LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji Lisansüstü Programı
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Çıkarma tarihi ile LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji Lisansüstü Programı'a göz atma
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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.
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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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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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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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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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ÖgeDADA2 hastalarının periferik kan mononükleer hücrelerinde total ada aktivitesinin analizi(Graduate School, 2022-02-09) Demirci, Turna ; Turanlı Tahir, Eda ; 521171122 ; Molecular Biology, Genetics, and BiotechnologyDeficiency of Adenosine Deaminase Type 2 is an autosomal recessive disease caused by biallelic mutations in the ADA2 gene. It was first defined as monogenic vasculitis syndrome in 2014 as a result of studies conducted by two different groups independently. Although it has been shown that the prevalence of ADA2 Deficiency maybe 4 in 100,000, the prevalence of the disease may differ between ethnic groups, depending on the degree of consanguinity and the presence of founding variants. Adenosine deaminase is an enzyme involved in the regulation of adenosine homeostasis and purine metabolism by converting adenosine to inosine and 2'-deoxyadenosine to 2'-deoxyinosine. There are two isoforms of adenosine deaminase in humans, and one of them, the 57-kDa homodimer ADA2 protein, is produced by the Adenosine Deaminase 2 (ADA2) gene. The N-terminal portion of the ADA2 protein is responsible for growth factor activity, while the C-terminal portion is responsible for adenosine deaminase activity. In addition to the catalytic domain, the ADA2 protein also has a protein dimerization domain and a cell surface binding domain. ADA2 proteins bind to different cell surfaces via glycosaminoglycan chains and to T cells via adenosine receptors. In this way, it shows both cytokine-like and autocrine-type growth factor properties. Although the ADA2 protein is involved in macrophage polarization, it also has an important regulatory function for neutrophil activation. In addition, it significantly reduces the formation of neutrophil extracellular traps, which are caused by extracellular adenosine and can lead to the activation of proinflammatory cytokines. Despite the clinical manifestations of DADA2 being very diverse, episodic clinical findings are usually observed in patients with fever and systemic inflammation. The most common type is vasculitis findings. In addition to dermatological and neurological symptoms, it is also rarely defined by renal involvement and gastrointestinal system findings. More than half of patients have attacks of non-infectious fever. Symptoms include recurrent oral and genital ulcers, musculoskeletal symptoms, recurrent abdominal pain, inflammatory bowel disease, and immunodeficiency. Hematologic findings include cytopenia, anemia, and rare bone marrow failure. The diagnosis of the disease is made based on the detection of pathogenic variants on the ADA2 gene or the measurement of ADA2 activity in serum/plasma. Treatment methods are selected depending on the symptoms and the severity of the disease. Currently, anti-TNF-α is the most common treatment modality, especially for patients with signs of vasculitis. Hematopoietic stem cell transplantation can be used in the treatment of hematological diseases. In addition, although it is not a suitable choice for long-term treatments, fresh frozen plasma infusions are also among the treatments applied. Enzyme-linked immunosorbent assay (ELISA) is a method used to detect and quantify protein in soluble substances, based on antigen-antibody interaction and measuring enzyme activity by colorimetric analysis. The purpose of this study was to compare the total adenosine deaminase (ADA) activity in peripheral blood mononuclear cells of patients diagnosed with DADA2 with the control group. 8 patients diagnosed with ADA2 deficiency and 5 healthy individuals were studied. Two of the patients are Syrian and have a G47R/G321E heterozygous mutation. 3 of the patients have G47R homozygous. Total ADA activity was measured in lysates prepared from subjects' peripheral blood mononuclear cells using a colorimetric ADA Activity Assay kit that is a commercial kit. ADA activity was calculated by following the protocol written in the kit, and then the statistical comparison of the results was analyzed by performing the t-test. The disease-causing variant p.G47R, which occurs in the dimerization domain, affects the stability of the homodimer required for enzyme activity of the ADA2 protein. Therefore, due to the decrease in ADA2 catalytic activity in patients with p.G47R mutation, it is expected that the total ADA activity will be lower than in the healthy group. As a result of statistical analysis, a significant difference was observed in ADA activity (p=0.0008). As expected, ADA activity was lower in the patient group compared to the healthy group. In addition, when patients with heterozygous mutations were compared with patients with homozygous mutations, lower ADA activity was observed in patients with heterozygous mutations. In this case, it can be said that the G321E mutation plays an important role in catalytic activity.
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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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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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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.
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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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ÖgeBiochemical characterisation of truncated amylopullulanase from Thermoanaerobacter brockii brockii(Graduate School, 2022-06-24) Kayrav, Aycan ; Karagüler Gül, Nevin ; 521201105 ; Molecular Biology-Genetics and BiotechnologyThe last point reached as a result of the chemical transformation carried out by nature is life. Enzymes are responsible biomolecules for the chemical transformation that makes life possible. People had benefited from the reactions carried out by enzymes to obtain various products in many fields throughout history, from the times when they did not know the existence of these catalysts. Over time, people had understood how great potential these biocatalysts have with the structure and working principles. Since then the characterisation of novel enzymes and the usage area of these enzymes have been expanded day by day. Today, more than 3.000 enzymes are used in biotechnological and industrial fields and it is reported that the size of this enzyme market has reached 6 billion USD in 2021. The enzymes that dominate this market with a share of 30 % are those used in the food and beverage industry and the ones that have the upper hand in this group are the enzymes used in starch processing applications. Starch, the main component of many agricultural products and the primary source of carbohydrates for most people, is the main source of carbon in the world. In addition to starch, oligosaccharides, disaccharides and monosaccharides obtained as a result of hydrolysis are also used in various fields such as food, pharmaceuticals and biofuels. To obtain these hydrolysis products, starch is subjected to a three-step process: gelatinisation, liquefaction and saccharification. The viscous solution which is obtained by hydrolysis of α-1,4- and α-1,6- glycosidic bonds in the starch structure and gelatinisation is achieved by using α-amylase, β-amylase, glucoamylase and pullulanase enzymes during the liquefaction. During all these processes to remain the enzymes stable for a long time and maintain their activity, pH adjustment and the addition of Ca2+ ionsare required . Remove both the formed salt as a result of pH adjustments and excess Ca2+ ions causes additional steps and all of these lead to an increase in the cost of production. To eliminate these steps and perform a one-step liquefaction-saccharification process can be