LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Doktora
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ÖgeA molecular dynamics study of the prion protein(Graduate School, 2023-05-12)Transmissible spongiform encephalopathies are caused by the conversion of the cellular prion protein PrPC into a misfolded form, PrPSc. In sheep populations there is a polymorphism at positions 136 (alanine/valine), 154 (arginine/histidine) and 171 (arginine/glutamine). While the A136-R154-R171 (ARR) variant confers highest resistance to scrapie, the V136-R154-Q171 (VRQ) variant leads to highest scrapie susceptibility. The A136-R154-Q171 (ARQ) variant with intermediate resistance is considered as wild type. To identify important conformational rearrangements at the initial steps of misfolding, microseconds long restrained and unrestrained molecular dynamics simulations have been prefomed at neutral pH, at 310 K and 330 K on naturally existing prion variants. Also, unfolding potentials of all three helicas of prion protein structure were also conducted at differentiated temperatures with the help of replica exchange molecular dynamic simulations. Moreover, at differentiated pH conditions unfolding potential of helix 1 and interaction of helix 1 with some other sequences were also conducted. Susceptibility of the disease might be related to hyrophobic side chain of the valine at position 136 which seemed to ease the unfolding process. While arginine at position 171 worked as a clamp to keep helix 2 and helix 3 of the cellular prion protein structure together. That might be the reason why VRQ is the most susceptable one where ARR is the most resistance. On the other hand, unfolding of helix 1 played the most critical role since it was the most stable helical structure in all conducted simulations. Inter- and/or intramolecular salt bridges of helix 1 were important to keep helix 1 stable in both helical structure and/or unfolded structure. Energy calculation showed that not high energy was needen to unwind helix 1. This helical structure of hydrophilic H1 might be broken by another hydrophilic sequence of the same prion protein, and its unwinding might be the key point to catalyze the complete unfolding of the protein
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ÖgeInvestigation of NFİB function and regulation of its putative target genes in human neural stem cell and SH-SY5Y neuroblastoma cell lines(Graduate School, 2023-03-13)The central nervous system comprises numerous neuronal and glial subpopulations that have unique identities. Molecular mechanisms that underlie the generation of this cellular diversity have been under investigation. During development, the formation of cell subclasses with particular features is determined by tissue-specific transcription factors (TF). TFs sequence-specifically bind to DNA, interact with other proteins, and affect the expression of target genes. One of the key TFs in the developing brain is the Nuclear Factor I (NFI) family. There are four members (A, B, C, and X) in vertebrates. NFI proteins comprise a highly conserved N-terminal DNA binding and dimerization domain and bind to a TTGGC(N5)GCCAA consensus sequence as homo or heterodimers. However, they have a less conserved C-terminal transcription modulation domain which may lead to differential transcriptional regulation of target genes. In the developing mouse, Nfia, Nfib, and Nfix are expressed in an overlapping but distinct expression pattern in different regions of the embryonic brain, while their expression is restricted to stem cell niches in the adult. In the central nervous system, deletion of each member leads to delayed glial and neuronal differentiation, aberrant cell migration and increased proliferation. In the developing hindbrain, only the absence of Nfib leads to delayed development of several precerebellar nuclei, indicating that Nfib may play a unique role in this system. Dysregulated expression of NFIs have also been linked to tumor growth and progression, however, with opposing effects. For example, NFIB is oncogenic