LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji Lisansüstü Programı
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ÖgeA molecular dynamics study of the prion protein(Graduate School, 2023-05-12) Tavşanlı, Ayşenaz ; Balta, Bülent ; 521152101 ; Molecular Biology-Genetics and BiotechnologyTransmissible 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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ÖgeComputational investigation of reaction mechanism of FET3 protein in yeast(Graduate School, 2023-02-17) Ahıshalı, Büşra ; Balta, Bülent ; 521191104 ; Molecular Biology-Genetics and BiotechnologyIn Saccharomyces cerevisiae, a yeast species, iron uptake into the cell takes place with the Reducing Iron Uptake Model. Ferric chelates (Fe3C-L) are degraded on the cell surface by being reduced from Fe3+ to Fe2+ by the cell surface reductases Fre1p and Fre2p. The free reduced Fe2+ ions are taken up by Fet3p-Ftr1p, a high-affinity oxidase-permease complex, or by Fet4, another metal carrier. In this study, the reaction mechanism and the role of Fet3p in reducing iron uptake are examined. Fet3p is a membrane-bound protein and a member of the multicopper oxidase protein family. It metabolizes iron uptake with a high affinity for Fe2+ and plays a role in iron uptake together with iron-permease Ftr1p. Since Ftr1p can only transport the oxidized form of iron, Fe2+ needs to be oxidized before entering the cell. Fet3p couples the four-electron reduction of O2 to H2O with the one-electron oxidation of four Fe2+. The oxidized iron leaves the iron-binding site in Fet3p and is transferred to Ftr1p. Thus, Fe3+ ions are transported into the cytoplasm by a permease, Ftr1p. The understanding of the mechanism of Fet3p is of great importance to shed light on other multicopper oxidase members such as laccases and human ceruloplasmin, some having wide industrial applications. When the active site structure of Fet3p is examined, it has 4 copper as a cofactor in the active site. These coppers are divided into 3 types according to their characteristics: Type 1 (T1), Type 2 (T2), and binuclear Type 3 (T3a and T3b). T2 and T3 coppers form the trinuclear cluster (TNC). Iron as a substrate is not observed in any of the crystal structures of Fet3p. However, according to the information obtained from mutation studies and comparing them with the crystal structures of other MCOs, especially copper efflux oxidase (CueO), the amino acids in the iron-binding region of Fet3p and the location of iron were determined. Fet3p couples four one-electron oxidations of 4 Fe2+ as a substrate to the four-electron reduction of dioxygen to water by taking four protons from the environment. This process is mediated by oxidation-reduction reactions of copper ions as cofactors and consists of two stages. In the first stage, the O2 molecule, which will be reduced to H2O during the reaction, enters the TNC through the solvent channel and binds to the TNC. The O-O bond is cleaved by taking two electrons from two coppers (T1 and one of T3 coppers). Proton donation of E481 to one of the oxygens bridging T3 coppers facilitates this cleavage. Finally, all coppers are oxidized to Cu2+, and one O2- and two OH- ions are formed. In the second stage, the four reductions from Cu2+ to Cu+ with oxidation of four Fe2+ to Fe3+, and four protonations occur, and OH- and O2- ions are converted to two water molecules. In the literature, most of the first stage of the reaction mechanism of MCOs, especially dioxygen-cleavage and peroxy intermediate structure are known. However, the exact mechanism of the second stage, the order of electron and proton transfer reactions is not known because this part occurs fast. Due to the rapidity of these reactions, they have not been studied before and the order of the reaction is unknown due to the difficulty of following the protonation order experimentally. In addition to examining the reaction scheme, it is known that D283 plays an important role in iron binding to substrate-binding site, and electron transfer (ET) is enhanced by D283. However, in the crystal structure, the loop containing D283 is oriented away from the active site, suggesting that it closes only after the binding of Fe2+. Thus, to find out the role of D283 on ET and reaction pathways, the geometries are separately examined when the loop containing D283 is open and closed. In order to elucidate the unknown parts, computational methods were used in this present study, so the possible reaction mechanism will be determined. Thus, it is aimed to understand the mechanisms of other multicopper oxidase members through Fet3p. The calculations and geometry optimizations were carried out using the Quantum Mechanics/Molecular Mechanics QM/MM approach. The M06-2X method, a Density Functional Theory (DFT) method, was