LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Doktora

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http://hdl.handle.net/11527/19402

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Protein engineering applications on industrially important enzymes

(Fen Bilimleri Enstitüsü, 2019) Özgün, Gülşah ; Karagüler, Nevin Gül ; Moleküler Biyoloji-Genetik ve Biyoteknoloji ; Molecular Biology-Genetics and Biotechnology

Büyüyen 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.
Investigation 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 Biyoteknoloji

Neurodegenerative 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.
Discovery of novel enzymes using proteomic approaches

(Graduate School, 2021-01-26) Kılınç Öztuğ, Merve ; Karagüler, Nevin Gül ; Akgöz, Müslüm ; 521142107 ; Molecular Biology-Genetics and Biotechnology

Thermophilic 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.
Investigating 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 Biyoteknoloji

The 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.
Investigation of familial multiple sclerosis genetics

(Graduate School, 2022-05-16) Everest, Elif ; Turanlı Tahir, Eda ; 521152104 ; Molecular Biology-Genetics and Biotechnology

Multiple 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.
Development 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 Biotechnology

Glioblastoma Multiforme (GBM) is the most malign form of glial tumors, which accounts for the majority of brain tumor cases worldwide. There have been different approaches to treat GBM effectively, and with the advancements made for the last decade molecular pathology, target driven therapy, and personalized medicine gained attraction. One of such promising targets for GBM is Bcl-2 induced intrinsic apoptosis pathway. Anti-apoptotic members of Bcl-2 induced intrinsic apoptosis pathway have an important role in the regulation of GBM cell death. In this thesis study, we screened seven potential Bcl-2 inhibitor compounds and evaluated their effects on proliferation of GBM cells as well as their inhibitory capacity of Bcl-2 protein. Of those, I further analyzed three of them namely 58, 243, and ind-199. 58 and ind-199 compounds did not show any significant anti-proliferation effect on GBM cells. Eventually, we decided to elucidate the mechanism of action of 243 compound, a thiazolidine derivative BH3 mimetic, which was the most promising one according to the in vitro proliferation experiments. I performed colony formation assay to assess proliferation of YKG1 GBM cells, additionally to the proliferation assay with A172 GBM cells. While 243 inihibited cell growth significantly compared to control group, Bcl-2 inhibitor ABT-199 did not inhibit cell proliferation. Moreover, I tested 243 on YKG1 tumorspheres to determine its effectivity on tumor initiating cancer stem cells (CSC). Both ABT-199 and 243 had inhibitory effect on CSC proliferation, however 243 was significantly more effective than ABT-199 when compared to control group. Since 243 is a Bcl-2 inhibitor, I analyzed key players of Bcl-2 family and intrinsic apoptosis pathway. I have analyzed gene expression levels of BCL2, BCLXL, BAX, CASP3, CASP7, and CASP9. Furthermore, I also analyzed genes related with cell death which are CASP8 and TP53. Time dependent quantitative RT-PCR results suggested that, GBM cells that are treated with Bcl-2 inhibitors ABT-263 and 243 acts differently in case of gene expressions related to apoptosis. Next, we wanted to show apoptotic cell death with Annexin V-PI assay. Interestingly, we did not detect significantly elevated apoptosis in A172 cells when they are treated with either ABT-199 or 243. Similarly, cell cycle analysis showed that 243 did not have any effect on cell cycle, altough ABT-199 induced G1 phase arrest. Moreover, I determined expression levels of apoptosis related proteins PARP, Caspase-3, and Caspase-9. I used staurosporine treatment as a positive control to induce apoptosis. None of the treatment groups apart from staurosporine increased cleaved-PARP expression. Similarly, I checked if there is a difference in expression of Pro-caspase-3 and Pro-caspase-9, and observed that only stauroporine treated group expressed lower levels of Pro-caspases, indicating that cleaved forms of both Caspase-3 and 9 were produced upon staurosporine treatment only. At this point, we hypothesized that both ABT-199 and 243 could only induce limited apoptotic cell death because BCL2 expression was relatively low in A172 cell line. Expectedly, when I compared gene expression levels among different cell lines, I observed that BCL2 expression was very low in A172 cells, and it was abundant in SH-SY5Y neuroblastoma cells. Therefore, I decided to analyze apoptosis of SH-SY5Y cells after a treatment with ABT-199 and 243. Within only 48 hours of treatment with both inhibitors, I observed apoptotic cell death of SH-SY5Y cells. Hence, we had a new hypothesis that when BCL2 expression is low, upon Bcl-2 inhibitor treatment, cells may die through autophagy since Bcl-2 forms a complex with autophagy related protein Beclin 1. I showed that 243 treatment significantly upregulated autophagy related genes such as BECN1, ATG5, and MAP1LC3B, whereas ABT-199 induced autophagy on limited level. Moreover, autophagy indicative LC3B-II expression was significantly upregulated on a protein level with the 243 treatment, when compared to control as well as ABT-199 treatment. Additionally, I determined protein expression level of p53, which has a role in the interplay between apoptosis, cell cycle, and autophagy. I observed that p53 protein expression was increased upon both ABT-199 and 243 treatment, when compared to control group. Expectedly, when we performed in silico computational analysis, Beclin 1:Bcl-2 interaction and binding of 243 to their BH3 binding domains, we observed that 243 binds to Bcl-2 through important interactions. Since 243 and Beclin 1 binds to Bcl-2 from the same domain, when cells are treated with 243, Beclin 1 cannot bind to Bcl-2 and therefore it is released to initiate autophagy. In addition, we demonstrated that 243 significantly reduced in vivo tumor growth and prolonged survival in orthotropic brain tumor models, compared to vehicle group as well as ABT-263 treated animals. Furthermore, I assessed the anti-proliferative effects of 243 on primary glial cell lines as well. 243 exerted anti-proliferative effect on all patient derived glioma cell lines that have different grades and histopathology, except OLG3 cell line which is a grade 2 oligodendroglioma. According to quantitative RT- PCR results of OLG3, OLG7, and GBM9 cell lines I observed that OLG3 has a lower expression level of BCL2. These results suggest that patients with high BCL2 expression might benefit from 243 treatment. Taken together, our results indicate that 243 disrupts Beclin 1:Bcl-2 complex, hence activates autophagic cell death, and may serve as a potential therapeutic for the treatment of GBM.
Targeting 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 Biotechnology

In 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.
Investigation 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 Biotechnology