and promotes metastasis in colorectal cancer, melanoma, gastric cancer, estrogen receptor (ER)-negative breast cancer, and small cell lung cancer; while it has a tumorsuppressive function in non-small cell lung cancer, glioblastoma, osteosarcoma, and cutaneous cell carcinoma. NFIs perform their context dependent, cell-type and tissue specific functions, via regulation of specific set of downstream transcriptional targets. Despite the fact that NFI binding motifs have been found in the promoter and upstream enhancer regions of many genes, only a few of them have been so far investigated as direct NFI targets. Further identification and characterization of downstream targets of NFIs in various tissues will help elucidate molecular mechanisms that regulate embryonic development and related diseases, as well as cancer pathologies. In an attempt to investigate how NFIB regulates neurogenesis in developing precebellar nuclei, differentially expressed genes in E14 Nfib knock-out mouse precerebellar neuroepithelium have been analyzed. The RNA profiling analysis revealed putative candidates for further research. Of these putative NFIB targets, we selected Cdon (Cell adhesion molecule related, down regulated by oncogenes) and Fgf15 (Fibroblast growth factor 15), since these genes have been implicated in neural development of the cortex. We examined NFIB-mediated transcriptional regulation mechanisms of CDON and FGF19, the human ortholog of mouse Fgf15, in human neural stem cells (hNSCs derived from H9 ESC, Gibco). Neural stem cell culture systems provide an in vitro model of human neural development. Since NFIs have been reported to regulate neuron production in diverse parts of the developing brain, they may have comparable functions in vitro. Understanding these processes and the underlying molecular mechanisms in vitro will also help understand how the brain develops in vivo as well as failures in this process. Thus, in the first chapter of the thesis, we set out to examine NFI function and regulation mechanisms of potential NFIB targets, CDON and FGF19, in neuronal differentiation of hNSCs in vitro. RT-qPCR analyses revealed that mRNA expression of NFIB, NFIC, and NFIX is downregulated, whereas NFIA is upregulated in differentiating hNSCs. Since NFIA levels are quite low in these cells, overall NFI expression levels decrease during neuronal differentiation in hNSCs. These cells express NFIB at much higher levels compared to the other NFI members. Therefore, this study focuses on NFIB's role in hNSCs. We analyzed cell proliferation and differentiation by BrdU incorporation assays and immunofluorescence staining of neural stem and neuronal marker proteins. However, NFIB overexpression or knockdown did not affect the proliferation or neuronal differentiation potential of hNSCs. Nevertheless, these data cannot preclude NFIB's potential role in differentiation and/or self-renewal of hNSCs since NFIB could be silenced only by 30–50% in these cells and analyses were performed in whole cell populations that might mask possible changes induced by NFIB loss. Moreover, in NFIB overexpression experiments, we may need other proteins acting as cofactors that are not supplied along with NFIB. This study identifies FGF19 as a novel downstream target of NFIB in hNSCs. Human FGF19 is preferentially expressed in the fetal brain, among other tissues. Recombinant human FGF19 treatment has been shown to enhance neuronal differentiation in mouse neuroepithelial and cortical cells. In accordance with these data, FGF19 expression increases in differentiating hNSCs. Moreover, FGF19 expression increases in NFIB silenced hNSCs while it is reduced in NFIB overexpressing cells, indicating that NFIB regulates FGF19 transcription in hNSCs. Indeed, NFIs directly repress FGF19 promoter-driven luciferase activity, confirming that NFIs transcriptionally target FGF19. Moreover, chromatin immunoprecipitation (ChIP) assays showed that NFI proteins occupy −777 (relative to the transcription start site) in hNSCs, indicating NFI interaction with the FGF19 promoter in vivo. Since