LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Doktora
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Sustainable Development Goal "Goal 3: Good Health and Well-being" ile LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Doktora'a göz atma
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ÖgeBiointerfacial cell/protein–polymer interactions investigated by quartz crystal microbalance with dissipation(Graduate School, 2023-08-01) Sert Özdabak, Ayşe Buse ; Kılıç, Abdülhalim ; 521162101 ; Molecular Biology-Genetics and BiotechnologyUnderstanding cell-surface interactions is required for the development of novel and functional biomaterials. The biocompatibility and in vivo performance of these biomaterials heavily depend on processes such as protein adsorption and subsequent cell adhesion on the material surface. Common experimental approaches used to assess these processes typically involve end-point assays. However, these assays often require cell fixation or disruption and pre-or post-labeling of the cells, potentially affecting cell physiology and leading to the loss of valuable information. Furthermore, these methods cannot distinguish interfacial interactions occurring at nanometer scales between cells and the surface. The physicochemical properties of the material surface also significantly impact the performance of potential biomaterials. The interactions taking place at the interface between cells/proteins and materials are intricate and must be comprehensively understood and carefully designed to meet specific application requirements. In this context, Quartz Crystal Microbalance with Dissipation (QCM-D) emerges as an alternative and complementary method. QCM-D serves as a powerful, noninvasive technique that enables real-time and label-free monitoring of cell-surface interactions at the nanoscale. QCM-D provides distinct data regarding specific interactions at the cell - material interface, thereby offering new insights into the cell adhesion / protein adsorption behaviors. The aim of the thesis is to investigate cell-polymer interactions and to monitor the entire process in real-time using QCM-D system. For this purpose, two commonly employed polymers in the biomaterials field, Polycaprolactone (PCL) and Chitosan (CH), as well as their blends (75:25 and 25:75), were employed to investigate real-time cell adhesion behavior. As surface topography, chemical composition and wettability have significantly influence on cell adhesion process, it is important to analyze cell adhesion on well-characterized surfaces. Two types of cell lines (hFOB and 3T3) were employed to monitor cell interactions. Complementary cell culture assays were also conducted to validate the outcomes obtained from QCM-D. In the first part of the thesis, the preparation and characterization of thin films on silicon substrates and silica sensor surfaces were completed. In order to achieve homogeneous films, various parameters were investigated, i.e., polymer ratio, solvent type, substrate surface characteristics (activated with oxygen plasma or hydrophobic treatment), polymer molecular weight, and polymer ratio. The homogeneity of the films was assessed using Atomic Force Microscopy (AFM). It was founded that blends prepared with a constant amount of chitosan yielded homogeneous coatings on the silicon substrate. The chemical composition of the constructed surface was further analyzed using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR). The spectra exhibited distinct peaks at 1725 cm-1 for PCL and 1645 cm-1 and 1584 cm-1 for chitosan, confirming the successful coating of both polymers onto the surface. Analysis of AFM images over a scanned area of 100 µm2 revealed that pure polymers produced morphologically homogeneous films, whereas the blend surfaces displayed visible domains. Particularly, 75:25 PCL/CH blend exhibited micrometer-scale domains. Comparatively, thin films prepared with pure polymers displayed smoother surfaces compared to the blends. In contrast, the blends prepared with a ratio of 75:25 exhibited the highest roughness value. In terms of zeta potential, pure PCL films exhibited the highest negative value (-88 mV), whereas pure chitosan films displayed the lowest negative value (-15 mV) at pH 7.4. Blend film zeta potential values were between these two extremes. Film thickness analysis revealed that pure chitosan films had the smallest thickness (ca. 10 nm), whereas 75:25 PCL/CH blend film had the greatest thickness (ca. 55 nm). The pure PCL film and the 25:75 blend film had thicknesses of approximately 38 nm and 14 nm, respectively. In addition, in situ spectroscopic ellipsometry was employed to determine swollen polymer thicknesses. It was observed that PCL, being a hydrophobic polymer, did not swell much in aqueous solutions. Chitosan thin films exhibited the highest degree of swelling. The blend films exhibited swelling degrees between those of the pure polymer films, while higher PCL amount (75:25) resulted in reduced swelling, as expected. Before the cell adhesion studies, protein adsorption studies onto the constructed films was conducted. Bovine Serum Albumin (BSA) adsorption was monitored in real-time using both QCM-D and spectroscopic ellipsometry at various pH values at room temperature. In the case of pure PCL film, BSA adsorption onto pure PCL film showed a consistent frequency change upon adsorption with QCM-D for all pH values investigated. However, for the other investigated films, the presence of chitosan led to pH dependent adsorption behavior. At pH 4.5, both BSA and CH were positively charged, resulting in adsorption under repulsive conditions. At pH 6.0, the electrostatic attraction between the polymer chains and BSA led to higher adsorption on films containing chitosan. The lowest frequency decrease, i.e., mass load, was observed at pH 7.4 compared to pH 4.5 and 6.0. These findings indicate that blend composition, pH and ion presence in the environment have a substantial influence on protein adsorption. To compare the adsorbed protein amounts determined by QCM-D and ellipsometry methods, diverse models were applied. When two methods are assessed, the protein quantity derived from QCM-D data was consistently higher than that obtained