used for quantum mechanical (QM) calculations. B3LYP, TPSS, and M06 methods were also used to investigate whether M06-2X is the most suitable method for energy calculations and geometry optimizations of Fet3p containing copper and iron metals. Although M06-2X is not recommended to be used on metals in the literature, all necessary electronic states and spin densities could be obtained only with M06-2X in this study. For this reason, the results were interpreted over the energies obtained with M06-2X. The determination of the QM region to be calculated during the QM/MM calculations is of great importance for the calculations to obtain more accurate results. While choosing the most ideal QM region, residues that have the potential to affect the reaction, electron transfer, and proton exchange, especially close to the region where the reaction took place, were determined. The proximity of amino acids that will contribute to electron transfer around copper and Fe was investigated; therefore, calculations were made accordingly by choosing different QM regions. Considering the computational costs, the most ideal QM region was determined. In the structure where the loop containing D283 is closed, the first Fe2+ oxidation occurs exothermically without protonation while T1 is reduced. Protonation of OH- or O2- ions are not needed due to the cost of protonation. It is examined whether the first electron transfers from T1 to TNC before the second iron binds; nevertheless, the structure could not be obtained without protonation. With the protonation of the TNC region, electron transfer to the TNC has yielded a stable structure. After the oxidized iron leaves, the second Fe2+ binds. Meanwhile, the electron already transferred from the first iron remains in the protonated TNC. Considering the necessity of a second proton transfer before oxidation, the proton taken from D94 returns back to D94 during the optimization, thus the second proton transfer is not necessary. The electron from Fe2+ transfers to T1 copper, and oxidation of the second iron takes place. The second oxidation, which was endothermic when D283 was open, is exothermic in the structure where the loop is closed. The results draw attention to the importance of the loop containing D283. After the second oxidation, the oxidized Fe3+ is replaced with the third Fe2+. For the third iron, structures with two protons, three protons, and four protons are examined. For the third and fourth Fe2+ the geometries when the loop with D283 is open are also examined. According to the results, even three protonations are not enough for third oxidation, and a fourth protonation is needed. When the loop containing D283 is open, the oxidation of the fourth Fe2+ is endothermic even in the presence of four protons in TNC, which is the maximum number of protons TNC can take. In the oxidation reactions protonation of TNC-O2- decrease the negativity of TNC; thus, electron transfer to TNC is more favorable. The protonation of TNC is important to reduce coppers at TNC (T2 and T3 coppers) and for transferring an electron from the substrate to TNC. Similarly, the transfer of an electron from the substrate to TNC and the reduction of TNC coppers force the TNC-O2- or T3-OH- to take proton.
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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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ÖgeDiscovery of novel enzymes using proteomic approaches(Graduate School, 2021-01-26) Kılınç Öztuğ, Merve ; Karagüler, Nevin Gül ; Akgöz, Müslüm ; 521142107 ; Molecular Biology-Genetics and BiotechnologyThermophilic microorganisms that survive and grow in extreme environments, above temperatures of 50 °C, have been well studied over the last decade allowing us to increase our knowledge of the compositional and functional potential of these microbial communities. These microorganisms are of great importance for industrial processes since they express heat-resistive enzymes with the potential to serve as a biocatalyst in the future. Developing proteomic and metaproteomic approaches to discover novel enzymes from environmental samples is growing research of interest owing to the advanced mass spectrometry (MS) based techniques. In this study, proteomics and metaproteomics approaches were applied to discover novel enzymes from harsh environmental conditions. Geothermal sources are among the habitats of thermophilic bacteria. In Turkey, there are many spas that have the potential habitat for numerous thermophilic bacteria, and this offers a good opportunity for the discovery of new thermophilic microorganisms. In this study, a thermophilic bacterial consortium of the Armutlu Hot Spring in the Yalova region of Turkey was investigated in a culture-dependent manner using proteomic approaches.