The central nervous system comprises numerous neuronal and glial subpopulations that have unique identities. Molecular mechanisms that underlie the generation of this cellular diversity have been under investigation. During development, the formation of cell subclasses with particular features is determined by tissue-specific transcription factors (TF). TFs sequence-specifically bind to DNA, interact with other proteins, and affect the expression of target genes. One of the key TFs in the developing brain is the Nuclear Factor I (NFI) family. There are four members (A, B, C, and X) in vertebrates. NFI proteins comprise a highly conserved N-terminal DNA binding and dimerization domain and bind to a TTGGC(N5)GCCAA consensus sequence as homo or heterodimers. However, they have a less conserved C-terminal transcription modulation domain which may lead to differential transcriptional regulation of target genes. In the developing mouse, Nfia, Nfib, and Nfix are expressed in an overlapping but distinct expression pattern in different regions of the embryonic brain, while their expression is restricted to stem cell niches in the adult. In the central nervous system, deletion of each member leads to delayed glial and neuronal differentiation, aberrant cell migration and increased proliferation. In the developing hindbrain, only the absence of Nfib leads to delayed development of several precerebellar nuclei, indicating that Nfib may play a unique role in this system. Dysregulated expression of NFIs have also been linked to tumor growth and progression, however, with opposing effects. For example, NFIB is oncogenic and promotes metastasis in colorectal cancer, melanoma, gastric cancer, estrogen receptor (ER)-negative breast cancer, and small cell lung cancer; while it has a tumorsuppressive function in non-small cell lung cancer, glioblastoma, osteosarcoma, and cutaneous cell carcinoma. NFIs perform their context dependent, cell-type and tissue specific functions, via regulation of specific set of downstream transcriptional targets. Despite the fact that NFI binding motifs have been found in the promoter and upstream enhancer regions of many genes, only a few of them have been so far investigated as direct NFI targets. Further identification and characterization of downstream targets of NFIs in various tissues will help elucidate molecular mechanisms that regulate embryonic development and related diseases, as well as cancer pathologies. In an attempt to investigate how NFIB regulates neurogenesis in developing precebellar nuclei, differentially expressed genes in E14 Nfib knock-out mouse precerebellar neuroepithelium have been analyzed. The RNA profiling analysis revealed putative candidates for further research. Of these putative NFIB targets, we selected Cdon (Cell adhesion molecule related, down regulated by oncogenes) and Fgf15 (Fibroblast growth factor 15), since these genes have been implicated in neural development of the cortex. We examined NFIB-mediated transcriptional regulation mechanisms of CDON and FGF19, the human ortholog of mouse Fgf15, in human neural stem cells (hNSCs derived from H9 ESC, Gibco). Neural stem cell culture systems provide an in vitro model of human neural development. Since NFIs have been reported to regulate neuron production in diverse parts of the developing brain, they may have comparable functions in vitro. Understanding these processes and the underlying molecular mechanisms in vitro will also help understand how the brain develops in vivo as well as failures in this process. Thus, in the first chapter of the thesis, we set out to examine NFI function and regulation mechanisms of potential NFIB targets, CDON and FGF19, in neuronal differentiation of hNSCs in vitro. RT-qPCR analyses revealed that mRNA expression of NFIB, NFIC, and NFIX is downregulated, whereas NFIA is upregulated in differentiating hNSCs. Since NFIA levels are quite low in these cells, overall NFI expression levels decrease during neuronal differentiation in hNSCs. These cells express NFIB at much higher levels compared to the other NFI members. Therefore, this study focuses on NFIB's role in hNSCs. We analyzed cell proliferation and differentiation by BrdU incorporation assays and immunofluorescence staining of neural stem and neuronal marker proteins. However, NFIB overexpression or knockdown did not affect the proliferation or neuronal differentiation potential of hNSCs. Nevertheless, these data cannot preclude NFIB's potential role in differentiation and/or self-renewal of hNSCs since NFIB could be silenced only by 30–50% in these cells and analyses were performed in whole cell populations that might mask possible changes induced by NFIB loss. Moreover, in NFIB overexpression experiments, we may need other proteins acting as cofactors that are not supplied along with NFIB. This study identifies FGF19 as a novel downstream target of NFIB in hNSCs. Human FGF19 is preferentially expressed in the fetal brain, among other tissues. Recombinant human FGF19 treatment has been shown to enhance neuronal differentiation in mouse neuroepithelial and cortical cells. In accordance with these data, FGF19 expression increases in differentiating hNSCs. Moreover, FGF19 expression increases in NFIB silenced hNSCs while it is reduced in NFIB overexpressing cells, indicating that NFIB regulates FGF19 transcription in hNSCs. Indeed, NFIs directly repress FGF19 promoter-driven luciferase activity, confirming that NFIs transcriptionally target FGF19. Moreover, chromatin immunoprecipitation (ChIP) assays showed that NFI proteins occupy −777 (relative to the transcription start site) in hNSCs, indicating NFI interaction with the FGF19 promoter in vivo. Since NFIB expression decreases upon neuronal differentiation, while FGF19 increases and NFIB directly represses FGF19 in hNSCs, future studies are required to address functional relevance of NFIBmediated FGF19 repression in the control of self-renewal and neural differentiation of these cells. In the absence of NFI, Cdon, a cell surface glycoprotein of the immunoglobulin (Ig) superfamily, is upregulated in the developing mouse brain. CDON is expressed in various tissues, primarily in the brain, muscle, and endocrine tissues during human and murine embryogenesis. Moreover, CDON is implicated in proliferation and differentiation control as it promotes myogenesis and neurogenesis in vitro and is essential for proper brain and skeletal-muscle development. However, in this study, CDON expression decreased in differentiating hNSCs and it did not change in NFIB overexpressed or silenced hNSCs, analyzed by RT-qPCR. These data indicate that CDON is not an NFIB target in this system. Recently, CDON has been described as a dependence receptor that induces apoptosis in the absence of its ligand SHH. During cancer progression, in an environment with limited SHH, tumorigenic tissue may downregulate CDON to eliminate its apoptotic activity. Indeed, CDON expression decreases in colon, lung, and neuroblastoma tumors, implicating a tumor suppressor role for CDON. As NFIs are involved in progression of various cancers, we examined whether NFIs regulate CDON transcription in SH-SY5Y human neuroblastoma cells. ChIP assays showed that NFIs bind to human CDON gene regulatory regions, -8 and -941 (relative to the transcription start site), in hNSCs and SH-SY5Y cells. Moreover, NFIs repress CDON promoter-driven luciferase expression via interacting with those NFI sites. Finally, CDON is upregulated in NFIB silenced SH-SY5Y cells, suggesting that the NFIB-CDON axis may be involved in neuroblastoma biology. On the other hand, silencing NFIB in SH-SY5Y cells decreases cell viability and proliferation, suggesting an oncogenic role for NFIB in neuroblastoma. Next, we tested the hypothesis that NFIB may affect SH-SY5Y cell survival by suppressing expression and thereby, pro-apoptotic activity of CDON. However, downregulation of CDON on its own could not rescue the phenotype induced by NFIB silencing, most likely because other NFIB downstream targets, which may include p21, are also involved. Further studies are required to investigate the functional consequences of NFIB mediated CDON repression in other developmental systems and disease models. NFIB's oncogenic effects in SH-SY5Y cells may involve inhibition of apoptosis and/or regulation of cell cycle components. Moreover, NFIB might promote differentiation of SH-SY5Y cells and/or contribute to the aggressive state of neuroblastoma tumorigenesis. However, these and underlying mechanisms need to be further investigated.
Gıda 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 Biyoteknoloji