possible with an alternative enzyme. Amylopullulanases (E.C. 3.2.1.1/41) are enzymes capable of hydrolysing both α-1,4- and α-1,6- glycosidic bonds, while those obtained from extremophilic organisms can maintain their activities under harsh industrial conditions. In this study, the amylopullulanase (TbbApu) enzyme belonging to the Thermoanaerobium brockii brockii organism, is one of the strong candidates for starch hydrolysis processes, is examined. Previously, for the recombinant production of TbbApu, the whole apu gene had been obtained by using the primary walking method by our group and the optimisation of expression studies was in progress. As a result of the studies, pET-28 a (+) vector and E. coli BL21 (DE3) were chosen as the appropriate expression vector and and E. coli BL21 (DE3) the appropriate host, respectively. Additionally, the variants TbbApuΔSH3 without SH3 domain and TbbApuΔCBM20 without CBM20 domain were constructed to investigate the effect of SH3 and CBM20 domains. In the scope of this thesis, apart from TbbApuΔSH3 and TbbApuΔCBM20 variants, four additional truncated constructs namely TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1 and TbbApuΔX25-2-CBM20 were also obtained to disclose the effects of X25 domain. The biochemical characterisation, raw starch binding ability and kinetic studies of TbbApu and its six variants have been accomplished. The function of the X25 domain on substrate binding, activity and stability of the enzyme has been revealed for the first time with this study. The genes belonging to each construct were amplified by PCR with specific forward and reverse primers that have SacI and NotI restriction sites at their 5' ends and using the whole apu gene as a template. As a result of the amplification, tbbApuΔX25-1-SH3, tbbApuΔX25-2-SH3, tbbApuΔX25-1-CBM20 and tbbApuΔX25-2-CBM20 genes with a length of 4065, 3750, 3375 and 3060 base pairs, respectively, were obtained. Then, the obtained genes and the pET-28 a (+) expression vector were cut with SacI and NotI enzymes to form sticky ends and the ligation reactions were set up for the insertion of the genes into the pET-28 a (+) expression vector. Then, transformation was performed using competent E. coli BL21 (DE3) host cells. For the determination of positive colonies from the colonies obtained as a result of the transformation, half of the colonies were inoculated on agar plates containing red pullulan. 1 µM IPTG and 40 µg/mL kanamycin were also included in the agar plate for induction and selection respectively. For the control of detected positive colonies from the PRR plate, the other half of the colonies were inoculated into Luria-Bertani (LB) medium and plasmid isolation was performed from these cells. Then, the isolated plasmids were cut with FastDigest SacI enzyme and linearised to determine the length of the plasmids. By linearisation, tbbApuΔX25-1-SH3 - pET-28 a (+) vector with a length of 9434 base pairs; 9119 base pairs long tbbApuΔX25-2-SH3 -pET-28 a (+) vector, 8744 base pairs tbbApuΔX25-1-CBM20- pET-28 a (+) vector and 8429 base pairs tbbApuΔX25-2-CBM20- pET-28 a (+) vector were obtained from all selected colonies. After the cloning, TbbApu, TbbApuΔSH3, TbbApuΔCBM20, TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20 and TbbApuΔX25-2-CBM20 were over-expressed using magic media. To purify these recombinant enzymes, purification was achieved in three steps by using metal affinity chromatography, ion exchange chromatography and heat purification respectively. Then, the pullulanase and α-amylase activities of TbbApu and its six variants were checked by the red pullulan and starch-containing polyacrylamide gel. The biochemical characterisation of the enzymes was completed. As a result of the studies, the optimum reaction temperature for pullulanase activities was determined as 70 °C for TbbApu, TbbApuΔSH3 and TbbApuΔCBM20, 75 °C for TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3 and TbbApuΔX25-1-CBM20 and also 80 °C for TbbApuΔX25-2-CBM20 domain. The optimum reaction temperature for α-amylase activities was specified as 75 °C for TbbApu, TbbApuΔSH3 and TbbApuΔCBM20, and 80 °C for TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20 and TbbApuΔX25-2-CBM20. The pure variants were incubated for 24 hours at variable temperatures to examine the effect of temperature on the stability of enzymes. It was observed that the most stable temperature of 60 °C for TbbApu, TbbApuΔSH3, TbbApuΔCBM20, TbbApuΔX25-1-SH3 and TbbApuΔX25-2-SH3, 50 °C for TbbApuΔX25-1-CBM20 and 40 °C for TbbApuΔX25-2-CBM20. Optimum pH values for both activities were found to be 6.5 for TbbApu, TbbApuΔX25-1-SH3 and TbbApuΔX25-1-CBM20, 6 for TbbApuΔSH3, TbbApuΔCBM20 and TbbApuΔX25-2-SH3, and 7 for TbbApuΔX25-2-CBM20. It has been determined that the enzymes reached 80 % of their α-amylase activities with pH 5 and protected it up to pH 8. When the effects of different pH values on the stability of the enzymes were examined, the most stable pH according to pullulanase activities was found as pH 4 for TbbApu, TbbApuΔSH3, TbbApuΔCBM20 and TbbApuΔX25-2-CBM20, pH 6 for TbbApuΔX25-2-SH3, pH 6.5 for TbbApuΔX25-1-SH3 and TbbApuΔX25-1-CBM20 after 24 hours of incubation. According to their α-amylase activities, the most stable pH was found as pH 3 for TbbApu, TbbApuΔSH3, TbbApuΔCBM20, pH 6 for TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20 and TbbApuΔX25-2-CBM20, pH 7 for TbbApuΔX25-1-SH3. In addition, it is represented that TbbApu and its variants can maintain their stability between pH 3 and 8 and TbbApu can be used in starch hydrolysis processes without pH adjustment. When the effect of metal ions was examined, while Mn2+ and Co2+ ions increased both pullulanase and α-amylase activities of TbbApu and its variants whereas, Mg2+, Zn2+ and Al3+ ions decreased both activities of all enzymes except pullulanase activity of TbbApuΔCBM20 variant. Although α-amylase enzymes used in starch processing are Ca2+ ion-dependent, TbbApu and its variants are not dependent on Ca2+ ion for activity and stability, making the enzyme and its variants a strong candidate for starch hydrolsis process. Also, in the presence of 20 % and 50 % hexane and acetone, with some exceptions, both activities of TbbApu and its variants were increased and 20 % and 50 % butanol, DMF and DMSO presence strongly inhibited both activities of the enzymes. When the effects of organic solvents on the stability of enzymes were examined, it was observed that, with some exceptions, the stability of the enzymes increased in the presence of 20 % and 50 % acetone and hexane, compared to both pullulanase and α-amylase activities, after 24 hours of incubation. In the case of inhibitors and detergents, it was observed that inhibitors moderately inhibited pullulanase and α-amylase activities of TbbApu and its variants with some exceptions, whereas all inhibitors increased the pullulanase activity of TbbApuΔCBM20 and nonionic and anionic enzymes inhibited both activities moderately with. However, the activities of all enzymes were strongly inhibited in the presence of CTAB, which is a cationic detergent. Then, the kinetic parameters of the enzymes were elucidated. The results imply that, truncation SH3 domain improves both α-amylase and pullanase activities of the enzyme. However, truncation of CBM20 and X25 domains from TbbApu caused to loss of affinity and specificity of the enzyme to soluble starch and to shift in the specificity of the enzyme to pullulan. The removal of the SH3 domain and also CBM20 domain, the carbohydrate-binding module in the enzymes did not have any effect on the raw starch binding capacity of TbbApu. The removal of the SH3 domain and also CBM20 domain, the carbohydrate binding module in the enzymes did not have any effect on the raw starch binding capacity of TbbApu. However, sequential removal of X25 and SH3 domains resulted in 27.3 % and 58.4 % reduction in raw starch binding ability, while removal of CBM20 and X25 domains resulted in a 90 % decrease in raw starch binding ability. Thus, it was revealed that the X25 domain is involved in binding raw starch. The results of TLC analysis represented that TbbApu and its variants released maltotriose and maltose as a result of pullulan hydrolysis and maltotriose starch hydrolysis, respectively. The results of TLC analysis represented that TbbApu and its variants released maltotriose and maltose in pullulan hydrolysis and maltotriose in starch hydrolysis, respectively.