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ÖgeExpression, purification, and characterization of recombinant human IL-2(Graduate School, 2022-01-18) Akgün, Buse ; Doğanay Dinler, Gizem ; 521181103 ; Molecular Biology – Genetics and BiotechnologyCytokines, which are small proteins secreted by the immune system, are in charge of directing the immune system. Through their formation, differentiation, and activation functions, cytokines govern the maintenance of innate and adaptive immune responses. They are primarily formed by mononuclear phagocytes, dendritic cells, and antigen-presenting cells. Interleukin (IL) is a kind of cytokine that acts as an immunomodulatory protein. It induces a variety of cell and tissue responses. Interleukins mediate the interaction of leukocytes (white blood cells) and initiate a response by attaching to high-affinity receptors on the surface of the cells. They play a critical role in the regulation of cellular formation, differentiation, and activation that occurs over the course of inflammatory and immunological responses. Each family is assigned an IL based on sequence homology, receptor chain similarity, and functional qualities. Interleukin-2 (IL-2) was the first cytokine discovered to stimulate the growth of T lymphocytes. T cells, B cells, natural killer (NK) cells, lymphokine-activated killer cells, and macrophages all require IL-2 to regulate their proliferation and differentiation. Mier et al. discovered the molecule and named it "IL-2" since it was produced by and acted on leukocytes. Its discovery is regarded as a milestone in immunology. However, there is one issue that is common to all lymphokines when it comes to the molecular and functional characterization of IL-2, and it is due to their production in small quantities. The cloning of cDNA for IL-2 was a significant turning point in 1983, precipitated by the discovery of IL-2. The Jurkat T cell leukemia cell line was employed for the IL-2 cDNA clone development. IL-2 is a 15.5 kDa glycoprotein that belongs to the cytokine family four α-helical bundles. There are 153 amino acid residues in a single polypeptide chain of IL-2. IL-2 binds to and communicates with a receptor complex composed of three different subunits known as IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2R (CD132). Different combinations of these three components bind to IL-2 with varying degrees of affinity. The αβγ heterotrimer, βγ dimer, and α chain monomer all bind to IL-2 with "high," "intermediate," and "low" affinity, respectively. Binding of IL-2 to the IL-2R heterodimer complex activates several pathways. In response to an interaction between interleukin-2 and its receptor, kinases connect to cytoplasmic areas of the receptor subunits, resulting in the tyrosine phosphorylation of many proteins and the activation of a number of signaling pathways, including JAK/STAT, PI-3K/AKT, and Ras/MAPK. IL-2 activity promotes cell survival, proliferation, cell cycle progression, and targeted gene transcription. Due to its ability to activate both T and NK cells, IL-2 was the first cytokine to be successfully used in cancer treatment. The US Food and Drug Administration authorized high-dose IL2 for the treatment of melanoma and renal cell carcinoma in xxii 1992 and 1998, respectively. Moreover, the use of recombinant IL-2 therapy may help researchers understand better the coronavirus disease 2019 (COVID-19), which is caused by a virus that leads to severe acute respiratory illnesses and has rapidly spread throughout the world. As a prospective treatment for this condition, the use of rIL2 may be beneficial for patients since it has the potential to accelerate disease recovery by increasing the number of lymphocytes in the body. A major difficulty is figuring out how to direct IL-2 activity toward Teffs and away from Tregs, which inhibit the immune system. IL-2 is available in two recombinant forms derived from E. coli, but only aldesleukin is FDA-approved. Recombinant IL-2 differs structurally from its natural version. IL-2 recombinant is not glycosylated and lacks N-terminal alanine. To avoid the formation of an incorrect disulfide bond, serine has been substituted with cysteine at amino acid position 125. The pharmacological actions of endogenous and recombinant human IL-2 are similar. In this study, E. coli Rosetta (DE3) was used as the host cell. Induction of protein expression was accomplished by the use of IPTG. Following that, inclusion bodies, which develop in the cell as a result of