Gıdalar besleyici ve metabolize edilebilen substratlar olarak değerlendirildiğinde bu özellikleri nedeniyle birçok mikroorganizmanın büyümesi için uygun ortam sağlamaları kaçınılmazdır. Kontamine yiyecek veya içeceklerin tüketilmesi ile gıda kaynaklı patojenlerin meydana getirebileceği birçok hastalık mevcuttur ve dünya üzerinde hem gelişmiş hem de gelişmekte olan ülkelerde halk sağlığı için yaygın ve ciddi bir tehdit olmaya devam etmektedir. Bu hastalıkların şiddeti basit akut semptomlarla ilgili olabileceği gibi ciddi ve yaşamı tehdit edici seviyelere erişebilir. Patojen mikroorganizma bulaşlarını önlemek ve dolayısıyla insan sağlığı açısından oluşabilecek ciddi durum ve ölümleri engellemek amacıyla gıdalar için üretim aşamasından tüketim aşamasına kadar gerekli kalite kontrol programlarının uygulanması son derece önemli bir süreçtir. Kalite kontrolde kullanılabilecek geleneksel yöntemler kültüre dayalıdır. Bu yöntemler basit, kolaylıkla adapte edilebilir ve genel olarak ucuzdur. Kültüre dayalı yöntemlerde sırasıyla bakterinin kültür ortamında büyütülmesi, bu ortamdan izole edilmesi, izolasyon sonrası biyokimyasal ve/veya serolojik tanımlama yapılması ve bazı durumlarda subspesifik tanımlama yapılma aşamaları gerçekleşmektedir. Bu nedenle kültüre dayalı tanımlama uzun zaman almaktadır. Bu yöntemlerin, fenotipik özelliklerin ortaya konmasındaki yetersizlikleri, kültüre alınmamış mikroorganizmaların belirlenememesi ve sonuca birkaç günde ulaşılabiliyor olması gibi dezavantajları insanları hızlı, daha güvenilir, hassas metotların geliştirilmesine yöneltmiştir. Bu yöntemlerin büyük çoğunluğu antikor ve nükleik asit tabanlı teknikleri kapsamakla birlikte geleneksel yöntemlerin modifikasyonu ile oluşan teknikleri de içermektedir. Patojen mikroorganizmaların tespitinde son yıllarda nükleik asit tabanlı teknikler oldukça önem kazanmıştır. Bunlardan en öne çıkanları Polimeraz Zincir Reaksiyonu (PCR) tekniklerine dayanmaktadır. Gıda patojenlerinin tespitinde gıda mikrobiyoloji laboratuvarları tarafından kullanılan PCR testleri mevcuttur. Geleneksel PCR yöntemi elektroforez gibi görüntüleme sistemlerine ihtiyaç duyar. İkinci nesil PCR olarak tabir edilebilecek Real Time-PCR tekniğinde, elektroforez gibi işlemlere gerek kalmadan çoğalan ürün aynı anda floresan sinyali ile görüntülenebilmesi yanında kantitasyona da olanak sağlayabilmektedir. Birçok doğrulanmış ve ticarileşmiş farklı özgüllüğe, doğruluğa ve hassasiyet limitine sahip ticari Real Time-PCR testi mevcuttur. Teorik olarak PCR, bir kopya nükleik asitten iki saatten az bir zamanda milyonlarca kopya üretebilmektedir. Fakat gıdalardaki inhibitörlerin varlığı hassasiyeti düşürmektedir. Sonuca ulaşmak 30-90 dakika gibi bir zaman almakla birlikte hassasiyet limitleri 103-104 kob/g civarındadır. Bu nedenle mikrobiyal zenginleştirme yöntemleri ile sistemi kombine etmek düşük patojen sayısı nedeniyle negatif sonuç alınması, fizyolojik olarak strese girmiş veya yaralı patojen hücrelerin tespit zorluğu ve ölü hücrelerden gelebilecek deoksiribonükleik asitin (DNA) elimine edilememesi gibi birçok limitasyonla baş edilebilmek mümkün olacaktır. Kısmen yapılacak bir ön zenginleştirme işlemi ile hassasiyet limitleri bu yöntemde düşürülebilmektedir. Fakat bu işlem sonuç alma süresini uzatmaktadır. Ön zenginleştirme işlemi mikroorganizmanın çeşidine göre 6-8 saatten 48 saate kadar uzayabilmektedir. Uluslararası Standardizasyon Örgütü'ne göre Salmonella için bu süre 18-20 saat, Listeria monocytogenes için ise 25-26 saat olarak belirtilmiştir. Teknolojinin gelişmesiyle bir öncekine göre daha avantajlı sistemler geliştirilmekte ve birçok farklı sistem kullanıcıların onayına sunulmaktadır. Nükleik asitlerin biyolojik ve moleküler biyolojik çalışmalarda doğru ve kesin bir şekilde kantite edilmesi, üzerinde çalışılan ve birçok alanda uygulaması olan önemli bir konudur. Dijital PCR bu iş için oldukça geniş kullanım yelpazesine ulaşmış yeni bir platform olarak görülmektedir. Bu yöntemle herhangi bir referans standarda veya dışsal kontrole ihtiyaç duyulmadan doğrudan nükleik asit kantitasyonu yapılabilmektedir ve yöntem Real Time-PCR sistemi ile aynı primer prob yapısını kullanmaktadır. Real Time-PCR yönteminden farklı olarak inhibitörlere daha dayanıklı olması ve yöntem gereği reaksiyon içerisindeki 1 kopyayı tespit edebilecek hassasiyete sahip olabilmesi dijital PCR yöntemini daha avantajlı duruma getirmiştir. Bu çalışmada yaygın kullanılan Real Time-PCR sistemi yerine sonuç alma süresini azaltacak bir yöntem araştırması yapılmıştır. 3. nesil PCR olarak da adlandırılan ve bir kopyaya kadar hassas sonuç verebilme yeteneğine sahip Droplet Dijital PCR (ddPCR) sistemi ile Salmonella spp. türlerini ve Listeria monocytogenes'i yüksek hassasiyette tespit edebilecek minimum zenginleştirme aşamasına sahip bir yöntem geliştirmek üzerine çalışılmıştır. Salmonella spp. için gıda matrisi olarak çiğ kıyma örnekleri, Listeria monocytogenes için süt örnekleri seçilmiştir. Çalışma sonucunda Salmonella spp. için 25 g örnekte 1.39 kob hassasiyette sonuç alınırken Listeria monocytogenes için 1.35 kob hassasiyette sonuçlar alınmıştır.
A molecular dynamics study of the prion protein

(Graduate School, 2023-05-12) Tavşanlı, Ayşenaz ; Balta, Bülent ; 521152101 ; Molecular Biology-Genetics and Biotechnology

Transmissible spongiform encephalopathies are caused by the conversion of the cellular prion protein PrPC into a misfolded form, PrPSc. In sheep populations there is a polymorphism at positions 136 (alanine/valine), 154 (arginine/histidine) and 171 (arginine/glutamine). While the A136-R154-R171 (ARR) variant confers highest resistance to scrapie, the V136-R154-Q171 (VRQ) variant leads to highest scrapie susceptibility. The A136-R154-Q171 (ARQ) variant with intermediate resistance is considered as wild type. To identify important conformational rearrangements at the initial steps of misfolding, microseconds long restrained and unrestrained molecular dynamics simulations have been prefomed at neutral pH, at 310 K and 330 K on naturally existing prion variants. Also, unfolding potentials of all three helicas of prion protein structure were also conducted at differentiated temperatures with the help of replica exchange molecular dynamic simulations. Moreover, at differentiated pH conditions unfolding potential of helix 1 and interaction of helix 1 with some other sequences were also conducted. Susceptibility of the disease might be related to hyrophobic side chain of the valine at position 136 which seemed to ease the unfolding process. While arginine at position 171 worked as a clamp to keep helix 2 and helix 3 of the cellular prion protein structure together. That might be the reason why VRQ is the most susceptable one where ARR is the most resistance. On the other hand, unfolding of helix 1 played the most critical role since it was the most stable helical structure in all conducted simulations. Inter- and/or intramolecular salt bridges of helix 1 were important to keep helix 1 stable in both helical structure and/or unfolded structure. Energy calculation showed that not high energy was needen to unwind helix 1. This helical structure of hydrophilic H1 might be broken by another hydrophilic sequence of the same prion protein, and its unwinding might be the key point to catalyze the complete unfolding of the protein
Investigation of novel genes and functional roles in MEFV negative FMF patients through next-generation sequencing

(Lisansüstü Eğitim Enstitüsü, 2023-06-20) Önen Özkılıç, Merve ; Turanlı Tahir, Eda ; 521142106 ; Molecular Biology-Genetics and Biotechnology

The p.Arg228Cys variant and the known pathogenic variant p.A230T are both located on the F-BAR domain, which is important for PSTPIP1 protein to form the functional trimeric complex with pyrin. The computational analyses of the xxi PSTPIP1 structure suggest that the p.Arg228Cys variant may cause a potential destabilization and change in the weak interaction network, leading to a stronger preference for certain interaction partners such as pyrin. Studies have shown that the p.Arg228Cys variant may lead to increased interaction with pyrin and reduced interaction with LYP phosphatase, which is normally required for the regulation of immune responses. The differences in symptoms between patients with the p.A230T or p.E250Q pathgoenic variants causing PAPA syndrome and patients with the p.Arg228Cys variant causing milder autoinflammatory symptoms in the patients in this study may be due to the diverse interaction strength of PSTPIP1 with different phosphatase proteins and pyrin protein. Within the scope of this thesis, it is found that p.Arg228Cys variant appears to increase the binding of PSTPIP1 to pyrin in PBMC samples of the patients. This was indicated by the higher levels of pyrin observed in the IP:PSTPIP1 lanes of our patient samples when compared to healthy controls. The interaction between PSTPIP1 and pyrin was increased in the patients as for P-III-2 1.43 fold and for P-II-1 patient 1.69 fold than HC1, and as for P-III-1 11.7 fold and for P-I-1 14.7 fold than HC2. This increased interaction may lead to hyperphosphorylation of PSTPIP1 and triggering the activation of pyrin inflammasome. Thus, the inflammation model in cultured PBMCs showed increased protein expression levels of pyrin, PSTPIP1, caspase-1, and IL1ß in cell lysates of patients with the p.Arg228Cys variant compared to the patient with a different heterozygote variant (p.A372V) and healthy control in the inflammation-induced condition (LPS+ATP) and secreted caspase1 and IL1ß levels were also found higher in supernatants of patients with p.Arg228Cys variant according to others in the inflammation-induced condition (LPS+ATP). Thus, it is important that other inflammation-related genes involvement should be considered in patients presenting with FMF phenotype negative for MEFV exon 10 recessive mutations. The targeted sequencing approach is useful for detecting rare pathogenic variations in patients with autoinflammatory phenotypes. Further functional analyses of the identified pathogenic variants could be helpful for better understanding the underlying molecular mechanism of FMF development.
Comprehensive transcriptomic and genomic analysis of oxidative stress-resistant saccharomyces cerevisiae