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ÖgeRecombinant production and characterization of chitinase enzyme from Pseudomonas mandelii KGI_MA19(Graduate School, 2022-06-24) Saraç Cebeci, Emine Tuğçe ; Karagüler Gül, Nevin ; 521201111 ; Molecular Biology-Genetics and BiotechnologyBiological catalysts, generally found in protein structure, that catalyze the biochemical reactions necessary for life are called enzymes. They increase the reaction rate by lowering the activation energy of the reaction catalysed. In recent years, enzymes have gained a large share in different industrial areas, like food, agriculture, pharmacy, cosmetics, waste removal, leather processing, detergent, and medical applications. Enzymes can minimize the formation of unwanted by-products, they are environmentally friendly and cheap, and have biodegradable properties. They are also considered safe for the cleaning, health, and food industries. In addition to their preferred properties, enzymes that remain active at low/high temperatures, in the presence of organic solvents, at different salt concentrations and pH values, and with an affinity for different substrates are attracting the attention of the industry. Meeting the increasing demand for the use of biomolecules with different properties in the industry is possible with the development of the physical and biochemical properties of the biocatalysts defined today with the help of protein engineering, metagenomics, advanced DNA technologies, nanotechnology, and finally, the discovery of new biocatalysts. Scientific research has been increasing in this direction, especially because enzymes from living things that live in extreme conditions can be used in a wide range of industries. Chitin (C8H13O5N)n is an inelastic, hard, white, non-elastic biopolymer formed by the bonding of N-Acetyl-D-glucosamine monomers (Glc-NAc) with β-1,4 glycosidic bonds, which ranks second after cellulose among the most abundant biopolymers in nature. It is a nitrogen-containing polysaccharide. Chitin is found in the cell wall of fungi, the outer shells of insects, the shells of sea creatures such as lobster, crab, shrimp, and the mouth areas of cephalopods such as cuttlefish and octopus which have a high annual production in the world. According to the Food and Agriculture Organization (FAO, 2019) data, 10.5 million chitin-rich shellfish (12.3%) are grown in aquaculture. The need for converting chitin wastes, which are rising in quantity by the day, into biologically useful products develops. In recent years, the employment of biocatalysts in the removal of chitin wastes instead of chemical methods, which are poisonous to the environment and expensive, has allowed for a safe conversion for the environment. Chitinase enzymes (EC 3.2.1.14), in the hydrolase class, catalyse the destruction of β-1-4 glycosidic bonds in chitin (C8H13O5N)n and separate N-acetyl-D-glucosamine molecules from the polysaccharide chain. The chitinase enzyme, which is one of the most common hydrolase enzymes in nature, is found in insects, plants, fungi and viruses. It is also commonly found in different bacterial genera such as Aeromonas, Arthrobacter, Bacillus, Chromobacterium, Flavobacterium, Pseudomonas, Sanguibacter, Serratia, and Streptomyces. With the increase in green and environmentally-friendly technology in recent years, the interest in chitinases is increasing day by day. Chitinase enzymes, which have agricultural importance, replace chemicals and pesticides used in agriculture with their ability to be used as biocontrol agents. It creates a positive effect on the marine ecosystem by recycling marine waste. Due to their pharmaceutically antifungal and antibacterial properties, they are used in the production of anti-inflammatory and anti-fungal drugs, anti-cancer and immune-enhancing agents, wound dressings, contact lenses, and surgical sutures. It is a bioprotective additive used to increase shelf life in the food industry. Antarctica with the highest elevation and lowest temperature has recently become a popular research area. 70% of the freshwater resources on the Antarctic continent, which has not been touched by human hands until lately, are in the form of ice. It also features powerful winds, extremely cold temperatures, and is exposed to low temperatures in the winter and high UV rays in the summer. It has a natural and distinct habitat as a result of these factors. Because of the harsh circumstances, it is unavoidable to uncover new species and enzymes and genes. Extremophiles are living systems that can thrive in harsh environments. Biocatalysts derived from extremophilic organisms and demonstrating catalytic activity even under adverse circumstances have been dubbed extremozymes. Psychrophiles and psychrotolerants are extremophilic microorganisms that can live at 0-20 °C and 0-30 °C, respectively. Because of their low energy needs, flexibility, and high catalytic activity, cold-compatible enzymes derived from psychrophilic and psychrotolerant organisms are crucial for commercial applications. Biotechnological approaches help the identification of novel and diverse extremophiles and extremozymes from Antarctica that has not been described in the literature yet. In the study, which was carried out using Antarctic sediment samples collected within the scope of the 2nd National Antarctic Science Expedition (TAE-2), 12 sediment samples taken from 8 different regions were cultured and enriched. Subsequently, freeze-thaw stress was applied to each cultured sample. After the applied freeze-thaw stress, it was observed that five cultures were resistant to stress. Two morphologically different colonies were selected from each stress-resistant sample, DNA isolations were made and 16S rRNA analyzes were carried out. The sample with the lowest similarity rate according to 16S rRNA analysis was sent for whole-genome analysis and according to the results of the analysis, a new strain of Pseudomonas mandelii, Pseudomonas mandelii KGI_MA19, was identified. Phenotypic and biochemical characterizations of the identified KGI_MA19 strain were performed. The next step was the recombinant production and the characterization of the chitinase enzyme of the psychrotolerant Pseudomonas mandelii KGI_MA19. The gene region of the chitinase enzyme of Pseudomonas mandelii KGI_MA19 was amplified by polymerase chain reaction (PCR) by designing gene-specific primers containing SacI and NotI restriction sites. The amplified gene region of interest and the pET-28a (+) vector were cut with SacI and NotI restriction enzymes. T4 DNA ligase enzyme was used for ligation of the cut PCR product and expression vector. Plasmid pET-28a (+) containing the PCR product was transformed into Escherichia coli BL21 (DE3) competent cells. The colony PCR method was applied to determine the colonies containing the relevant gene as a result of the