excessive protein expression, were separated and solubilized from cell lysates and refolded by step-wise dialysis. Anion exchange chromatography was used to separate the target protein from the rest of the protein mixture. After purification, the yield was determined to be 0.114 mg per liter of cell culture. SDS-PAGE and immunoblotting methods were used to validate the effectiveness of the purification. The molecular weight is estimated using intact mass analysis through LC/MS. The CE-SDS analysis revealed that rIL-2 has a purity of around 80%. In addition, the pI value of the protein was determined as 7.31 using the capillary isoelectric focusing method. The peptide mapping on LC-MS/MS is used to figure out the main structure of the protein that has been purified. The secondary structure of pure human interleukin-2 (hIL-2) was investigated using circular dichroism (CD), and the results revealed that it included a high concentration of alpha helices. The biological action of our IL-2 is determined by phosphorylation of one of the MAPK pathway proteins, extracellular signal-regulated kinase 1/2 (ERK), on human monocytic cells, THP-1. An active protein has been produced as a result of this work. The experimental results indicate that the procedures established for generating and purifying the rIL-2 protein may be employed to create a pure product that maintains its bioactivity.
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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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ÖgeInverse metabolic engineering of KCl-resistant Saccharomyces cerevisiae(Graduate School, 2022-06-20) Morkoç, Ogün ; Çakar, Zeynep Petek ; 521181116 ; Molecular Biology - Genetics and BiotechnologyBu çalışmada, KCl tuzu kaynaklı hiperosmotik strese dayanıklı S. cerevisiae suşları elde etmek için, tersine metabolik mühendislik yaklaşımı olan evrimsel mühendislik kullanılmıştır. Evrimsel mühendislik ile elde edilen suşların fizyolojik ve metabolik analizleri yapılmıştır. Evrimsel mühendislik için seçilim deneyi, kademeli olarak artan KCl stresi altında gerçekleştirilmiştir.
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ÖgeInvestigating protein variations of Bag-1 in wild type and Bag-1 knockout MCF-7 breast cancer cells(Lisansüstü Eğitim Enstitüsü, 2022) Kılbaş, Pelin Özfiliz ; Doğanay, Gizem Dinler ; Arısan, Elif Damla ; 723988 ; Moleküler Biyoloji-Genetik ve BiyoteknolojiThe multifunctional Bag-1 protein which is known for its anti-apoptotic role has many critical direct or indirect interaction partners in the cell. According to these interactions, the Bag-1 protein plays an important role in the decision mechanism between survival or death of cancer cells. In previous studies determining the chromosomal location of the Bag-1 gene, it has been stated that this gene is associated with neurodegenerative diseases such as Alzheimer's and Parkinson's, and cancer. It has been shown that the change in the expression level of Bag-1 protein in the cell is associated with many different cancer types. In addition, it has been demonstrated that Bag-1 may provide clinical benefit as a prognostic marker in the determination of breast cancer, which is the first among women with its incidence. Elevated levels of Bag-1 are generally associated with breast cancer growth, development, and aggressiveness. In particular, interactions with the Hsc70/Hsp70 chaperone family are effective in the long-term survival of breast cancer cells under stress conditions. Our previous studies showed that silencing the Bag-1 protein increases drug-induced apoptosis in breast cancer. Although there are several studies on the association of changes in the expression level of the Bag-1 protein with cancer, the biological aspect of the complete deletion of the Bag-1 gene in breast cancer cells is not detailed explained. Therefore, this thesis study consists of two parts covering the molecular function of the deletion of the Bag-1 gene in MCF-7 breast cancer cells. The first part of the thesis consists of the generation of Bag-1 knockout cells with the CRISPR/Cas9 system, the validation and characterization, and the investigation of the effect of Bag-1 deficiency on the viability and cellular response of MCF-7 cells. The second part of the thesis includes the profiling of differentially expressed microRNAs and the determination of the molecular targets of these significantly differentially expressed miRNAs in wild-type and Bag-1 knockout MCF-7 breast cancer cells.