NFIB expression decreases upon neuronal differentiation, while FGF19 increases and NFIB directly represses FGF19 in hNSCs, future studies are required to address functional relevance of NFIBmediated FGF19 repression in the control of self-renewal and neural differentiation of these cells. In the absence of NFI, Cdon, a cell surface glycoprotein of the immunoglobulin (Ig) superfamily, is upregulated in the developing mouse brain. CDON is expressed in various tissues, primarily in the brain, muscle, and endocrine tissues during human and murine embryogenesis. Moreover, CDON is implicated in proliferation and differentiation control as it promotes myogenesis and neurogenesis in vitro and is essential for proper brain and skeletal-muscle development. However, in this study, CDON expression decreased in differentiating hNSCs and it did not change in NFIB overexpressed or silenced hNSCs, analyzed by RT-qPCR. These data indicate that CDON is not an NFIB target in this system. Recently, CDON has been described as a dependence receptor that induces apoptosis in the absence of its ligand SHH. During cancer progression, in an environment with limited SHH, tumorigenic tissue may downregulate CDON to eliminate its apoptotic activity. Indeed, CDON expression decreases in colon, lung, and neuroblastoma tumors, implicating a tumor suppressor role for CDON. As NFIs are involved in progression of various cancers, we examined whether NFIs regulate CDON transcription in SH-SY5Y human neuroblastoma cells. ChIP assays showed that NFIs bind to human CDON gene regulatory regions, -8 and -941 (relative to the transcription start site), in hNSCs and SH-SY5Y cells. Moreover, NFIs repress CDON promoter-driven luciferase expression via interacting with those NFI sites. Finally, CDON is upregulated in NFIB silenced SH-SY5Y cells, suggesting that the NFIB-CDON axis may be involved in neuroblastoma biology. On the other hand, silencing NFIB in SH-SY5Y cells decreases cell viability and proliferation, suggesting an oncogenic role for NFIB in neuroblastoma. Next, we tested the hypothesis that NFIB may affect SH-SY5Y cell survival by suppressing expression and thereby, pro-apoptotic activity of CDON. However, downregulation of CDON on its own could not rescue the phenotype induced by NFIB silencing, most likely because other NFIB downstream targets, which may include p21, are also involved. Further studies are required to investigate the functional consequences of NFIB mediated CDON repression in other developmental systems and disease models. NFIB's oncogenic effects in SH-SY5Y cells may involve inhibition of apoptosis and/or regulation of cell cycle components. Moreover, NFIB might promote differentiation of SH-SY5Y cells and/or contribute to the aggressive state of neuroblastoma tumorigenesis. However, these and underlying mechanisms need to be further investigated.
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ÖgeGıda patojenleri için dijital PCR ile hızlı tespit yöntemlerinin geliştirilmesi(Lisansüstü Eğitim Enstitüsü, 2023-03-20)Gıdalar besleyici ve metabolize edilebilen substratlar olarak değerlendirildiğinde bu özellikleri nedeniyle birçok mikroorganizmanın büyümesi için uygun ortam sağlamaları kaçınılmazdır. Kontamine yiyecek veya içeceklerin tüketilmesi ile gıda kaynaklı patojenlerin meydana getirebileceği birçok hastalık mevcuttur ve dünya üzerinde hem gelişmiş hem de gelişmekte olan ülkelerde halk sağlığı için yaygın ve ciddi bir tehdit olmaya devam etmektedir. Bu hastalıkların şiddeti basit akut semptomlarla ilgili olabileceği gibi ciddi ve yaşamı tehdit edici seviyelere erişebilir. Patojen mikroorganizma bulaşlarını önlemek ve dolayısıyla insan sağlığı açısından oluşabilecek ciddi durum ve ölümleri engellemek amacıyla gıdalar için üretim aşamasından tüketim aşamasına kadar gerekli kalite kontrol programlarının uygulanması son derece önemli bir süreçtir. Kalite kontrolde kullanılabilecek geleneksel yöntemler kültüre dayalıdır. Bu yöntemler basit, kolaylıkla adapte edilebilir ve genel olarak ucuzdur. Kültüre dayalı yöntemlerde sırasıyla