transformation, and plasmid isolation of two colonies selected from among the colonies thought to be positive was performed. Then, to understand whether the colonies contain the gene product or not, the plasmid was cut with the EcoRV endonuclease enzyme, which has a fast-cutting feature. The DNA sequencing results showed that the chitinase gene-specific PCR product was correctly inserted into the pET-28a (+) plasmid. The Magic MediaTM (Invitrogen), which is commercially available and used in the expression of E.coli cells, was used to monitor the expression level of the chitinase gene in E.coli BL21 cells. At the end of Magic Media incubation, cells were treated with lysis (0.1M Tris-HCl, pH 8.0, 0.3M NaCl) buffer, and homogenization was performed by ultrasonication using the M73 probe. A high amount of produced chitinase enzyme was purified by the His-Tag affinity chromatography method using the His-Trap column. The activity of the chitinase enzyme obtained in high purity was determined by the 3,5-Dinitrosalicylic acid (DNS) method which allows one to determine the amount of reducing sugars expected to be released as a result of the reaction performed by the usage of colloidal chitin as a substrate. Biochemical characterization including optimum pH, pH stability, and optimum temperature, thermal stability has been completed. A new, psychrotolerant strain, Pseudomonas mandelii KGI_MA19, was identified from sediment samples from the Antarctic King George Island, and its molecular, phenotypic, morphological, and biochemical characterization was completed. In addition, the chitinase enzyme which has an increasing impact in the industrial field has been successfully produced and biochemically characterized by using recombinant DNA methods. The cold-adaptive chitinase enzyme of the Pseudomonas mandelii KGI_MA19 strain, which was obtained for the first time within the scope of this thesis, is thought to be a promising potential biocatalyst for industrial applications.
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ÖgeExpression, purification, and characterization of soluble recombinant TNFR1(Graduate School, 2022-06-28) Hatipoğlu, Derya ; Dinler Doğanay, Gizem ; 521191107 ; Molecular Biology – Genetics and BiotechnologyTumor Necrosis Factor Alpha (TNF-α) is a trimeric cytokine secreted by macrophages and monocytes. It belongs to type II transmembrane protein family and is involved in both innate and adaptive immune system. TNF-α exists in two forms: transmembrane TNF (tmTNF), which is synthesized as a precursor form, and solubilized TNF (sTNF), which is created after further processing. TNF-α functions in a variety of biological processes by interacting with specific receptors namely as TNFR1 and TNFR2. TNFR1 consists of intracellular, transmembrane, and extracellular domains. The extracellular part consists of four cysteine-rich parts. When interacting to TNFR1, TNF-α can activate various signaling pathways for instance inflammation and apoptosis. TNF-α level is undetectable in normal individuals, yet under inflammation conditions the protein concentration in serum increases proportionally to the inflammation level in the body, thus making the protein detectable. Controlled production of TNF-α is crucial for tissue healing and fight against infection, but a constantly high level of TNF-α can cause various diseases including rheumatoid arthritis (RA), ankylosing spondylitis (AS), psoriasis/psoriatic arthritis, and Chron's disease. TNF-α inhibitors are designed and used in treatment of diseases caused by overexpression of TNF-α. An alternative pathway includes employment of TNFR1 extracellular domain as a biotherapeutic tool to inhibit the effects of overexpressed TNF-α. The latter approach seems to be way more preferred than other therapeutics due to its targetselectivity and high affinity. Production of proteins like TNFR1 are characterized by cysteine-rich domains by using bacterial systems is quite advantageous in terms of cost, yield, and time. However, lack of an effective translation system in Escherichia coli (E. coli) may hinder production of such disulfide bond containing proteins mainly in terms of generating mismatched cysteine residues or failing in acquiring a sulfide bridge formation thus leading to protein aggregation and inclusion body formation. Recovering proteins from inclusion bodies is time-consuming and expensive while considering the protein loss and incorrectly folded proteins due to the wrongly formed disulfide bonds. Dsb protein family including DsbA, DsbB, DsbC, DsbD, and DsbG, is responsible for the formation of disulfide bonds in bacteria. The DsbA-DsbB complex plays role in the oxidative pathways, while DsbC-DsbD complex functions in the isomerization pathway. Among all Dsb proteins, DsbA and DsbC are the most extensively studied and widely used in the field of biotechnology. Our previous studies showed that the extracellular portion of TNFR1 forms inclusion bodies in E. coli, and in vitro refolding steps are needed to obtain the correct xxvi conformation. Protein aggregations during the refolding process also caused a decrease in yield. The purpose of this study is to obtain the extracellular part of TNFR1 as a soluble protein in E. coli. Initially, a SHuffle T7 strain with a cytoplasmic DsbC copy was selected as the host cell and expression experiments were carried out accordingly. However, the target protein formed an inclusion body. To assess the effect of DsbC in the correctly folded-soluble TNFR1 production, a expression vector containing fusion protein DsbC-TNFR1 was constructed. Formation of DsbC-TNFR1 fusion protein was held on the basis of plasmid design conferring the order of a 6x histidine tag, DsbC, TEV cleavage site, and TNFR1 from N to C terminus respectively. It was assured that periplasm target sequence was removed from DsbC gene, allowing its production within the bacterial cytoplasm. DsbC-TNFR1 production was verified by SDS-PAGE and immunoblotting analyses, which revealed that some conditions led to the production of the fusion protein in soluble form. In order to detect the optimum conditions to produce DsbC-TNFR1, four different E. coli strains as host cells were employed, namely as Rosetta (DE3), Rosetta-gami 2, SHuffle T7, and BL21 (DE3). However, since production of the Rosetta (DE3) strain was higher than others, the research was conducted on that particular strain. Induction conditions including IPTG induction and autoinduction medium were also assessed to get higher yield from the selected cells. As a result of induction trials, the target protein was obtained in soluble form when autoinduction was utilized. Following sonication and centrifugation of the cells, the affinity chromatography was performed for separation of the recombinant protein from other host cell proteins. Anion exchange chromatography (AEX) was used as a second purification