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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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ÖgeInvestigation of the effect of ATP13A2 (PARK9) frameshift mutation on the protein function(Lisansüstü Eğitim Enstitüsü, 2021) Kırımtay, Koray ; Karabay Korkmaz, Arzu ; 692806 ; Moleküler Biyoloji-Genetik ve BiyoteknolojiNeurodegenerative diseases occur due to deformations in the nervous system as a result of structural and functional disorders in nerve cells. In practice, neurodegenerative diseases are classified according to clinical and pathological findings. Genetic and epigenetic factors play essential roles on the basis of the disease pathology. Mutations that occur in different genes or at various points in the same gene act at the onset of the disease by acting on different molecular mechanisms. In many neurodegenerative diseases, overlapping clinical findings at the beginning and progression of the disease are the most critical limiting factors in making the correct diagnosis in these diseases. Rare neurodegenerative disorders, such as complex Hereditary Spastic Paraplegia (HSP), Spino-cerebellar Ataxia (SCA), and Kufor-Rakeb syndrome (KRS), have many common symptoms. Spastic Paraplegia-78, a subtype of HSP, and KRS have been associated with mutations in the ATP13A2 gene. In these two diseases that are inherited in autosomal recessive manner, different mutations in the ATP13A2 gene cause many common symptoms, making clinical diagnosis difficult. Genetic and functional molecular studies are important for the accurate diagnosis of the disease. Within the scope of this thesis, two cases in a consanguineous Turkish family were examined genetically and cellularly. Clinically, patients were diagnosed with "autosomal recessive spastic paraparesis" and/or "autosomal recessive ataxia" due to the observation of spasticity, cerebral and cerebellar atrophy. In this thesis, gene mutations associated with the disease were screened by whole-exome sequencing in the family. Three variants of the ATP13A2 gene ("c.1422_1423del: p.P474fs", "c.G1426T: p.A476S" and "c.1429_1430insAAA: p.M477delinsKM") were detected as a result of whole-exome sequencing. The bioinformatics analysis determined that the "c.1422_1423del: p.P474fs" variant caused a frameshift and a premature termination codon. It was determined that the premature termination codon formed by the effect of frameshift causes the loss of N and P regions necessary for phosphorylation, which is important in the function of the ATP13A2 protein, resulting in the formation of a truncated protein of 519 amino acids. Following these data, ATP13A2 expression in primary fibroblast cells obtained from patients, their parents, and a healthy control was examined by qRT-PCR and Western blot method. According to the qRT-PCR result, it was determined that ATP13A2 mRNA was reduced by 60-70% in patients' fibroblasts compared to control fibroblasts. ATP13A2 mRNA was observed to decrease by 5-20% in the fibroblasts of the parents. When protein expression was examined by Western blot method, wild-type ATP13A2 protein with a molecular weight of 150 kDa was detected in the fibroblasts of control and parents. In comparison, the expected truncated protein around 50 kDa was not detected in the patient fibroblasts. The reason for not observing the truncated protein could possibly be to the fact that the produced truncated protein might have been degraded by