin düzenlenmesindeki rolünün zebra balığı modelinde araştırılması
Wnt/β-catenin signalling pathway is crucial for developmental processes and tissue homeostasis. Abnormal regulation of this signalling transduction might lead to several disorders. Hence, the understanding mechanisms underlying Wnt-related diseases might provide benefits for novel therapeutic approaches. For this purpose, elucidation of the roles of the modulators in this pathway has a significant importance for discovering novel drug targets. In the context of this thesis, the role of the neurotrophin receptor associated death domain protein (Nradd), which is a homolog of p75 neurotrophin receptor (p75NTR), in regulation of Wnt/ß-catenin signaling was investigated using the zebrafish model. Since the Nradd is a transmembrane protein with an intracellular death domain, it is able to induce apoptosis in healthy and cancerous cells. Therefore, the relationship between regulatory role as part of the Wnt/ß-catenin signaling and induction of apoptosis in cell culture and zebrafish model has been aimed to be unravelled. As a result of this modulation mechanism, the interaction between Nradd and other Wnt components was determined as a indicator of the dual role of this novel death domain protein. Based on our biochemical and molecular biological results, Nradd was identified as a novel feedback regulator of the Wnt/β-catenin signalling pathway. Consequently, we propose that targeting Wnt signalling via modulation of Nradd in novel treatment strategies could be beneficial for eradicating neurological disorders.
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.
TGF-b ve NF-kB sinyal yolaklarının çapraz konuşmasında Malt1 geninin rolü
Transforming growth factor-b (TGF-b) is a cytokine superfamily expressed in a variety of cell types. Through contextual regulation of a large number of genes, TGF-b ligands serve critical functions in key cellular and molecular mechanisms, including cancer cell survival and proliferation. Notably, canonical (Smad-dependent) and non-canonical (Smad-independent) signaling pathways are involved in its activity. Furthermore, TGF-b signaling is known to interact with other intracellular pathways, including MAPK, JNK, JAK/STAT and NF-kB. Importantly, the nuclear factor kappa-B (NF-kB) pathway constitutes a small family of transcription factors found in virtually every cell type and is involved in multiple aspects of biological functions relevant to physiological and pathophysiological processes. The activity of NF-kB pathway is also modulated by both canonical and non-canonical signals. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1), a paracaspase first identified in a subset of MALT lymphoma, is a critical component of NF-kB pathway in several biological contexts. Yet, the role of Malt1 in TGF-b and NF-kB interaction is unknown. Here we report that Malt1 protein potentiates the crosstalk between TGF-b and NF-kB signaling pathways. Mechanistically, through extensive molecular studies in A549 and Huh7 cancer cell lines using RNAi and CRISPR/Cas9 perturbations, we find that Malt1 is a Smad3-dependent target gene of canonical TGF-b signaling. More significantly, TGF-b-mediated Malt1 induction facilitates NF-kB activation. Collectively, our pioneering work provides mechanistic insights into how TGF-b/Smad signaling regulates Malt1 expression and reveals a novel mode of crosstalk between TGF-b and NF-kB signaling pathways.