(Graduate School, 2023-07-25) Özşen Kocaefe, Nazlı ; Çakar, Zeynep Petek ; 521122107 ; Molecular Biology-Genetics and Biotechnology

Oxidative stress occurs as the oxidant and antioxidant balance in an organism is disrupted in the direction of oxidants, and oxidants come to a position that can potentially cause damage. All aerobic organisms use oxygen for respiration and oxidation of nutrients for producing energy. For this reason, while oxygen is necessary for aerobic organisms to live, it is also harmful because of the reactive oxygen species (ROS) that emerge during these processes that are necessary for life. Lipid, protein and DNA molecules, which form the basis of life, can be damaged directly or indirectly due to oxidative stress caused by ROS. Aerobic organisms have developed various mechanisms to protect themselves from the harmful effects of oxidative stress. These mechanisms involve enzymatic and non-enzymatic (antioxidants) systems. In addition to these mechanisms, organisms use various repair mechanisms, especially to repair damaged DNA. Oxidative stress from ROS is biomedically important and widely researched, as it causes many neurodegenerative and autoimmune diseases, as well as cardiovascular diseases, cancer, and aging in mammals. The yeast Saccharomyces cerevisiae is used as a model organism, because of its beneficial characteristics. S. cerevisiae, which is a eukaryotic organism, has a short-life span. It is a single cell organism and can be found as haploid, diploid or polyploid in nature. Thus, S. cerevisiae is an important model organism for elucidating the processes in eukaryotic complex organisms. The aim of this study was to characterize an oxidative stress-resistant mutant S. cerevisiae strain at genetic and transcriptomic levels. The main idea, however, was to comprehensively examine the complex molecular infrastructure of oxidative stress resistance. In addition, as a physiological analysis, the cell wall properties of the mutant strain was also tested and information was obtained about the effect of stress on the cell wall structure. In this thesis study, transcriptomic analysis of the mutant S. cerevisiae was performed by comparing the transcriptomic profiles of the oxidative stress-resistant, evolved S. cerevisiae and the reference strain. Transcriptomic data of the strains were obtained by microarray analysis. As a genomic analysis, the entire genomes of the oxidative stress-resistant strain and the reference strain were sequenced and the differences in the genome were determined to find the mutations in the evolved srain that are related to oxidative stress resistance. In addition, cell wall analyses of the reference strain and the mutant strain were performed, using lyticase susceptibility test. As a result of transcriptomic analysis, it was observed that the expression level of 869 genes changed by at least 2-fold, 349 genes changed by at least 3-fold, 144 genes changed by at least 4-fold, and 67 genes changed by at least 5-fold in the oxidative stress-resistant mutant. Among these 869 genes that were differentially regulated by 2 times or more, 459 genes were upregulated and 410 genes were downregulated. The genes whose expression were decreased are generally related with ribosomal RNA, nuclear transport, organelle integration, tRNA cell cycle, mitosis and RNA polymerase. Expression levels of stress response genes, carbohydrates, lipid, protein, precursor metabolites and ion/metabolite transport-related genes were generally increased in the mutant strain. According to the ESR analysis results, a positive correlation was observed between the ESR-induced genes and the genes of the oxidative stress- resistant mutant with increased expression, according to the database. In addition, a positive correlation was observed between ESR-suppressed genes and genes of the mutant with decreased expression. The expression levels of two genes related to oxidative stress decreased and twenty three of them increased in the oxidative stress-resistant, evolved strain. The expression levels of six autophagy-related genes in the oxidative stress-resistant mutant decreased and 30 of them increased. According to whole genome sequencing results, 34 missense, 2 nonsense, 1 deletion and 12 silent mutations were found in the genes of the mutant strain, and 13 mutations were detected in chromosomal regions outside the coding regions. A nonsense mutation in the NRG1 gene, which is a transcriptomic regulator, results in the formation of a truncated protein. Further genomic and proteomic studies would be necessary to clarify the role of these genes and mutations in the oxidative stress resistance.
Biointerfacial cell/protein–polymer interactions investigated by quartz crystal microbalance with dissipation

(Graduate School, 2023-08-01) Sert Özdabak, Ayşe Buse ; Kılıç, Abdülhalim ; 521162101 ; Molecular Biology-Genetics and Biotechnology