proteasome machinery due to misfolding. In this context, truncated protein expression was investigated by inhibiting the proteasome mechanism with MG132. According to the Western blot result, mutant protein expression was not observed after MG132 application. Besides, BiP and PDI, which are endoplasmic chaperone proteins involved in protein folding, expressions were also examined, and no difference was observed in the expression of these two proteins. These results highlighted the possibility that the mutant ATP13A2 mRNA was targeted and degraded by NMD. To examine whether NMD degraded the mutant ATP13A2 mRNA, 5-azacytidine, which was shown to cause NMD inhibition by c-myc, was applied to fibroblasts. After 5-azacytidine administration, it was observed that the mutant ATP13A2 protein was expressed in patients' fibroblasts. However, the increase in both mRNA and protein levels in control fibroblast cells that do not carry mutations suggested that 5-azacytidine acts by a different mechanism independent of NMD. It is known in the literature that 5-azacytidine increases c-myc expression. Accordingly, after the 5-azacytidine treatment, c-myc expression was examined by Western blot, and an increase in c-myc expression was observed. In addition, the mutant ATP13A2 expression was detected in fibroblast cells differentiated from the patient's induced pluripotent stem cells in which c-myc had been expressing due to the presence of c-myc expressing Sendai virus. The reason for the increased ATP13A2 expression in both patient fibroblasts and control fibroblasts after 5-azacytidine suggested that the gene expression by c-myc may be transcriptionally regulated. When the ATP13A2 gene's promoter region was examined, the putative c-myc transcription factor binding sites were determined. These findings strengthen the possibility that the ATP13A2 gene can be regulated by c-myc. After 5-azacytidine treatment, NMD-independent increase in ATP13A2 expression was observed, fibroblasts were treated with NMDI-14, a specific NMD inhibitor, and a 2-fold increase in ATP13A2 mRNA level was observed in patient fibroblasts. In addition, mutant protein expression was detected in patients' fibroblasts. After determining that ATP13A2 protein was not expressed as a result of the mutation in the patients, based on the hypointense basal ganglion images on MRI, iron accumulation was examined by Prussian blue staining in the patient fibroblasts. Iron accumulation was found in the patient fibroblasts after staining. Later, the effect of iron deposition on cell viability was investigated by the MTT assay. It was found that patient fibroblasts were more sensitive to iron overload. It is known that increased intracellular iron increases reactive oxygen species and triggers oxidative stress. In the presence of oxidative stress, the Nrf2 transcription factor is localized to the nucleus and provides the expression of several genes involved in oxidative stress defense. For this reason, the intracellular localization of Nrf2 was examined by immunocytochemistry. It was observed that Nrf2 was localized in the nucleus in the patient's fibroblasts, while it was mostly cytoplasmic in the parents' fibroblasts. These data show that patients have iron overload and oxidative stress due to accumulation.