step to remove remaining impurities. The efficiency of purification was evaluated by using SDS-PAGE and immunoblotting analyses. Then, protein characterization studies were performed. The isoelectric point of the pure protein was calculated by isoelectric focusing via capillary electrophoresis device, and the purity of the protein was checked using the purity determination method. The secondary structure of the fusion protein was analyzed by circular dichroism spectroscopy and it was determined to be in the α-helix structure. Intact mass analysis through LC/MS was also performed to calculate the molecular weight of the protein. In addition, the peptide mapping was used to identify the amino acid sequence. The native structure of the pure protein was investigated by using blue native polyacrylamide gel electrophoresis (BN-PAGE), which revealed that the protein exists in both dimers and different oligomer structures. The functionality of the fusion protein was assessed by performing a pull-down assay, which showed that DsbC-TNFR1 is functional as it was capable of binding to TNFR1 ligand TNF-α. As a consequence of the research, it was revealed that the implementation of such a fusion protein is a successful tool for expressing TNFR1 in its soluble form in bacterial cells. After analysis of the pure protein generated as the outcome of downstream steps, it was shown that the TNFR1 produced by using the implemented fusion method is functional.
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ÖgeCharacterization of BAG-1S/C-raf interaction targeting peptide(Graduate School, 2022-07-25) Çebi, Ecenur ; Doğanay Dinler, Gizem ; 521201110 ; Molecular Biology-Genetics and BiotechnologyERK/MAPK cascade is one of the most important cellular pathways, which regulates many distinct physiological functions such as cell proliferation, migration, differentiation, and apoptosis. Since it has vital roles in many cellular functions, dysregulation of its members usually results in uncontrolled proliferation and development of cancer cells such as breast cancer. Ras and Raf serine/threonine protein kinases are mostly deregulated components of this pathway and mutations in both proteins have been identified approximately one-third of all human cancer types. Although there are several inhibitors against oncoproteins, because of the improving drug resistance, there is a need for the development of new drugs. One of the emerging interest is to target protein-protein interactions (PPIs) related with cancers. Human Raf kinase family has three isoforms: A-Raf, B-Raf, and C-Raf. C-Raf and B-Raf are the important members of ERK pathway. Although B-Raf is dysregulated mostly, C-Raf has ability to suppress apoptosis and hence, both of the Raf kinase is vital for the cancer progression. The Raf kinases are regulated by several phosphorylation events and also, interactions with some proteins like BAG-1. Bcl-2 associated athanogene 1 (BAG-1) which is an anti-apoptotic co-chaperone protein is usually overexpressed in several cancer types making it possible oncoprotein. Moreover, its known that the interaction between BAG-1 and C-Raf promotes the C-Raf activation and stabilization. Therefore, targeting this interaction with small molecules and/or peptides would be effective therapeutics against different cancer types. In our previous studies, the interaction surface between BAG-1S and C-Raf was determined and a peptidomimetic which coded as Pep3 was designed against the BAG-1S from natural sequence of C-Raf. The aim of this study was to characterize the interaction between BAG-1S and Pep3 and also, the interaction between BAG-1S and TPep3 which is cell penetrating form of Pep3. Firstly, His-tagged BAG-1S proteins were produced in mammalian and bacterial cells. Then, BAG-1S proteins were purified by Ni-NTA affinity purification in two steps. After first step, His-tag was cleaved with TEV protease and tagless proteins were eluted as flow-through in second step while the impurities and TEV proteases were remained bound to resin. Then, the purities of both proteins (produced in mammalian and bacterial) were calculated as >80% by SDS-PAGE. Secondary structure analysis of proteins was performed with circular dichroism and both of the proteins has been folded and showed primarily ɑ-helix characteristics. After BAG-1S proteins were characterized. Pep3 and TPep3 characterization were performed by mass spectrometry and circular dichroism analysis. Pep3 and TPep3 has been synthesized by Fmoc-based solid phase peptide synthesis and they were dissolved in 20 mM AMBIC and water respectively since the Pep3 shows hydrophobic characteristics. Molecular weights of peptides were measured as 2.066 kDa for Pep3 and 3.653 kDa for TPep3 which were compatible with the theoretical calculations. Both of the peptides showed β-sheet characteristics approximately 30% combined with random coils. The interaction between BAG-1S and peptides were confirmed by crosslinking reaction with medium length crosslinker DSS (disuccinimidyl suberate). BAG-1S proteins were incubated with peptides separately to form interaction. After they formed interaction, the crosslink reaction was performed with 50-fold molar excess of DSS. The reactions were analyzed by immunoblotting. BAG-1S was shifted 2 kDa when interacts with Pep3 and 4 kDa with TPep3 comparing to the BAG-1S only reaction. These results were confirmed the interaction of BAG-1S with two peptides. The binding kinetics of BAG-1S and Pep3 was measured by Surface Plasmon Resonance (SPR) with multi-cycle kinetics method. BAG-1S protein was captured on Protein G chip with Anti-BAG-1 and different concentrations of Pep3 was injected. Binding kinetics was calculated as 68.56 nM by 1:1 binding model showing that Pep3 binds BAG-1S with high affinity. The effects of TPep3 on MAPK pathway and cell viability of MCF-7 breast cancer cells were analysed. MCF-7 cells were treated with different concentrations (0 uM to 50 uM) of TPep3 and total proteins were analysed with immunoblotting. C-Raf and p-C-RAf (S338) levels were decreased with the increasing concentration of TPep3. As a result of p-C-Raf inactivation, p-MEK levels were also decreased which showed the decrease in ERK pathway. Moreover, B-Raf and p-B-Raf (S446) levels were decreased. So, it can be said that peptide does not only affect the activitation of C-Raf but also B-Raf. To see the effects of peptide on other cell survival pathways, Akt and p-Akt (S473) levels were analysed and it was seen that their levels remained unchanged even with the highest peptide concentration. Lastly, the IC50 value was calculated as approximately 18 uM from the cell viability MTT assay. Together with all the results, TPep3 and Pep3 peptides have potential to be a promising therapeutics for cancer types relying on ERK pathway since the activity of pathway was decreased with the peptide treatment.