Understanding cell-surface interactions is required for the development of novel and functional biomaterials. The biocompatibility and in vivo performance of these biomaterials heavily depend on processes such as protein adsorption and subsequent cell adhesion on the material surface. Common experimental approaches used to assess these processes typically involve end-point assays. However, these assays often require cell fixation or disruption and pre-or post-labeling of the cells, potentially affecting cell physiology and leading to the loss of valuable information. Furthermore, these methods cannot distinguish interfacial interactions occurring at nanometer scales between cells and the surface. The physicochemical properties of the material surface also significantly impact the performance of potential biomaterials. The interactions taking place at the interface between cells/proteins and materials are intricate and must be comprehensively understood and carefully designed to meet specific application requirements. In this context, Quartz Crystal Microbalance with Dissipation (QCM-D) emerges as an alternative and complementary method. QCM-D serves as a powerful, noninvasive technique that enables real-time and label-free monitoring of cell-surface interactions at the nanoscale. QCM-D provides distinct data regarding specific interactions at the cell - material interface, thereby offering new insights into the cell adhesion / protein adsorption behaviors. The aim of the thesis is to investigate cell-polymer interactions and to monitor the entire process in real-time using QCM-D system. For this purpose, two commonly employed polymers in the biomaterials field, Polycaprolactone (PCL) and Chitosan (CH), as well as their blends (75:25 and 25:75), were employed to investigate real-time cell adhesion behavior. As surface topography, chemical composition and wettability have significantly influence on cell adhesion process, it is important to analyze cell adhesion on well-characterized surfaces. Two types of cell lines (hFOB and 3T3) were employed to monitor cell interactions. Complementary cell culture assays were also conducted to validate the outcomes obtained from QCM-D. In the first part of the thesis, the preparation and characterization of thin films on silicon substrates and silica sensor surfaces were completed. In order to achieve homogeneous films, various parameters were investigated, i.e., polymer ratio, solvent type, substrate surface characteristics (activated with oxygen plasma or hydrophobic treatment), polymer molecular weight, and polymer ratio. The homogeneity of the films was assessed using Atomic Force Microscopy (AFM). It was founded that blends prepared with a constant amount of chitosan yielded homogeneous coatings on the silicon substrate. The chemical composition of the constructed surface was further analyzed using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR). The spectra exhibited distinct peaks at 1725 cm-1 for PCL and 1645 cm-1 and 1584 cm-1 for chitosan, confirming the successful coating of both polymers onto the surface. Analysis of AFM images over a scanned area of 100 µm2 revealed that pure polymers produced morphologically homogeneous films, whereas the blend surfaces displayed visible domains. Particularly, 75:25 PCL/CH blend exhibited micrometer-scale domains. Comparatively, thin films prepared with pure polymers displayed smoother surfaces compared to the blends. In contrast, the blends prepared with a ratio of 75:25 exhibited the highest roughness value. In terms of zeta potential, pure PCL films exhibited the highest negative value (-88 mV), whereas pure chitosan films displayed the lowest negative value (-15 mV) at pH 7.4. Blend film zeta potential values were between these two extremes. Film thickness analysis revealed that pure chitosan films had the smallest thickness (ca. 10 nm), whereas 75:25 PCL/CH blend film had the greatest thickness (ca. 55 nm). The pure PCL film and the 25:75 blend film had thicknesses of approximately 38 nm and 14 nm, respectively. In addition, in situ spectroscopic ellipsometry was employed to determine swollen polymer thicknesses. It was observed that PCL, being a hydrophobic polymer, did not swell much in aqueous solutions. Chitosan thin films exhibited the highest degree of swelling. The blend films exhibited swelling degrees between those of the pure polymer films, while higher PCL amount (75:25) resulted in reduced swelling, as expected. Before the cell adhesion studies, protein adsorption studies onto the constructed films was conducted. Bovine Serum Albumin (BSA) adsorption was monitored in real-time using both QCM-D and spectroscopic ellipsometry at various pH values at room temperature. In the case of pure PCL film, BSA adsorption onto pure PCL film showed a consistent frequency change upon adsorption with QCM-D for all pH values investigated. However, for the other investigated films, the presence of chitosan led to pH dependent adsorption behavior. At pH 4.5, both BSA and CH were positively charged, resulting in adsorption under repulsive conditions. At pH 6.0, the electrostatic attraction between the polymer chains and BSA led to higher adsorption on films containing chitosan. The lowest frequency decrease, i.e., mass load, was observed at pH 7.4 compared to pH 4.5 and 6.0. These findings indicate that blend composition, pH and ion presence in the environment have a substantial influence on protein adsorption. To compare the adsorbed protein amounts determined by QCM-D and ellipsometry methods, diverse models were applied. When two methods are assessed, the protein quantity derived from QCM-D data was consistently higher than that obtained by ellipsometry. The amount of protein calculated from ellipsometric data was similar for the blend films and pure chitosan films for all pH values investigated. However, higher values were evident in QCM-D method due to the inclusion of coupled water in the calculations. In addition, fibrinogen adsorption presented composition dependent behavior on thin films. The highest adsorbed fibrinogen amount was monitored on pure PCL films. In contrast, no significant protein adsorption was monitored on pure chitosan films. Consequently, the adsorbed amount of fibrinogen decreased with an increasing percentage of chitosan in the films, which predominantly showed an inverse correlation with the surface hydrophilicity. Following the comprehensive characterization of the films and conduction of the protein adsorption experiments, the cell adhesion behavior of two cell lines, human fetal osteoblastic (hFOB) and mouse fibroblast (NIH/3T3), was monitored on constructed films using QCM-D. For this purpose, the cells were introduced into the QCM-D chamber and allowed to flow for 1 hour. Initial cell sedimentation after 1 h resulted in reduced cell deposition as the chitosan ratio increased in the film. This trend was consistent for the both cell lines in the first hour. Subsequently, changes in frequency and dissipation were monitored over an 18-hour period. Complementary cell culture assays were performed to validate the observations of QCM-D. For this purpose, fluorescence images and live cell images at various time intervals were captured. Distinct QCM-D signal patterns were found for the investigated cell lines, indicating the influence of the varying interfacial properties on cell adhesion, which is also dependent on the specific cell type. In the case of hFOB cells, fully spreading was observed on pure PCL films, with elongated morphologies as confirmed by fluorescence microscopy and scanning electron microscopy (SEM). Corresponding QCM-D signals showed the highest frequency drop and the highest dissipation. Blend films supported hFOB cell adhesion, but with lower dissipation values compared to the PCL film. This might be attributed to higher rigidity at the hFOB cell−blend interface, because these cells did not progress to the further stages of spreading after secretion of their extracellular matrix (ECM) proteins. Variations in the QCM-D data obtained from the blend films could be attributed to differences in the morphology of the films. Pure chitosan films showed limited hFOB cell adhesion, accompanied by low frequency drop and low dissipation. The initial sedimentation of 3T3 cells onto the constructed surfaces similarly showed dependence on the surface composition. Unlike the behavior of hFOB cells, 3T3 cell lines did not adhere to pure chitosan surfaces, evident from consistent positive frequency signals. The highest frequency change was observed on reference silica surface, with dissipation gradually decreasing. This behavior indicated an average number of cells remaining in ECM remodeling stage. The ΔD signal shape was similar for 75:25 PCL/CH blend to the reference silica surface, however a slight decrease in the frequency was observed after 10 h. This suggests the stronger attachment to the surface while cells lacked further spreading stages on 75:25 PCL/CH blend. 3T3 cells on 25:75 PCL/CH blend showed substantial frequency drop after 10 h, which accompanied by an increase in dissipation. This behavior corresponded to the later stages of cell adhesion, implying that cells probably underwent actin remodeling and fully spreading on the surface. In conclusion, distinct QCM-D signal patterns were evident in the adhesion of hFOB and 3T3 cell lines. These distinctive patterns were attributed to the variations in the strength of cell adhesion, which are influenced by both cell type and surface chemical properties. The real-time and label-free data collected through QCM-D gave us a more profound comprehension of the dynamic adhesion behavior of the cells on constructed thin films. This knowledge and understanding holds the potential to provide valuable insights for the design of novel biomaterials tailored to diverse applications.
Bacterial cellulose production using enzymatic hydrolysate of olive pomace

(Graduate School, 2023-10-25) Öztan Sağdıç, Ceren ; Karagüler, Nevin Gül ; Tüter, Melek ; 521152107 ; Molecular Biology-Genetics and Biotechnology

Bacterial cellulose is a biopolymer which has the identical chemical and structural properties with plant cellulose. However, it is recently of great scientific interest due to its superior characteristics such as high degree of polymerization, water retention capacity, and biocompatibility, compared to its plant-originated counterpart. Additionally, while plant cellulose is often bound to lignin, pectin and hemicellulose polymers, BC is obtained as a pure product. By virtue of these features, bacterial cellulose has been successfully employed in many applications among which are food, paper, biosensors, electronics, drug delivery systems, cosmetics, and wound healing materials. Along with some bacteria genera such as Acetobacter, Pseudomonas, Rhizobium and Escherichia, this polymer can also be formed by some algae (Valonia, Chaetamorpha) and fungi. It has been considered to procure the microorganisms an evolutionary advantage by protecting them against environmental factors such as UV radiation, desiccation and, contamination. There are several standardized growth media for the production of bacterial cellulose. Nonetheless, high cost of these laboratory-grade ingredients obstructs the large scale use of bacterial cellulose. Heretofore, numerous strategies have been proposed to minimize the medium cost. Molasses, rotten fruit, orange peel can be counted among the alternative media which gave promising results for bacterial cellulose generation. In this study, use of lignocellulosic material, particularly of olive pomace as carbon source for bacterial cellulose production was demonstrated. Lignocellulose, being the most abundant biopolymer in the world, has a great potential to serve as a substitute for fossil fuels. In fact, bioethanol and biofuel generation from lignocellulose by means of microorganims has been widely applied in industry. Similarly, in this work, it was aimed to obtain monomeric sugars from lignocellulose which in turn was used as carbon source for the cellulose producing bacteria- Novacetimonas hansenii. On the other hand, olive pomace (OP) is a by-product of the olive oil industry. Olive pomace is made up of olive pulp, stones, and skin of the fruit. It may pose environmental problems if not conducted properly. Approximately 35 - 45 kg dry olive pomace is obtained from 100 kg olive during oil production. This material is generally sorted and burned for energy production Nevertheless, it has been shown that OP is high in lignocellulosic content and the need for a more efficient way to use this material is apparent. In the first part of the study, two strains were compared according to their cellulose production yields. In addition, several cultivation conditions were performed to determine the most effective method. N. hansenii (ATCC 53582) in static growth condition gave the best results and therefore applied on the next steps. Moreover, three agricultural waste products; meat-bone flour, fish flour, and olive pomace were investigated for their efficiency to function as growth media. While no cellulose was formed with meat-bone flour and fish flour media, little cellulose was obtained in the medium prepared with olive pomace and lactose. After that, several trials with the use of olive pomace as nitrogen source while examining the performance of lactose and glucose as carbon sources were realized. However, elementary analysis revealed that the nitrogen content of olive pomace was not sufficient to supply the growth medium as nitrogen source. Besides, cellulose produced in olive pomace medium had poor mechanical qualities which is not suitable for any further application. On the other hand, olive pomace was shown to possess high organic component with approximately 45 % carbon. Therefore, in the second part of the study, generation of monomeric sugars by degradation of the lignocellulose of olive pomace was aimed. For this, acidic pretreatment and enzymatic hydrolysis were applied respectively. Acidic pretreatment breaks down the complex organization of lignocellulosic material to expose cellulose and hemicellulose for enzymatic degeneration. Subsequent enzymatic hydrolysis generates monomeric sugars which can be used by microorganisms. In this study, olive pomace was pretreated with 1 % phosphoric acid at 170 oC at 8 bar in order to separate cellulose from hemicellulose and lignin, thus unveiling the hydrolysable ends and producing oligosaccharides. Consecutive enzymatic reaction was conducted at 50 oC for 72 h with enzyme:substrate concentrations varying from 1.5 to 30 % (w/w) in static and agitated conditions. The reducing sugar concentration of the liquid part following the acidic pretreatment was determined by glucose hexokinase assay and found to be too low to lead to any microbial growth. Furthermore, reducing sugar concentration of each enzymatic hydrolysate was detected by dinitrosalicylic acid (DNSA) assay. Among varying enzyme:substrate concentrations, 30 % enzyme reaction in static condition resulted in the highest reducing sugar yield with 9.31 g/l. Enzymatic hydrolysates were scanned for the presence of galactose, glucose, mannose, arabinose, xylose, rhamnose, lactose, fructose, maltose and cellobiose by HPLC and distribution of these sugars along different hydrolysates was determined. For each hydrolysate, glucose was found to be the major monosaccharide. Growth media were prepared from selected hydrolysates with the ingredients of Hestrin-Schramm medium, except the carbon source, however, no cellulose formed. Therefore, the hydrolysates were detoxified to eliminate the inhibitory molecules generated in course of pretreatment. Among the methods attempted, Ca(OH)2 treatment was shown to be the most effective. From the media prepared with detoxified hydrolysates, the highest amount of bacterial cellulose production was 0.68 g/l. In addition, Hestrin-Schramm conventional medium and the medium with enzymatic hydrolysates were compared according to the substrate conversion ratio, cellulose production rate and yield. Sugar consumption in the control and test media was also detected. In the third part of the study, bacterial cellulose produced in the alternative medium was characterized with X-ray Diffraction Analysis, Fourier-Transform Infrared Spectroscopy (FTIR), and Scanning Electron Microscopy (SEM). X-ray diffraction demonstrated that the new BC posssesed the typical cellulose I peaks. However, the BC had a small amount of phosphate salts which caused HAP signals on the diffratogram. The FTIR analysis showed that the new bacterial cellulose had the characteristic spectrum of cellulose and no impurities were found. Similarly, with SEM analysis, it was demonstrated that the new material had nano-sized fibrillary structure very similar to the control material.
Exploration of novel serine protease do-like HtrA from acigöl