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ÖgeProtein engineering applications on industrially important enzymes(Fen Bilimleri Enstitüsü, 2019) Özgün, Gülşah ; Karagüler, Nevin Gül ; Moleküler Biyoloji-Genetik ve Biyoteknoloji ; Molecular Biology-Genetics and BiotechnologyBüyüyen biyoteknoloji marketi, beraberinde biyokatalizörlerin geliştirilmesi ve yeni özellikler kazandırılmasına yönelik çalışmaların artmasına yol açmıştır. Endüstriyel ortamlar genellikle doğal enzimlerin kullanımı için uygun olmayan ekstrem şartlar barındırmaktadır, yüksek sıcaklık, basınç ve çok yüksek veya düşük pH gibi koşullarda, çoğunlukla doğal enzimler istenilen performansı sağlayamamaktadır. Başta protein mühendisliği olmak üzere birçok disiplin farklı endüstriyel alanlarda enzimlerin etkili bir şekilde kullanımına yönelik stratejiler geliştirmektedirler. Protein mühendisliği, endüstriyel sektörün özelliklerine ve enzimin kullanılacağı ortama bağlı olarak istenilen özelliklerin geliştirilmesinde, örneğin aktivite ve stabilitenin arttırılması, substrat veya koenzimin spesifisitesinin değiştirilmesi veya geliştirilmesi, optimum pH nın değiştirilmesi gibi stratejilere yönelik rasyonel tasarım, yönlendirilmiş evrim ve kombinasyonel uygulamaları kullanmaktadır. Protein mühendisliği stratejilerinin belirlenmesinde aynı zamanda enzime ait (yapı-fonksiyon) bilgilerin varlığı veya yokluğu etkili olmaktadır. Protein mühendisliği, moleküler biyoloji temeline dayanarak proteinin yapı fonksiyon ilişkisinin anlaşılmasına olanak sağlayarak, proteinin genetik düzeyde yeniden dizayn edilmesini ve istenilen özellikte biyokatalizörlerin oluşturulmasını mümkün kılar. Bu kapsamda tez, endüstriyel öneme sahip Bacillus subtilis lipazA (bsLipA) ve Candida methylica format dehidrogenaz (cmFDH) enzimlerinin üç farklı stratejinin kullanıldığı protein mühendisliği uygulamalarına odaklanmıştır. i) Optimum pH nın ii) ve koenzim spesifitesinin değiştirilmesi iii) termal stabilitenin arttırılması hedeflenen stratejiler olmuştur. Deneysel çalışmalar üç bölümde detaylandırılmıştır. Tezin birinci kısmında, bsLipA enziminin optimum pH'sının değiştirilmesi amacıyla rasyonel tasarım uygulamalarından bölgeye özel mutasyon yöntemi uygulanmıştır. Lipazlar (EC 3.1.1.3) trigliseridlerin serbest yağ asitleri ve gliserole hidrolizini gerçekleştirirlerken, aynı zamanda transesterifikasyon, aminoliziz ve asidoliziz reaksiyonlarını da katalizlerler. Mikrobiyal lipazların susuz ve az-sulu ortamlardaki potansiyeli, enzimin çok yönlü biyoteknolojik bir araç haline gelmesini sağlamıştır. Farklı endüstriyel alanlarda uygulama imkanı bulan lipazlar, son yıllarda sıvı veya süperkritik karbon dioksit (LCO2/SCCO2) gibi hijyen sistemlerinde hidrolitik enzim katkısı olarak kullanım potansiyeline sahiptir. LCO2/SCCO2 sistemlerinin temizleme etkisinin hidrolitik enzim ilavesi ile arttırılması mümkün olmasına karşın, çözgen olarak kullanılacak olan LCO2 / SCCO2' in polar olmayan bir çözgen olması ve düşük su içeriği sebebiyle, kullanılacak enzim sisteminin az sulu ve düşük pH' ya sahip çözgen sistemlerinde aktif olması beklenmektedir. Bacillus subtilis lipaz (bsLipA), geniş bir pH (4-11) aralığına sahip olmasına karşın optimum pH'sı 10'dur, bu sebeple LCO2 / SCCO2 çözgen sistemlerinde kullanımı için modifikasyonu gerekmektedir. Bacillus subtilis lipaz A (PDB ID: 1ISP) kristal yapısı ve Insight II programı kullanılarak enzimin optimum pH değişimini sağlayacak hedef mutantlar belirlenmiştir. Proteinin aktif bölgesinde katalitik özellikteki amino asitlerin pKa değerlerini etkileyebileceği düşünülen, yaklaşık 9 Å'luk mesafe içerisinde olan ve katalitik amino asitler ile doğrudan ilişkili olan amino asitlerin, bölgeye