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ÖgeImmunoreceptors modulate eosinophilic functions in viral immunity(Graduate School, 2022-11-09) Durmuş, Lübeyne ; Muğan Çıracı, Ceren ; 521201116 ; Molecular Biology-Genetics and BiotechnologyImmunity a term used as resistance to pathogens, also referred to reactions of the body to noninfectious compounds such as tumors, harmless environmental substances and even sometimes host's components, each of which are called as tumor immunity, allergy and autoimmunity, respectively. The whole components like cells, tissues and molecules that generate this immunity is defined as the immune system and the immune response is coordinated by these components to foreign substances. Providing protection against infections or eradication of the infectious compounds from the body is the major physiological function of the immune system. Besides, growth of some tumors and cancers can be inhibited by stimulating immune reactions against cancer cells. The human defense system against pathogens can be divided into 3 levels: physical and chemical barriers, innate immunity and adaptive immunity. The innate immunity relies on a finite number of receptors for detecting the invading pathogens. These limited number of receptors target large groups of pathogens by recognizing conserved microbial patterns. Furthermore, activation of adaptive immune response is achieved by innate immune response. Innate immune cells include both hematopoietic and nonhematopoietic origin which make them different from adaptive immunity which relies on T and B lymphocytes. Innate immune response development involves hematopoietic cells that differentiate into monocytes, macrophages, mast cells, dendritic cells, natural killer cells, natural killer T cells, basophils, neutrophils and eosinophils. Moreover, hematopoietic cells include skin and epithelial cells reside in respiratory, genitourinary and gastrointestinal tracts. Innate immunity depends a few germline encodedreceptors which sense pathogen specific structural motifs. These receptors are collectively called pattern recognition receptors (PRRs) and microbial components recognized by PRRs are called pathogen associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). PRRs can exist in various cellular compartments like cell membrane and endosomal membranes, or cytosol. Moreover, they can be found in extracellular environment such as bloodstream or interstitial fluids. They are mainly divided into four major types: 1) Toll-like receptors (TLRs), 2) Nucleotide binding and oligomerization domain-like receptors (NLRs), 3) Retinoic acid-inducible gene 1-like receptors (RLRs), 4) C-type lectin receptors (CLRs). Activation of these receptors of the innate immune cells result with induction of adaptive immune cells. Hence, PRRs help to generate immune response to eradicate infectious agents, for example they induce the death of the infected cells. Humans are constantly under the threat ofinvasive opportunistic microorganisms including viruses, fungi, bacteria and parasites. The detection and development of an appropriate immune response against invading viruses are crucial for the outcome of virus infections. Recognizing viral nucleic acids is the first step for sensing virus infection. This recognition mainly depends on the genetically predetermined and germline encoded PRRs. There are TLR protein family members recognize viral genetic material such as TLR3, TLR7, TLR8 and TLR9. Upon recognition of viral nucleic acids or proteins by PRRs, production of type I IFN is induced resulting in the activation of target cells in both autocrine and paracrine manners. TLR7 and TLR8 have the ability to detect the existence of various single stranded RNA (ssRNA) viruses. These viruses include influenza, vesicular stomatitis virus (VSV), HIV, Sendai virus (SV) and a number of coronaviruses and flaviviruses. TLR3 is an endosomal nucleotide sensor which is located on endosomes and activated by double stranded RNA (dsRNA). As dsRNA is the genome for many viruses or they synthesize dsRNA during their life cycles, TLR3 also detects the existence of dsRNA and DNA viruses. Unlike TLRs, RLRs are RNA detectors locolized in the cytosol. RLR protein family includes three members: RIG-I, MDA5 and LGP2. Upon viral RNA association and oligomerization, RLRs bind to mitochondrial antiviral signaling protein (MAVS) through its CARD. MAVS has an essential role in RLR signal transduction as an adaptor protein whichinduces TBK1 and IKKε leading to the activation of IRF3 and IRF7. These transcription factors and NF-κB, together, activate the production of type 1 IFNs. Moreover, NLRs are important part of the cytosolic innate immunity and have a role in various key pathways such as inflammasome, MAPK, NF-κB and type 1 IFN signaling. Activation of inflammasome complexes is essential because it induces inflammation by leading to the production and secretion of IL1β and IL18 also known as inflammasome dependent cytokines. Eosinophils attract considerable attention with their identified roles in many pathological processes such as acute and chronic infections, cancer and thrombosis. Although their accepted roles in parasitic infections, involvement of eosinophils in fungal, bacterial and viral infections is an on-going research topic in the field of immunology. These granulocytes contain and generate substances with antiviral functions such as RNases and they may also involved in adaptive immunity with their potential antigen-presenting ability. Together, findings about eosinophils indicate potential antiviral role for eosinophils which need to be explored further. Despite the studies on animal models and primary human eosinophils demonstrating the importance of eosinophils against viral infections, the question of how eosinophilic functions are regulated following he viral infections is still ambiguous. Thus, the aim of the study is to investigate the changes in eosinophilic functions upon activation of TLR3, TLR7 and TLR8 with proper ligands, poly(I:C), R848, and ssRNA40, respectively. low number of eosinophils in blood (1-6%) makes them difficult to study in vitro. Therefore, we utilized EoL-1 human eosinophil line as a model in our study which was previously shown to serve an ideal model due to the expression of eosinophilic markers In this study we initially determined the activation of Eol-1 cells upon treatment with viral ligands, eosinophilic PRRs and immune receptors. Secondly, surface markers of eosinophils were determined to further understand the eosinophilic functions. Also, cytokines that they released were analyzed to understand the potential involvement in the induction of adaptive immunity. Moreover, production of matrix metalloproteinases was measured. We observed significant increase in IL-6 and IFN-γ secretion upon TLR8 activation. Also, the number of IL5Rα and PDL1 expressing Eol-1 cells were augmented with TLR3, TLR7/8 and TLR8 inductions. Our data suggested roles for TLR3, TLR7 and TLR8 in eosinophilic functions. We then investigated the changes in granule content of eosinophils during viral infections. We showed that ECP mRNA levels were upregulated upon the ssRNA40 treatment in Eol-1 cells. This data also, demonstrate the possible roles of ECP on the regulation of antiviral eosinophilic functions. MMPs are matrix-degrading enzymes which have roles