(Graduate School, 2023-12-06) Kılıç, Meryem Menekşe ; Karagüler, Nevin Gül ; Balcı Çelik, Nurgül ; 521112113 ; Molecular Biology-Genetics and Biotechnology

Enzymes involved in industrial biotechnological processes take place in conditions of extremely high temperature, high pH, and high salinity or when there are organic solvents that have made it necessary to discover enzymes resistant to these conditions. Microorganisms in extreme environments adapt to varying levels of stress such as very high pH, temperature, salt, and pressure. For the last 20 years, researchers have focused especially on extreme environments for the discovery of enzymes that are resistant to extreme conditions, with the hypothesis that the enzymes of microorganisms adapted to these conditions can also work under extreme conditions. In this context, microorganisms can be isolated from their environment and their enzymes can be characterized by traditional microbiological methods. Besides this, new enzymes have been discovered by the method called 'metagenomics', which is not based on culture. Environments with high salt concentration are divided into two in terms of their ionic compositions. Many high salt concentration environments were formed by the evaporation of seawater, also called 'thalassohaline'. Their salt content is similar to seawater and the pH varies from basic to slightly acidic. Environments with high salt concentrations, called 'Athalassohaline', are completely different from seawater in terms of ionic composition. Acıgöl, which is our study area, is a lake with high salt concentration, which is included in the 'Athalassohaline' state group. In this study, samples from Acıgöl were employed. Acıgöl is located between the provincial borders of Denizli and Burdur in the Aegean Region of our country. Looking at the chemical composition of the lake, it is seen that Na+, K+, Cl−, and SO4 2− ions are dominant. The salinity of the Acıgöl changes between 5.8%-13%, pH between 7.8-8.2, and temperature varies seasonally between 8 °C and 32 °C. These changing extreme conditions force the microorganisms in the lake to cellular and enzymatic adaptation. These organisms adapted to high salt concentration are called 'Halophilic' microorganisms, meaning salt-loving. Based on this information, the main subject of the study is the discovery of enzymes of halophilic microorganisms that can be used in difficult industrial processes. The primary objective of this study is to obtain new proteases, which are of industrial importance, by function-based screening of culturable microorganisms. In line with this goal, firstly, soil samples taken from Acıgöl were diluted in Nutrient Broth and spread on nutrient agar petri dishes containing 10% NaCl and 1% skim milk, and the species containing protease activity were determined. It was determined by the transparent region around the colonies that the isolate had protease activity, resulting from the breakdown of skim milk. With this screening method, halophilic species in Acıgöl, which actively produce protease, were determined. Sixmorphologically different species were determined. Twoshowed protease activity, and the species with t huge zones were chosen for further studies. In the second part of the study, the whole genome of the determined species was sequenced with the New Generation Sequencing method (Illumina HiSeq 2500 platform), and its serine proteases and other biotechnologically potential enzymes were determined. According to the sequencing results, it was determined that the entire genome of the isolated species was 4,708.499 bp (base pair) in length, had a G+C ratio of 36.66%, and had 4536 gene-coding sequences. In addition, it was revealed that 99.81% ratio similarity to Virgibacillus marismortui species according to 16S rDNA sequence similarity. The whole-genome average nucleotide identity (ANI) value was obtained as 99.44% and digital DNA-DNA hybridization was computed as 88.8%. The average amino acid identity ratio (AAI: Average Amino acid Identity) was calculated as 98.69%. In addition to genomic analyses, the isolated species was also examined phenotypically and biochemically. It was determined that the species was gram positive (Gr+), both alkaliphilic and moderately halophilic, motile, endospore-forming, and protease-producing bacterium. The isolated strain shows optimum growth at 37 °C with salinity and pH ranging from 5-10% and 6 and 9, respectively. As a result of this polyphasic analysis, it was conclueded that the isolate was a subspecies of Virgibacillus species, and it has been brought to the literature with the name Virgibacillus sp. AGTR. All genome information is stored in the NCBI database. Accession number JAJERH000000000. The last step of the study aimed to produce by recombinantly and characterize the serine protease from a new isolate. Among the four serine proteases determined by whole genome analysis, the Serine protease Do-like HtrA with the lowest sequence similarity rate and fewer studies in the literature was selected for recombinant production. The Serine protease Do-like HtrA is a member of the Trypsin-like serine protease superfamily (Tryp_SPc Superfamily) and S1-C subfamily. HtrA (high-temperature requirement A), a periplasmic heat-shock protein, it has two different functions. While it shows molecular chaperone properties at low temperatures, it shows proteolytic activity at high temperatures. The structure of this kind of protease differs slightly from other commercial and well-studied proteases. Due to these properties, it could be used specifically in the pharmaceutical industry. For the recombinant production of Serine protease Do-like HtrA, primers that contain EcoR I and SacI restriction sites were designed to be specific to the start and end sequences of the gene of interest (targeting the 5' and 3' ends). By using the genome of the isolated Virgibacillus sp. AGTR strain as a template, the target protease gene was amplified and ligated into the pET-28-a(+)expression vector. The cloned vector was inserted into E. coli BL21, E. coli C43 (DE3), and RosettaTM 2 expression cells to determine the best expression host cell. As a result of the purification study, the RosettaTM 2 cell was selected for expression. Expression studies were performed with 0.1 mM, 0.5 mM, and, 1 mM IPTG concentrations at 30 ºC and 37 ºC for up to 6 hours. The highest level of expression was achieved with 0.1 mM IPTG in 4 hours at 30 °C. Successfully expressed protease gene was purified by the His-tag method. The estimated molecular weight of the protein was 42100 Da and the isoelectric point was 4.53 which is calculated using the ExPASy program. As a result of purification, the molecular weight of the enzyme (42.1 kDa) was compatible with the predicted value, according to SDS-PAGE and Western blot tests.
Investigation of the spastin's role in the invasion capacity of glioblastomas

(Graduate School, 2024-01-05) İmanç Temizci, Benan ; Karabay, Arzu ; 521142102 ; Molecular Biology-Genetics and Biotechnology