bakterinin kültür ortamında büyütülmesi, bu ortamdan izole edilmesi, izolasyon sonrası biyokimyasal ve/veya serolojik tanımlama yapılması ve bazı durumlarda subspesifik tanımlama yapılma aşamaları gerçekleşmektedir. Bu nedenle kültüre dayalı tanımlama uzun zaman almaktadır. Bu yöntemlerin, fenotipik özelliklerin ortaya konmasındaki yetersizlikleri, kültüre alınmamış mikroorganizmaların belirlenememesi ve sonuca birkaç günde ulaşılabiliyor olması gibi dezavantajları insanları hızlı, daha güvenilir, hassas metotların geliştirilmesine yöneltmiştir. Bu yöntemlerin büyük çoğunluğu antikor ve nükleik asit tabanlı teknikleri kapsamakla birlikte geleneksel yöntemlerin modifikasyonu ile oluşan teknikleri de içermektedir. Patojen mikroorganizmaların tespitinde son yıllarda nükleik asit tabanlı teknikler oldukça önem kazanmıştır. Bunlardan en öne çıkanları Polimeraz Zincir Reaksiyonu (PCR) tekniklerine dayanmaktadır. Gıda patojenlerinin tespitinde gıda mikrobiyoloji laboratuvarları tarafından kullanılan PCR testleri mevcuttur. Geleneksel PCR yöntemi elektroforez gibi görüntüleme sistemlerine ihtiyaç duyar. İkinci nesil PCR olarak tabir edilebilecek Real Time-PCR tekniğinde, elektroforez gibi işlemlere gerek kalmadan çoğalan ürün aynı anda floresan sinyali ile görüntülenebilmesi yanında kantitasyona da olanak sağlayabilmektedir. Birçok doğrulanmış ve ticarileşmiş farklı özgüllüğe, doğruluğa ve hassasiyet limitine sahip ticari Real Time-PCR testi mevcuttur. Teorik olarak PCR, bir kopya nükleik asitten iki saatten az bir zamanda milyonlarca kopya üretebilmektedir. Fakat gıdalardaki inhibitörlerin varlığı hassasiyeti düşürmektedir. Sonuca ulaşmak 30-90 dakika gibi bir zaman almakla birlikte hassasiyet limitleri 103-104 kob/g civarındadır. Bu nedenle mikrobiyal zenginleştirme yöntemleri ile sistemi kombine etmek düşük patojen sayısı nedeniyle negatif sonuç alınması, fizyolojik olarak strese girmiş veya yaralı patojen hücrelerin tespit zorluğu ve ölü hücrelerden gelebilecek deoksiribonükleik asitin (DNA) elimine edilememesi gibi birçok limitasyonla baş edilebilmek mümkün olacaktır. Kısmen yapılacak bir ön zenginleştirme işlemi ile hassasiyet limitleri bu yöntemde düşürülebilmektedir. Fakat bu işlem sonuç alma süresini uzatmaktadır. Ön zenginleştirme işlemi mikroorganizmanın çeşidine göre 6-8 saatten 48 saate kadar uzayabilmektedir. Uluslararası Standardizasyon Örgütü'ne göre Salmonella için bu süre 18-20 saat, Listeria monocytogenes için ise 25-26 saat olarak belirtilmiştir. Teknolojinin gelişmesiyle bir öncekine göre daha avantajlı sistemler geliştirilmekte ve birçok farklı sistem kullanıcıların onayına sunulmaktadır. Nükleik asitlerin biyolojik ve moleküler biyolojik çalışmalarda doğru ve kesin bir şekilde kantite edilmesi, üzerinde çalışılan ve birçok alanda uygulaması olan önemli bir konudur. Dijital PCR bu iş için oldukça geniş kullanım yelpazesine ulaşmış yeni bir platform olarak görülmektedir. Bu yöntemle herhangi bir referans standarda veya dışsal kontrole ihtiyaç duyulmadan doğrudan nükleik asit kantitasyonu yapılabilmektedir ve yöntem Real Time-PCR sistemi ile aynı primer prob yapısını kullanmaktadır. Real Time-PCR yönteminden farklı olarak inhibitörlere daha dayanıklı olması ve yöntem gereği reaksiyon içerisindeki 1 kopyayı tespit edebilecek hassasiyete sahip olabilmesi dijital PCR yöntemini daha avantajlı duruma getirmiştir. Bu çalışmada yaygın kullanılan Real Time-PCR sistemi yerine sonuç alma süresini azaltacak bir yöntem araştırması yapılmıştır. 3. nesil PCR olarak da adlandırılan ve bir kopyaya kadar hassas sonuç verebilme yeteneğine sahip Droplet Dijital PCR (ddPCR) sistemi ile Salmonella spp. türlerini ve Listeria monocytogenes'i yüksek hassasiyette tespit edebilecek minimum zenginleştirme aşamasına sahip bir yöntem geliştirmek üzerine çalışılmıştır. Salmonella spp. için gıda matrisi olarak çiğ kıyma örnekleri, Listeria monocytogenes için süt örnekleri seçilmiştir. Çalışma sonucunda Salmonella spp. için 25 g örnekte 1.39 kob hassasiyette sonuç alınırken Listeria monocytogenes için 1.35 kob hassasiyette sonuçlar alınmıştır.