in leukocyte recruitment to chemokines during microbial infections. In addition to their functions in immune regulation, MMPs can also lead to tissue damage as a result of persistant pathogen infections or spread. Furthermore, CD147 is a matrix metalloproteinase inducer that plays critical roles in various viral infections. Recently, CD147 was shown as an alternative receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Therefore, we also investigated the MMPs and MMP inducer CD147to better understand their roles in eosinophilic antiviral immune responses. The results from RT-qPCR showed a relationship between both CD147 and TLR activation as well as the data obtained from gelatin zymography clearly indicated the relationship between MMP-9 and TLR activation in Eol-1 cells. All stimulants tested in this study elevatedthe CD147 at mRNA level in Eol-1 cells. Additionally, TLR3 and TLR8 ligands significantly increased the Pro-MMP9 production at 24 hours post-induction in EoL-1 cells. Upregulation of TLR8 led us investigate the potential adaptor molecules which may have roles in downstream signaling pathways through TLRs in eosinophils. CARD9 is a multifunctional adaptor protein which participates in different phases of the immune system including fungal, bacterial and viral infections. Thus, we investigated the CARD9 expression in Eol-1 cells upon TLR3, TLR7/8 and TLR8 inductions. Despite the decreased mRNA expressions of CARD9, there was a significant increase in CARD9 expression at protein level in Eol-1 cells. Next, we measured the expression levels of cytosolic PRR RIG-I at protein level because of its well-known roles in antiviral immunity. We observed 1.49-fold increase in the cells stimulated with TLR8 agonist (ssRNA40, 2 μg/ml). Then, we measured NOD2 as it is already known as the interaction partner for CARD9. Numerous studies formerly showed the NOD2 involvement in response to many RNA virus infections such as parainfluenza virus, VSV, RSV and IAV. Here we showed that NOD2 protein expression was significantly increased after induction with TLR7/8 and TLR8 ligands in Eol-1 cells. Overall, of all the stimuli we tested, ssRNA40 (TLR8 signaling) was the most potent in inducing the mRNA expressions of ECP and CD147, and protein expression of immune receptors, surface markers of eosinophils and cytokines. Interestingly, CARD9 which is a critical adaptor against fungi infections was significantly increased after induction with TLR7/8 ligands, suggesting an important role for TLR7 and TLR8 rather than TLR3 in antiviral immune response generated by eosinophils. Our findings will pave the way for future studies focusing on eosinophil related infectious diseases.
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ÖgeControlled release of tetracycline hydrochloride from silica based polycaprolactone nanohybrides(Graduate School, 2022-12-23) Cengiz, Aybüke ; Güvenilir, Yüksel ; 521191130 ; Molecular Biology - Genetics and BiotechnologyBiomaterials can be synthetic or natural materials which is designed for interact with the biological systems. There are various type of biomaterials such as metals, seramics and polymers. Biodegradable biomaterials that naturally degrade or completely dissolve in their physiological environments have gained attention for both invasive and noninvasive health monitoring due to providing an unique opportunity for therapeutic field. Biodegradable polymers are classified into two main categories as natural and synthetic polymers. Polymers offer high adjustability in terms of their chemical structure and morphology and they contain hydrolysable bonds and these bonds makes them prone to chemical degradation via hydrolysis or enzymecatalyzed hydrolysis. Biodegradable polymers are using in controlled drug delivery, anticancer drug delivery, protein and peptide delivery, gene delivery, and enzyme immobilization at industry and researches. Biodegradable polymers can degrade in two main ways; partially or fully degrade to monomeric units. Polymers' degradation rate is heavily affected by various factors. Examples of these factors are the following such as the morphology, molecular weight, its distribution, crystallinity temperature and environmental conditions. Polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid (PLGA) and poly(ε-caprolactone) (PCL) are some of the most used synthetic biodegradable polymers. These are polyester polymers that have chemical structure with ester bond linkages and these polymers most used in industry are aliphatic polyesters. Polycaprolactone (PCL) is a linear aliphatic polyester hydrophobic semi-crystalline. PCL can synthesis with two different methods. These are the condensation of epsilon-hydroxycaproic acid and the ringopening polymerisation (ROP) of e-CL. In the condensation of epsilon-hydroxycaproic acid which is a polycondensation, 6-hydroxycaproic acid is polymerising by using lipase from Candida antarctica. ROP is the most preferred route and it gives a polymer with a higher molecular weight and a lower polydispersity. PCL is mainly synthesized by ring-opening polymerization (ROP) of ε-caprolactone (ε-CL) monomer. There are three different ROP catalytic system and metal-based catalysts are the most used catalysts for ROP of ε-CL. The metal based compounds takes part in the ROP of lactones can be describe as catalysts, initiators, initiating systems or catalytic systems. PCL have wide applicability and advantage such as biocompatibility, controlled degradability, miscibility with other polymers, and if its properties can be controlled and it can be made inexpensively, it can be a very useful polymer. Drug delivery systems (DDSs) are developed to prevent problems such as reducing therapeutic efficacy and causing unwanted side effects for improving drug safety and helping to improve patient compliance and convenience. DDS have many important applications in every field of medicine such as cancer, pain, diabetes and ischemia, myocardial treatment. Release pattern of DDS mainly effect by the delivery vehicle , the drug properties, and the environmental conditions. Drug delivery systems varies based on their route, mechanism and materials used in and there are several approaches of DDS. One of these approaches is polymers that used in a variety of fields in pharmaceutical applications. Polymers have advantages in DDSs as providing controlled release of therapeutic agents in constant doses over long periods and their cyclic dosage, and tunable release of both hydrophilic and hydrophobic drugs. In the first part of this study performed RHA preparation and activation of RHA, immobilization followed by polymerization to obtain nanohybrid polymers. Rice husk ash (RHA) is obtained by burning husks of rice. prepared and then silanized with 3-Glycidoxypropyltrimethoxysilane (3-GPTMS). RHA silanized by using 3-GPTMS as support material to immobilize free enzyme Candida antarctica lipase B. Enzymatic ring-opening polymerization (eROP) of ɛ-caprolactone was provided by immobilized lipase enzyme Candida antarctica lipase B (CALB). eROP reaction started along with ε-caprolactone and immobilized enzyme, then G-PCL/RHA nanohybrid obtained after terminating the reaction with chloroform followed by the precipitation. In the second part of the study microspheres prepared by trying different conditions to find best efficiency ones and followed by