Glioblastoma Multiforme (GBM) is the most lethal form of glioma, which are the most frequent brain tumors. Even though multimodal therapy is employed to treat GBM, tumor recurrence makes treatment almost impossible due to its robust migration/invasion potential. For the improvement of GBM therapy, it is critical to identify the proteins involved in the disease's migration/invasion process. Tumor cells require special cellular extensions controlled by cytoskeletal components to achieve migration/invasion capabilities. Spastin, a microtubule-severing protein, is mainly expressed in neurons and controls dendrites and axonal extensions of neurons. Given that the formation mechanism of these extensions in post-mitotic cells is comparable to that of specialized cell protrusions in mitotic cells, Spastin might have roles in tumor cell migration/invasion. Interestingly, Spastin has been discovered to be co-localized with actin filaments in GBM cells, suggesting that it may play a role in GBM migration ability. However, this topic has not been investigated in the literature until this study. This thesis aims to clarify the molecular mechanism underlying the shift in the intracellular localization of Spastin in GBM cells, and the potential significance of this mechanism in GBM migration/invasion ability. This study discovered for the first time that Spastin takes an active role in GBM migration. Furthermore, Spastin was discovered to interact with Pin1 via phosphorylation of Pin1 recognition motifs located in its microtubule-binding domain. Moreover, this interaction was found to direct of Spastin towards actin filaments, which promotes migration/invasion ability of GBM cells. These findings suggest that Spastin might be a therapeutic target for several tumors with a high migration/invasion capacity, like GBM.
Clinical and molecular collaboration studies: i)comparison of CNV by NGS&MLPA in hereditary breast and ovarian cancer ii)effective covid-19 sampling and storage

(Graduate School, 2024-04-26) Yıldız, Jale ; Doğanay Dinler, Gizem ; 521182106 ; Molecular Biology-Genetics and Biotechnology

HBOC syndrome, as the focus of the study in chapter one, represents a significant concern in cancer genetics. HBOC is primarily associated with mutations in the BRCA1 and BRCA2 genes. In this context, the study aims to enhance the detection of CNVs, crucial genetic alterations often observed in malignancies, including HBOC. The research assesses the efficacy of NGS for CNV detection, comparing it against the established Multiplex Ligation-Dependent Probe Amplification (MLPA) method. In this study, 1276 cases were examined using targeted NGS panels. Of these, 691 cases were further validated through MLPA, encompassing 61 calls in 58 NGS-CNV positive and 630 NGS-CNV negative cases. The findings revealed a considerable disparity: 46% of NGS-CNV positive calls were consistent with MLPA results, while 54% displayed discrepancies. Two cases identified as single nucleotide variations (SNVs) by NGS were found to be CNVs by MLPA. Interestingly, 2-3% of the cases showed MLPA-confirmed CNV regions in the BRCA1/2 genes. Notably, while the NGS-CNV algorithm had a high rate of false positives, it did not yield false negatives. The instances where NGS indicated negative but MLPA showed positive was due to SNVs at MLPA probe binding sites. The study concludes that NGS-CNV analysis shows promising diagnostic capabilities in detecting CNVs, specifically for negative CNV cases. However, false positives and the necessity for confirmation through alternative methods highlight the need for an integrated approach in clinical diagnostics. This ensures more accurate and reliable detection of CNVs, which is crucial for understanding and treating HBOC. Chapter Two focuses on PCR tests, which have been widely adopted as an essential diagnostic tool for identifying SARS-CoV-2 infections during the COVID-19 pandemic. However, proper handling and storage of collected samples are essential to confirm the precision and reliability of test results. Understanding the impact of sample storage conditions on PCR assay performance is critical to maintaining the effectiveness of test protocols. Moreover, this thesis investigates the effectiveness of using saliva samples as an alternative to oro-nasopharyngeal swabs for the detection of SARS-CoV-2, the virus responsible for COVID-19, through reverse transcription-polymerase chain reaction (RT-PCR) testing.
Variant detection in inflammatory diseases

(Graduate School, 2024-04-30) Alkurt, Gizem ; Doğanay Dinler, Gizem ; 521162104 ; Molecular Biology-Genetics and Biotechnology

Cancer remains a public health concern, with a significant number of reported cases worldwide in 2020. 9.3 million cases were diagnosed in men and around 8.8 million cases were diagnosed in women according to 2022 data. From a healthcare perspective, cancer is an influenced by factors such as genetics, environment and inflammation. Cancer is widely recognised as an inflammatory disease, as it is closely linked to an interaction of processes within the body. Inflammation plays a role in the initiation, progression and promotion of cancer. It involves the activation of cells, the release of cytokines and the creation of an inflammatory environment. All of these contribute significantly to stages of cancer development. Therefore it can be deduced that cancer is not caused by mutations, but is also strongly influenced by inflammations. Beyond the events, genetic predisposition adds another layer of complexity that affects not only how the disease manifests, but also how it is treated. The development of cancer is the result of an interplay between hereditary mutations. Tumors associated with cancer syndromes provide insights into genetic conditions. One such syndrome is Lynch syndrome, an inherited disorder that increases the risk of colorectal and endometrial cancers, among others. Mutations in genes such as BRCA1 and BRCA2 are associated with breast and ovarian cancer syndromes, also significantly increase the likelihood of developing these cancers. Our research has explored the link between genetics and cancer by focusing on genes beyond BRCA1 and BRCA2. We examined genes such as ATM, PTEN and CHEK2 that play a role in cancers. For example, ATM mutations increase the risk of breast cancer. PTEN mutations are associated with Cowden syndrome, while CHEK2 mutations increase the risk for both breast and colorectal cancer. We have also investigated Lynch syndrome associated mutations in genes involved in mismatch repair (MLH1, MSH2, MSH6 and PMS2). These mutations are known to make people more susceptible to cancer. Recent studies have highlighted the importance of inherited mutations in determining cancer susceptibility. Therefore, in the first part of this thesis, our aim was to analyze variations in 25 genes associated with cancer susceptibility in a population through a large collaborative effort. The study included 732 breast cancer patients, 189 colorectal cancer patients and 490 elderly cancer-free controls who were free of cancer. The analysis revealed 119 likely pathogenic variations in a total of 149 individuals. Interestingly, around 22.7% of these variations were newly discovered, indicating genetic risks. In addition, 16.0% of the variations were found recurrently among individuals suggesting shared genetic predispositions. In high-risk breast cancer patients, harmful variations in BRCA1 and BRCA2 genes accounted for 61.3% of all identified mutations. Other high-risk breast cancer genes like PALB2, TP53, STK11 and CDH1 also contributed to the mutation spectrum. We also found mutations in genes with penetrance such as CHEK2 and ATM. In cancer cases, Lynch syndrome-related genes accounted for 62.5% of all disease-causing mutations observed. In addition, MUTYH pathogenic variants were present in about 4.4%of high-risk colorectal cancer patients. Interestingly, 2.9% of the control group had a genetic predisposition to cancer based on their family background, but none of them actually had any mutations. It's worth noting that individuals with these mutations also had no family history of cancer. This is possible because of the interaction of proteins that ensure cell survival with these genes and provides preliminary data for a viable project in the future. In the second part of this thesis, we analysed the expression levels of microRNAs in wild type and BAG-1 knockout breast cancer cells. The BAG-1 protein is known for its ability to prevent cell death. It interacts with various other molecules in the cell. These interactions play a role in determining whether cancer cells survive or die. Studies have shown that changes in the levels of BAG-1 protein are associated with types of cancer. In addition, BAG-1 has shown promise as a marker for breast cancer, which is the most common type of cancer in women. Higher levels of BAG-1 are generally associated with increased growth, development and aggressiveness of breast cancer. The presence of miRNAs plays a crucial role in regulating gene expression. To identify these miRNAs, we performed high-throughput sequencing and also determined the target genes and molecular signaling pathways associated with these expressed miRNAs. Of the 25 miRNAs sequenced, we found that 11 were significantly upregulated and14 were downregulated. We experimentally validated 14 out of the 25 miRNAs, among which hsa-miR-429 stood out as a miRNA in BAG-1 KO MCF-7 cells due to its downregulation observed through bioinformatics analysis and other assays. Shifting focus to the global health crisis, the final phase of the thesis examined antibody levels in healthcare workers in the context of the COVID-19 pandemic. The COVID-19 pandemic has highlighted the relationship between infections and the body's immune response particularly inflammation. When someone contracts COVID-19 from the SARS-CoV-2 virus, they may experience levels of inflammation. This natural defense mechanism is the bodys' way of fighting off the virus. Understanding and effectively managing this response is critical to treating COVID-19. Researchers and health professionals are focusing on strategies to regulate how the immune system responds to the virus. The aim is to prevent inflammation that could lead to illness. During the pandemic, we conducted a study of antibody levels in healthcare workers related to COVID-19. As infections continue to increase, it is important to research aspects of the disease. One aspect is to access the extent to which healthcare workers have been exposed to SARS-CoV-2 by testing for immunoglobulin G (IgG) antibodies. This study took place in three hospitals to determine whether healthcare workers (HCWs) had contracted SARS-CoV-2. The main objective was to determine the percentage of HCWs who tested positive for antibodies indicating their exposure to the virus. To measure this we used an microparticle immunoassay that detects antibodies. By analysing the prevalence of IgG antibodies, this research provides insights into HCWs' exposure and immune response to SARS-CoV-2. This information is critical for making decisions on vaccination programs, the need for personal protective equipment (PPE) and implementation of infection control measures in healthcare settings such as hospitals.
Molecular and functional investigation of disease-associated cytoskeletal proteins protrudin and MYO1H