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ÖgeDevelopment of novel BCL-2 inhibitors for glial tumors by using in vitro and in vivo systems(Graduate School, 2022-05-31)Glioblastoma Multiforme (GBM) is the most malign form of glial tumors, which accounts for the majority of brain tumor cases worldwide. There have been different approaches to treat GBM effectively, and with the advancements made for the last decade molecular pathology, target driven therapy, and personalized medicine gained attraction. One of such promising targets for GBM is Bcl-2 induced intrinsic apoptosis pathway. Anti-apoptotic members of Bcl-2 induced intrinsic apoptosis pathway have an important role in the regulation of GBM cell death. In this thesis study, we screened seven potential Bcl-2 inhibitor compounds and evaluated their effects on proliferation of GBM cells as well as their inhibitory capacity of Bcl-2 protein. Of those, I further analyzed three of them namely 58, 243, and ind-199. 58 and ind-199 compounds did not show any significant anti-proliferation effect on GBM cells. Eventually, we decided to elucidate the mechanism of action of 243 compound, a thiazolidine derivative BH3 mimetic, which was the most promising one according to the in vitro proliferation experiments. I performed colony formation assay to assess proliferation of YKG1 GBM cells, additionally to the proliferation assay with A172 GBM cells. While 243 inihibited cell growth significantly compared to control group, Bcl-2 inhibitor ABT-199 did not inhibit cell proliferation. Moreover, I tested 243 on YKG1 tumorspheres to determine its effectivity on tumor initiating cancer stem cells (CSC). Both ABT-199 and 243 had inhibitory effect on CSC proliferation, however 243 was significantly more effective than ABT-199 when compared to control group. Since 243 is a Bcl-2 inhibitor, I analyzed key players of Bcl-2 family and intrinsic apoptosis pathway. I have analyzed gene expression levels of BCL2, BCLXL, BAX, CASP3, CASP7, and CASP9. Furthermore, I also analyzed genes related with cell death which are CASP8 and TP53. Time dependent quantitative RT-PCR results suggested that, GBM cells that are treated with Bcl-2 inhibitors ABT-263 and 243 acts differently in case of gene expressions related to apoptosis. Next, we wanted to show apoptotic cell death with Annexin V-PI assay. Interestingly, we did not detect significantly elevated apoptosis in A172 cells when they are treated with either ABT-199 or 243. Similarly, cell cycle analysis showed that 243 did not have any effect on cell cycle, altough ABT-199 induced G1 phase arrest. Moreover, I determined expression levels of apoptosis related proteins PARP, Caspase-3, and Caspase-9. I used staurosporine treatment as a positive control to induce apoptosis. None of the treatment groups apart from staurosporine increased cleaved-PARP expression. Similarly, I checked if there is a difference in expression of Pro-caspase-3 and Pro-caspase-9, and observed that only stauroporine treated group expressed lower levels of Pro-caspases, indicating that cleaved forms of both Caspase-3 and 9 were produced upon staurosporine treatment only. At this point, we hypothesized that both ABT-199 and 243 could only induce limited apoptotic cell death because BCL2 expression was relatively low in A172 cell line. Expectedly, when I compared gene expression levels among different cell lines, I observed that BCL2 expression was very low in A172 cells, and it was abundant in SH-SY5Y neuroblastoma cells. Therefore, I decided to analyze apoptosis of SH-SY5Y cells after a treatment with ABT-199 and 243. Within only 48 hours of treatment with both inhibitors, I observed apoptotic cell death of SH-SY5Y cells. Hence, we had a new hypothesis that when BCL2 expression is low, upon Bcl-2 inhibitor treatment, cells may die through autophagy since Bcl-2 forms a complex with autophagy related protein Beclin 1. I showed that 243 treatment significantly upregulated autophagy related genes such as BECN1, ATG5, and MAP1LC3B, whereas ABT-199 induced autophagy on limited level. Moreover, autophagy indicative LC3B-II expression was significantly upregulated on a protein level with the 243 treatment, when compared to control as well as ABT-199 treatment. Additionally, I determined protein