Drug release experiment to the highest effiency microspheres. Drug loaded microspheres were prepared by W/O/W double-emulsion-evaporation method which is water-in-oil method. Since the drug used in study is a water-soluble drug active substance, have to find the right ratio for both PVA and drug amount. Because of this, tried to find best PVA percentage by using different PVA percentages. Here found the best ratio is %1 PVA. Then looked at the drug amounts by preparing microspheres in %1 and different drug amounts. G-PCL/RHA drug-loaded microspheres with best drug loading efficiency we can get were obtained. Drug release experiments were carried out at pH 7.4 condition The release profiles of the drug-loaded G-PCL/RHA microspheres were determined and afterwards the drug release percentages are calculated. According to drug release results, the highest cumulative drug release percentage was %56 at pH 7.4 in %0.5 PVA, 10mg drug amount conditions microspheres with 72 hours burst level and %29 drug loading. Meanwhile the highest drug loading, efficiency, was in %1 PVA and 10mg drug amount conditions with %54 drug loading. In the last part of study, experiments has been finalized with charactization of microspheres to analyse microspheres by using Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). FTIR spectra of G-PCL/RHA nanohybrid and drug-loaded G-PCL/RHA microspheres were compared along with literature tetracycline hydrochloride FT-IR spectra and the presence of tetracycline hydrochloride in the micropheres demonstrated. FT-IR results showed both G-PCL/RHA nanohybride and tetracycline FT-IR spectra on G-PCL/RHA microspheres FT-IR results. This proved the tetracycline hydrochloride presence on G-PCL/RHA nanohybrid microspheres. TGA results showed the decomposition temperatures and organic weight losses of drug-loaded G-PCL/RHA microspheres and tetracycline hydrochloride. According to DSC analysis, showed the melting and crystallization points of PCL polymer. Microsphere structures were observed by SEM analysis. Particles of the G-PCL/RHA microspheres were seen to be spherical and sphere-like structures with various size but they were more disadvantage cause of not being as highly porous compare to the literature microspheres. For better understanding drug-polymer interactions and higher how to get higher drug efficiency of microspheres on drug release mechanism, analysis methods can be developed in further studies. Beside drug release of hydrophylic drugs loaded microspheres can be carried out in vivo. Such studies exist in the literature.
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ÖgeThe synthesis, SLIC based labeling, and characterization of microbial rhodopsins by using custom build spectroscopic methods(Graduate School, 2023-01-26) Çavdar, Cansu ; Bayraktar, Halil ; 521191105 ; Molecular Biology-Genetics and BiotechnologyType I opsins (also known as microbial opsins) are seven transmembrane-domain proteins with retinal chromophore absorbing incoming light. Most of them are ion channels or pumps although they do not directly bind to G protein complexes. They are found in all three domains of life. Numerous homologous forms of rhodopsins have been identified in the microorganisms, including light sensors (sensory rhodopsins), transmembrane chloride pumps (halorhodopsins), and energy saving transmembrane proton pumps (bacteriorhodopsin or proteorhodopsin). Rhodopsin proteins are widely used in the field of biotechnology. For example, it is used to determine the membrane voltage level in neurons. The use of rhodopsins as tools to control membrane potential with light is another technique for transformative optogenetics technology. Membrane voltage is present in all cells, and it creates an electric signal to carry the signal across the cell membrane and provides cell-cell communication. Since the absorption methods are not sufficient to measure voltage signal due to low signal to noise ratio in cells, more sensitive fluorescent methods based on rhodopsin are strongly preferred for a wide spectrum of applications. An understanding of the dynamics of the microbial rhodopsin proteins is essential to tune the photophysical properties of rhodopsins. It is also necessary to label them with fluorescent proteins and characterize their localization in detail. For the fluorescent signal to vary with the amount of light absorption in the membrane protein, the linker peptide between the proteins has to be optimized for various applications. For electrochromic fluorescent energy transfer, it is necessary to select the appropriate fluorescent protein. The emission signal of the fluorescent protein must overlap with the absorption signal of the membrane protein. After the most suitable fluorescent proteins are selected by calculating the amount of overlap, the structure of the peptide that binds the membrane and the fluorescent protein should be determined. Since both the length and the bending ratio of the selected peptide are important, it is necessary to optimize by testing different constructs. Here we have synthesized, purified, and studied the rhodopsin by using molecular biology and various spectroscopy methods. After the synthesis of rhodospins in BL21 cells, it was purified with his-tag affinity column chromatography and reconstituted with a detergent solution. The color tuning of rhodopsin as a function of pH was investigated by using absorption spectroscopy. We found that BPR undergoes a large red shift under acidic conditions. A pH value was increased the color turned from orange to red at the basic solution. We concluded that the deprotonation of the retinal at the rhodopsin center results in a significant change in the color of BPR. We have also measured the transient absorption changes of BPR by using a custom home built spectrometer that was equipped with two laser lines and an op amp light detector. The data acquisition and the control of lasers were performed an by arduino and field programmable gate array device programmed with arduino and labview respectively. Our results indicate that BPR underwent an absorption change after stimulated with a 532 nm diode laser. Finally, the fluorescent proteins were also cloned into the SRII gene by using SLIC cloning method and expressed in BL21 cells to determine the changes in fluorescent emission. Sensory rhodopsin was similarly characterized by using absorption spectroscopy. As conclusion, BPR undergoes a large spectral shift due to deprotonation upon decreasing pH and alters the color of the protein. SLIC method provides a cost-efficient method to prepare fluorescently labeled rhodopsin proteins. Contrary to the standard cloning techniques used in molecular biology, the SLIC method, which is convenient in terms of time and cost, has been studied and the method has been optimized. The optimized SLIC method can be used as an alternative to other molecular cloning techniques. The custom build pump-probe system can also be used for the characterization of fluorescently labeled other rhodopsin proteins in future studies.
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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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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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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.