by ellipsometry. The amount of protein calculated from ellipsometric data was similar for the blend films and pure chitosan films for all pH values investigated. However, higher values were evident in QCM-D method due to the inclusion of coupled water in the calculations. In addition, fibrinogen adsorption presented composition dependent behavior on thin films. The highest adsorbed fibrinogen amount was monitored on pure PCL films. In contrast, no significant protein adsorption was monitored on pure chitosan films. Consequently, the adsorbed amount of fibrinogen decreased with an increasing percentage of chitosan in the films, which predominantly showed an inverse correlation with the surface hydrophilicity. Following the comprehensive characterization of the films and conduction of the protein adsorption experiments, the cell adhesion behavior of two cell lines, human fetal osteoblastic (hFOB) and mouse fibroblast (NIH/3T3), was monitored on constructed films using QCM-D. For this purpose, the cells were introduced into the QCM-D chamber and allowed to flow for 1 hour. Initial cell sedimentation after 1 h resulted in reduced cell deposition as the chitosan ratio increased in the film. This trend was consistent for the both cell lines in the first hour. Subsequently, changes in frequency and dissipation were monitored over an 18-hour period. Complementary cell culture assays were performed to validate the observations of QCM-D. For this purpose, fluorescence images and live cell images at various time intervals were captured. Distinct QCM-D signal patterns were found for the investigated cell lines, indicating the influence of the varying interfacial properties on cell adhesion, which is also dependent on the specific cell type. In the case of hFOB cells, fully spreading was observed on pure PCL films, with elongated morphologies as confirmed by fluorescence microscopy and scanning electron microscopy (SEM). Corresponding QCM-D signals showed the highest frequency drop and the highest dissipation. Blend films supported hFOB cell adhesion, but with lower dissipation values compared to the PCL film. This might be attributed to higher rigidity at the hFOB cell−blend interface, because these cells did not progress to the further stages of spreading after secretion of their extracellular matrix (ECM) proteins. Variations in the QCM-D data obtained from the blend films could be attributed to differences in the morphology of the films. Pure chitosan films showed limited hFOB cell adhesion, accompanied by low frequency drop and low dissipation. The initial sedimentation of 3T3 cells onto the constructed surfaces similarly showed dependence on the surface composition. Unlike the behavior of hFOB cells, 3T3 cell lines did not adhere to pure chitosan surfaces, evident from consistent positive frequency signals. The highest frequency change was observed on reference silica surface, with dissipation gradually decreasing. This behavior indicated an average number of cells remaining in ECM remodeling stage. The ΔD signal shape was similar for 75:25 PCL/CH blend to the reference silica surface, however a slight decrease in the frequency was observed after 10 h. This suggests the stronger attachment to the surface while cells lacked further spreading stages on 75:25 PCL/CH blend. 3T3 cells on 25:75 PCL/CH blend showed substantial frequency drop after 10 h, which accompanied by an increase in dissipation. This behavior corresponded to the later stages of cell adhesion, implying that cells probably underwent actin remodeling and fully spreading on the surface. In conclusion, distinct QCM-D signal patterns were evident in the adhesion of hFOB and 3T3 cell lines. These distinctive patterns were attributed to the variations in the strength of cell adhesion, which are influenced by both cell type and surface chemical properties. The real-time and label-free data collected through QCM-D gave us a more profound comprehension of the dynamic adhesion behavior of the cells on constructed thin films. This knowledge and understanding holds the potential to provide valuable insights for the design of novel biomaterials tailored to diverse applications.
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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) Çalış, Şeyma ; Turanlı Tahir, Eda ; Avşar, Timuçin ; 521162117 ; Molecular Biology – Genetics and BiotechnologyGlioblastoma 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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ÖgeGıda patojenleri için dijital PCR ile hızlı tespit yöntemlerinin geliştirilmesi(Lisansüstü Eğitim Enstitüsü, 2023-03-20) Öz Yücel, Yeliz ; Karataş Yazgan, Ayten ; 521092078 ; Moleküler Biyoloji-Genetik ve BiyoteknolojiGıdalar 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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ÖgeInvestigation of familial multiple sclerosis genetics(Graduate School, 2022-05-16) Everest, Elif ; Turanlı Tahir, Eda ; 521152104 ; Molecular Biology-Genetics and BiotechnologyMultiple sclerosis (MS) is a chronic, neuroinflammatory, neurodegenerative disease of the central nervous system. Several lines of evidence have shown that the primary pathophysiological mechanism of MS is the infiltration of autoreactive lymphocytes through the blood-brain barrier, attacking central nervous system components such as myelin and resulting in oligodendrocyte death. This process has been thought to be responsible for axonal pathology and neuronal loss, which result in progressive neuronal dysfunction in some patients. Over the recent years, the roles of astrocytes, microglia, and pericytes have also been increasingly shown in MS pathology. To date, several studies have revealed disease-related cellular pathways that emphasize the different pathological components of the disease; however, underlying mechanisms in MS development and progression are yet to be elucidated. Consistently with its heterogeneous clinical presentation and complex pathophysiology, MS also has a complex inheritance pattern and develops in genetically susceptible individuals under environmental influences. Many studies have been carried out using different approaches and methods to identify genomic regions and variants that cause genetic