expression level of p53, which has a role in the interplay between apoptosis, cell cycle, and autophagy. I observed that p53 protein expression was increased upon both ABT-199 and 243 treatment, when compared to control group. Expectedly, when we performed in silico computational analysis, Beclin 1:Bcl-2 interaction and binding of 243 to their BH3 binding domains, we observed that 243 binds to Bcl-2 through important interactions. Since 243 and Beclin 1 binds to Bcl-2 from the same domain, when cells are treated with 243, Beclin 1 cannot bind to Bcl-2 and therefore it is released to initiate autophagy. In addition, we demonstrated that 243 significantly reduced in vivo tumor growth and prolonged survival in orthotropic brain tumor models, compared to vehicle group as well as ABT-263 treated animals. Furthermore, I assessed the anti-proliferative effects of 243 on primary glial cell lines as well. 243 exerted anti-proliferative effect on all patient derived glioma cell lines that have different grades and histopathology, except OLG3 cell line which is a grade 2 oligodendroglioma. According to quantitative RT- PCR results of OLG3, OLG7, and GBM9 cell lines I observed that OLG3 has a lower expression level of BCL2. These results suggest that patients with high BCL2 expression might benefit from 243 treatment. Taken together, our results indicate that 243 disrupts Beclin 1:Bcl-2 complex, hence activates autophagic cell death, and may serve as a potential therapeutic for the treatment of GBM.
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ÖgeInvestigation of novel genes and functional roles in MEFV negative FMF patients through next-generation sequencing(Lisansüstü Eğitim Enstitüsü, 2023-06-20)The p.Arg228Cys variant and the known pathogenic variant p.A230T are both located on the F-BAR domain, which is important for PSTPIP1 protein to form the functional trimeric complex with pyrin. The computational analyses of the xxi PSTPIP1 structure suggest that the p.Arg228Cys variant may cause a potential destabilization and change in the weak interaction network, leading to a stronger preference for certain interaction partners such as pyrin. Studies have shown that the p.Arg228Cys variant may lead to increased interaction with pyrin and reduced interaction with LYP phosphatase, which is normally required for the regulation of immune responses. The differences in symptoms between patients with the p.A230T or p.E250Q pathgoenic variants causing PAPA syndrome and patients with the p.Arg228Cys variant causing milder autoinflammatory symptoms in the patients in this study may be due to the diverse interaction strength of PSTPIP1 with different phosphatase proteins and pyrin protein. Within the scope of this thesis, it is found that p.Arg228Cys variant appears to increase the binding of PSTPIP1 to pyrin in PBMC samples of the patients. This was indicated by the higher levels of pyrin observed in the IP:PSTPIP1 lanes of our patient samples when compared to healthy controls. The interaction between PSTPIP1 and pyrin was increased in the patients as for P-III-2 1.43 fold and for P-II-1 patient 1.69 fold than HC1, and as for P-III-1 11.7 fold and for P-I-1 14.7 fold than HC2. This increased interaction may lead to hyperphosphorylation of PSTPIP1 and triggering the activation of pyrin inflammasome. Thus, the inflammation model in cultured PBMCs showed increased protein expression levels of pyrin, PSTPIP1, caspase-1, and IL1ß in cell lysates of patients with the p.Arg228Cys variant compared to the patient with a different heterozygote variant (p.A372V) and healthy control in the inflammation-induced condition (LPS+ATP) and secreted caspase1 and IL1ß levels were also found higher in supernatants of patients with p.Arg228Cys variant according to others in the inflammation-induced condition (LPS+ATP). Thus, it is important that other inflammation-related genes involvement should be considered in patients presenting with FMF phenotype negative for MEFV exon 10 recessive mutations. The targeted sequencing approach is useful for detecting rare pathogenic variations in patients with autoinflammatory phenotypes. Further functional analyses of the identified pathogenic variants could be helpful for better understanding the underlying molecular mechanism of FMF development.