özel mutasyon tekniği ile tekli G11E, N18R, L102R, G103R, G104R, I157R mutantları oluşturulmuştur. N18R ve G103R mutantlarının template olarak kullanılması ile ikili (G11E-N18R, G103R- N18R, G103R- G11E, and G103R- G104R) mutantların da, bölgeye özel mutasyon tekniği ile oluşturulması planlanmıştır. Tezin ikinci kısmı cmFDH enziminin koenzim spesifitesinin değiştirilmesine yönelik yarı-rasyonel tasarım uygulamalarından bölge saturasyon mutagenez yöntemini kapsamaktadır. Tezin üçüncü kısmında ise cmFDH enziminin termal stabilitesinin arttırılmasına yönelik, , rasyonel tasarım uygulaması olan bölgeye özel mutasyon yöntemi uygulanmıştır. NAD+-bağımlı format dehidrogenaz enzimi (EC 1.2.1.2, FDH), format iyonunun karbondiokside dönüşümünü katalizlerken, NAD+ molekülünün NADH'e indirgenmesini sağlamaktadır. Gerçekleşen reaksiyonun basitliği, kullanılabilirliği, düşük maliyeti, termodinamik özellikleri ve son ürün olan CO2' in reaksiyondan kolaylıkla uzaklaştırılabilmesi gibi avantajlarından dolayı FDH, kimya endüstrisindeki kiral bileşiklerin sentezi için çok önemli olan NAD(P)H rejenerasyonunda potansiyel bir sistemdir. Fakat, doğada bulunan FDH'lerin çoğunluğunun NAD+ koenzimine spesifik olması ve düşük termal stabiliteye sahip olması, FDH'in kullanımını kısıtlamaktadır. Bu sınırlandırmaların aşılması amacıyla, tezin ikinci kısmında, cmFDH enziminin koenzim spesifitesinin değiştirilmesi için koenzim bağlama bölgesinde, koenzim spesifitesinden sorumlu amino asitler, Pseodomonas. sp.101 ve Candida boidinii FDH kristal yapıları baz alınarak, Insight II (Accelrys) programı ile oluşturulan cmFDH homoloji modeli yardımıyla belirlenmiştir. Belirlenen D195, Y196 ve Q197 bölgelerine ait dejenere primerler ile uygulanan bölge saturasyon mutagenez çalışması sonucunda her bölge için mutant kütüphaneleri oluşturulmuştur. NADP+ koenzimi ile aktivite gösteren aday mutantlar kolorimetrik tarama metoduyla belirlenmiştir. İki nesil oluşturulan adayların protein üretimi ve saflaştırılması neticesinde NADP+ koenzimine karşı olan ilgisi test edilmiştir. Yapılan çalışma sonucunda ikili mutantlardan D195S / Q197T ve D195S / Y196L mutant cmFDH enzimlerinin NADP+` ye karşı katalitik etkinlikleri yabanıl tip cmFDH enzimine kıyasla, sırasıyla 56000 ve 50000 kat artmıştır. Çalışmanın son kısmında, cmFDH enziminin termal stabilitesinin geliştirilmesi amacıyla, protein yüzeyindeki esnekliği yüksek olan oyuklar ve bu oyuklarda bulunan esnek amino asit kalıntıları hedef alınmıştır. Pseodomonas. sp.101 ve Candida boidinii FDH kristal yapıları baz alınarak, ExPASy programı ile cmFDH homoloji modeli elde edilmiştir. Oluşturulan model üzerinde FIRST algoritması kullanılarak, proteinin esnek oyukları ve hedef amino asit kalıntıları belirlenmiştir. Yapılan bilgisayar çalışmaları neticesinde belirlenen 12 aday; M131A, V133I, V139W, P140R, D158N, I162V, F186L, V219M, F247A, E272W, R277N ve K301R bölgeye özel mutasyon yöntemi ile oluşturulmuştur. Mutant adayların protein üretimi ve saflaştırılması neticesinde gerçekleştirilen kinetik ve sıcaklık çalışmaları sonucunda, birinci oyukta yer alan M131A mutant enziminin, rekombinant yabani tip cmFDH enzimine kıyasla yarı ömründe 4 °C'lik bir artışla diğer mutantlar arasında en iyi termal stabilite profili gösterdiği belirlenmiştir. Bu tez kapsamında yapmış olduğumuz tüm çalışmalar, endüstriyel kullanım için enzimlerin modifikasyonunda, doğru stratejiler kullanıldığında protein mühendisliği uygulamalarının, başarı sağladığını göstermektedir.