(Graduate School, 2024-07-13) Şahin Selçuk, Ece ; Korkmaz Karabay, Arzu ; 521122113 ; Molecular Biology-Genetics and Biotechnology

Hereditary Spastic Paraplegia (HSP) is a rare, inherited and progressive neurodegenerative disorder characterized by muscle weakness and spasticity, especially in the lower extremities. In general, HSP pathology is defined as degeneration of axons in the corticospinal pathway starting from the distal end and progressing towards the cell body. HSP can be inherited as autosomal recessive, autosomal dominant, X chromosome-linked or mitochondrial. HSP is classified into two subgroups as pure and complex according to clinical findings. In pure HSP, symptoms are limited to weakness and spasticity in the lower extremities, bladder-related disorders, and deep sensory loss. In complex HSP, neurological disorders such as seizures, dementia, cerebellar ataxia, optic atrophy, etc. may be observed in addition to the symptoms of pure form. For these reasons, HSP is both clinically and genetically heterogeneous. Moreover, many parameters such as age of onset, symptoms, severity or progression of the disease may vary due to these heterogeneities. Identification of the causative gene has great importance for understanding the mechanism of the disease or providing a target molecule to be used for its treatment. Although there are 85 genes or loci associated with HSP so far, mutations in SPAST (SPG4) and ATL (SPG3) genes account for 50% of all cases. When the cellular roles of proteins encoded by other genes whose mutations have been found to cause HSP are analyzed, it is seen that most of them are involved in the regulation of intracellular transport and in the morphogenesis of the endoplasmic reticulum (ER). The remaining HSP proteins have roles in pathways such as mitochondrial regulation, myelination, lipid biosynthesis and nucleotide metabolism. Within the scope of this thesis, identification of the candidate causative gene and mutation by whole exome sequencing method in a Turkish family diagnosed with pure HSP and identification of the possible cellular effects of the mutation to be identified were aimed to be investigated. This study consists of two parts: The whole exome sequencing data were first screened for 80 loci or genes HSP-related so far. Among these genes, the c.244G>A (p.V82I) variation in the ZFYVE27 gene was detected in all patients but not in healthy individuals. ZFYVE27 gene encodes a protein called Protrudin which is localized in the ER membrane. Protrudin is involved in cellular mechanisms such as the regulation of bidirectional membrane trafficking and recycling of endosomes, formation of cellular extensions, regulation of tubular structure formation for ER morphogenesis, neuronal elongation, and neurite growth. In the first report in the literature associating the ZFYVE27 gene with HSP, the ZFYVE27 gene product Protrudin was identified as an interaction partner of Spastin protein encoded by the SPAST gene. In the same study, it was shown that a different mutation from what I detected in the family involved in this thesis disrupted this interaction between Protrudin and Spastin protein.
Investigation of cobalt resistance in Rhodobacter sphaeroides at molecular level

(Graduate School, 2024-10-11) Atay, Güneş ; Çakar, Zeynep Petek ; 521142112 ; Molecular Biology – Genetics and Biotechnology

Rhodobacter sphaeroides, a Gram-negative α-proteobacterium, is able to perform photosynthesis and possesses a diverse array of metabolic capabilities. These capabilities include lithotrophy, respiration in both oxygen-rich and oxygen-poor environments, nitrogen-fixation, as well as the production of tetrapyrroles, chlorophylls, vitamin B12 and heme. R. sphaeroides possesses the ability to modify its metabolism, its metabolism can adapt to changes in environmental conditions and nutrient availability. This metabolic flexibility, combined with its fully sequenced genome and its ability to thrive under normal growth conditions, makes it a desirable choice for various biotechnological applications. It serves as a valuable model organism for the development and investigation of different protein expression systems. Particularly, it excels in the study of membrane protein complexes that convert light energy into electrical energy. This bacterium offers various advantages, including a fully sequenced genome, having an adaptive and well-known metabolism, and an enhanced membrane surface area when cultivated in oxygen-depleted environments. In a previous study, using evolutionary engineering, a R. sphaeroides mutant population resistant to cobalt chloride stress was obtained. This was achieved by subjecting the initial population to batch selection under gradually increased cobalt chloride stress conditions, without applying random mutagenesis prior to the selection process. Remarkably, the final mutant population exhibited resistance to cobalt levels as high as 15 mM, which has not been previously observed in R. sphaeroides. Seven mutant individuals selected from the final population were investigated, and mutant individuals were physiologically characterized. After characterization, the most resistant individual mutant (G7) with superior resistance properties was selected for more detailed analysis. Additional analysis of the G7 individual mutant from the final population revealed its ability to exhibit cross-resistance against various compounds like nickel (ІІ) chloride (2.2 mM, 2.4 mM), ethanol (8% v/v), sodium chloride (0.5 M), and aluminum chloride (5 mM), magnesium chloride (750 mM, 1M), iron (ІІ) chloride (5 mM), boric acid (30 mM, 50 mM), caffeine (20 mM) and ammonium iron (II) sulfate (5 mM). However, the underlying genomic causes of this physiological resistance capability remained unknown. The main aim of this study was to detect and analyze specific variations in the genetic makeup of the cobalt-resistant R. sphaeroides strain, known as single nucleotide polymorphisms (SNPs), which have the potential to significantly influence their resistance to cobalt chloride stress. By comprehensively exploring the interplay between these SNPs and their potential role in cobalt chloride stress resistance, this study aimed to shed light on the underlying mechanisms and pathways involved, ultimately contributing to a deeper understanding of bacterial adaptation and the development of effective strategies to combat cobalt chloride stress resistance. In this study, Flame Atomic Absorption Spectrometry (FAAS) method was used as a first step to gain insight into the cobalt resistance mechanism of G7 to determine if the mutant individual G7 retains cobalt ions inside the cell or not. As a result of the FAAS analyses, it was found that G7, which can survive in the presence of cobalt stress conditions, takes cobalt ions into/onto the cell. Moreover, to gain a detailed understanding of the underlying mechanisms behind cobalt tolerance, comparative Whole Genome Re-sequencing analysis was performed with G7 to identify and determine single nucleotide polymorphisms (SNPs) in this strain. Specifically, the G7 mutant individual and the Reference Strain (RS) were sequenced which allowed the identification of specific SNPs that potentially play a crucial role in the ability of G7 to resist cobalt chloride. By delving into the genetic variations and their potential implications, this approach aims to unravel the intricate mechanisms that contribute to cobalt resistance, thus paving the way for targeted interventions and strategies to combat this stress. According to whole genome re sequencing results, 11 missense mutations were found in various genes of the G7 mutant individual which were not present in the RS. Known mutated genes include mviN, hutC, rpoD, nifB and nhaD. Further genomic and proteomic studies would be necessary to understand the role of these genes and mutations in the cobalt chloride stress resistance of R. sphaeroides. To summarize, the comprehensive evaluation of cross-resistance tests, growth physiology observations, and genome sequencing data yielded significant insights into the genetic basis of cobalt stress resistance observed in the G7 strain. Through the identification of variations in different genes, this investigation has provided valuable information regarding the underlying mechanisms that contribute to the G7 strain's ability to withstand cobalt and other heavy metal stresses. These findings contribute to the understanding of the genetic background of heavy metal resistance and offer potential avenues for further research and targeted interventions in this field that involve R. sphaeroides.

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