predisposition to MS, identifying hundreds of common variants as well as candidate rare variants that increase the risk of MS. Today, MS associations of 233 common variants, as well as hundreds of suggestive associations, have been identified. However, all significant common variants, together with the suggestive effects, can cumulatively explain approximately half of MS heritability. Meta-analyses have shown that rare variants can further explain up to 5% MS heritability, still leaving a large proportion of MS genetics unknown. In this thesis study, it was aimed to reveal novel information on MS genetics and pathogenesis. Multiplex MS families with more than two affected family members were collected to identify possible novel genes that contribute to the high MS aggregation in these families. Seven multiplex MS families with the highest number of affected individuals and parental consanguinities were selected, and SNP genotyping (710K or 2.5M, Illumina) was performed (N=41). Candidate MS-associated genomic regions were identified through linkage analysis and homozygosity mapping. Exome sequencing (N=56) revealed that there were no fully penetrant, homozygous, rare, exonic variants segregating within the families. However, two variants were found to be segregated with the disease with an autosomal dominant inheritance pattern in the LRRC6 gene (rs139131485) in family FMS01 and RNF217 (rs73580047) gene in family FMS05, which may increase the risk of MS in corresponding families. Additionally, many incompletely penetrant, rare and low-frequency variants were identified. Subsequently, a weighted sum score analysis including previously identified common MS-associated risk variants and polygenic risk score (PRS) analysis were conducted in MS families (24 affected, 17 unaffected), 23 sporadic MS cases, 63 individuals in 19 non-MS control families, and 1272 independent, ancestry-matched controls to determine whether an increased burden of known MS-associated common variants explain the increased MS risk in these families. Logistic regression analyses showed that familial MS cases had higher sum scores (OR=2.16, P=0.002; OR=2.4, P=0.014) and PRS (OR=1.84, P=0.0077; OR=2.27, P=0.049) compared with the population controls and control families, respectively. Moreover, affected individuals in the MS families had higher weighted sum score and PRS values compared with the unaffected family members; however, the differences were not significant after Bonferroni correction. When individual families were observed, it was seen that the higher sum score and PRS trends in MS cases were evident in only three of the families, and in others, there were no apparent differences in the sum score and PRS values between the affected and unaffected family members or the unaffected individuals had higher sum score and PRS values compared with their relatives with MS, further supporting the polygenic inheritance of MS. Sporadic MS cases had significantly higher PRS compared with both affected and unaffected individuals in MS families, control families, and population controls (P=0.02, P=0.0055, P=0.003, and P=0.0008, respectively), supporting the presence of higher rare risk variation loading in the familial cases. There was no significant difference in the sum scores of familial and sporadic MS cases, possibly due to the high degree of convergence between common and rare risk variation in significant loci for MS. As part of this thesis study, we also performed an integrated bioinformatic analysis using genomic and proteomic data of an unrelated MS group. For this, first, SNP genotyping (300K, Illumina) was performed for 11 unrelated MS cases selected from our MS family cohort whose cerebrospinal fluid samples had been previously included in our proteomic study, in which 2D-gel electrophoresis, mass spectrophotometry, and pathway analyses had been conducted, revealing 151 differentially expressed proteins between MS cases with different clinical MS phenotypes and non-MS controls. To integrate the genomic and proteomic datasets of this patient group to reveal the most relevant disease pathways, pathway enrichment analyses of MS-associated SNPs and differentially-expressed proteins were conducted using the functional enrichment tool, PANOGA. Nine shared pathways were detected between the genomic and proteomic datasets after merging and clustering the enriched pathways. Among those, complement and coagulation cascade was the most significantly associated pathway (hsa04610, P=6.96×10−30). Other pathways involved in neurological or immunological mechanisms included adherens junctions (hsa04520, P=6.64 × 10−25), pathogenic Escherichia coli infection (hsa05130, P=9.03×10−14), and prion diseases (hsa05020, P=5.13×10−13). We conclude that despite the overall increased genetic burden in familial MS cases, weighted sum score and PRS distributions among affected and unaffected family members within individual families revealed that known susceptibility alleles can explain disease development in some high-risk multiplex families, while in others, additional genetic factors remain to be identified through more detailed genomic analyses such as genome sequencing. Additionally, integrating multiple omics datasets of the same patients helps reduce false negative and positive results of genome-wide SNP associations and highlights the most prominent cellular players among the complex pathophysiological mechanisms in MS.
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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) Uluca, Betül ; Kumbasar, Aslı ; 521132101 ; Molecular Biology-Genetics and BiotechnologyThe 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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ÖgeInvestigation of novel genes and functional roles in MEFV negative FMF patients through next-generation sequencing(Lisansüstü Eğitim Enstitüsü, 2023-06-20) Önen Özkılıç, Merve ; Turanlı Tahir, Eda ; 521142106 ; Molecular Biology-Genetics and BiotechnologyThe 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.
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ÖgeTargeting 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 BiotechnologyIn 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.