FBE- Moleküler Biyoloji-Genetik ve Biyoteknoloji Lisansüstü Programı - Yüksek Lisans
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ÖgeRecombinant expression, purification and characterization of TNFR1(Fen Bilimleri Enstitüsü, 2020)Receptor/ligand interactions are the mechanisms that form the basis of many signaling pathways. Establishing the correct interaction is crucial for ensuring regulation at the cell level and for the cell's proper functioning. Cytokines are low-molecular-weight glycoproteins that are secreted by different cells in the body, mainly cells of the immune system and which coordinate and propagate immune responses within the body. They have specific effects on the interactions and communications between cells. Cytokines can be divided into pro-inflammatory (such as tumor necrosis factor-alpha (TNFα), interleukin-6) or anti-inflammatory (interleukin-10). The cytokine TNF-α is expressed by a great variety of cells and forms homo-trimers in circulation that have wide biological effects. It is known that TNF-α has important effects, especially in autoimmune diseases. TNF-α acts by binding to specific receptors on the cell surface, stimulating secondary signal pathways. As a result of the stimulation, the signal cascade continues in three main ways: two of them are effective in the inflammation related to nuclear factor (NF) and the third is effective in the process of apoptosis. Today, the use of TNF-α receptors as inhibitors in the treatment of diseases that develop due to the increase of TNF-α is one of the approaches used in the clinical field. TNF-α has two forms in the body, the first of which is the membrane-anchored form (26 kDa), while the second is the secreted form (17 kDa). Firstly, TNF-α is synthesized as a trimeric molecule in which each subunit is corded to the cell surface through a membrane anchor. These membrane tethers are cut by the TNF-α converting enzyme (TACE / ADAM17) which is major sheddase. TNF-α achieves all its different cellular effects by its binding to either the TNF-α receptor 1 and 2 (TNFR1 and TNFR2). TNFR1 and TNFR2 are transmembrane I type proteins located on the cell surface. TNFR1 can interact with both soluble and membrane-bound forms of TNF-α, while TNFR2 can only be stimulated by membrane-bound TNF-α. TNFR1 consists of three main domains: extracellular, transmembrane, and intracellular domain. Signaling proceeds by the recognition of TNF-α trimers by endogenic TNF receptors (TNFR) 1 and 2, which form trimers themselves before complex formation with TNF-α. The intracellular downstream signal pathway begins as a result of conformational changes occurring in the intracellular domain by the interaction of TNF-α in the trimer structure with the extracellular domain of TNFR1. It has been proven that anti-inflammatory antibodies and antigen-binding fragments (Fab) against TNF-a successfully suppress TNF-a induced inflammation in inflammatory autoimmune diseases. In the field of biotherapeutic medicine, there are approaches regarding the use of TNFR1's extracellular domain as a therapeutic tool. In light of these approaches, we aimed at bacterial production, purification, and characterization of the extracellular domain of TNFR1 (hereafter TNFR1). In our study, the plasmid containing the TNFR1 gene was transferred into the host cell of which is Escherichia coli (E. coli) BL21DE3. Protein expression was achieved by IPTG induction. Inclusion bodies formed in the cell as a result of high protein expression were isolated from cell lysates and solubilized. The target protein was purified from the protein mixture by immobilized metal affinity chromatography (IMAC). Purification efficiency was confirmed by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) and immunoblotting techniques. Blue native polyacrylamide gel electrophoresis (BN-PAGE) was used to understand the native structure of the pure protein and it was found that the protein was in dimer structure. In order to secondary structure estimation of the purified protein, circular dichroism (CD) which is defined as the unequal absorption of left-handed and right-handed circularly polarized light was used. According to measurements in the far-UV range, beta structures were found to be dominant in the protein structure. In the measure of protein purity, capillary electrophoresis (CE-SDS) and high-performance liquid size exclusion chromatography (HPLC-SEC) methods, which are more sensitive techniques for protein characterization, were used. The purified protein was determined to be> 95% purity by CE-SDS. It was confirmed that the protein in the HPLC-SEC was dimeric according to the molecular weight and size of the pure protein. A Pull-down assay was utilized for the determination of the functionality of the recombinant protein. Binding experiments were performed with both the receptor in the cell lysate and the purified ones. As a result of the pull-down assay, the receptor was found to bind to TNF-α. The sample of the binding assay with purified receptor was also analyzed by the HPLC-SEC method and complex formation was observed. Based on the result of the experimental findings, it was found that the methods developed for the engendering and purification of the recombinant TNFR1 protein can be used to obtain the pristine product that maintains its bioactivity.
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ÖgePhysiological investigation of Rhodobacter sphaeroides(Institute of Science and Technology, 2013)Rhodobacter sphaeroides is an ?-3 purple non-sulfur eubacterium with an extensive metabolic organization. Under anaerobic conditions it is able to grow by performing photosynthesis, respiration and fermentation. Photosynthesis occurs only under anaerobic conditions since photosynthetic apparatus of R. sphaeroides is harmed in the presence of oxygen. Photosynthesis may be both photoheterotrophic and photoautotrophic. While R. sphaeroides performs photoheterotrophic photosynthesis using organic compounds as both a carbon and a reducing source, R. sphaeroides performs photoautrophic photosynthesis using carbon dioxide as the sole carbon source and hydrogen as the source of reducing power. In addition, R. sphaeroides can grow both chemoheterotrophically and chemoautotrophically. Regarding biotechnology applications, R. sphaeroides is used both in industry and in medicine. Industrial application of R. sphaeroides comprises hydrogen gas and polyhydroxybutyrate (PHB) production and bioremediation of heavy metals. R. sphaeroides can also be used in the production of vitamin B12, coenzyme Q10, 5-aminolevulinic acid (ALA) and porphyrin. Like other microorganisms, R. sphaeroides has different defence mechanisms to give response against different stress types that occurs frequently in its microenvironment. Defence mechanisms of R. sphaeroides vary according to the types of stress. For instance, while under osmotic stress condition, R. sphaeroides gives response by increasing cardiopilin levels which is an anionic phospholipid playing an important role in energy conversion in cells of R. sphaeroides, as for, under oxidative stress condition it gives response increasing its carotenoid levels.
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ÖgeDisease gene identification using linkage and exome analysis(Institute of Science And Technology, 2020-06-15)Rare disorders are described as conditions with prevalence less than 1 in 2000 people. Approximately 300 million people are affected with rare disease worldwide and in Turkey this number is estimated as 5 million. More than 6000 rare diseases are identified to date and 80% of these are monogenic disorders. Disease onset is usually during childhood with chronic, progressive and debilitating nature. Patients life expectancy is lower than unaffected population. Extensive clinical examination is required for definite diagnosis. For these reasons it is crucial to reveal underlying mechanisms of rare disorders. Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, chronic autoinflammatory disorder that affects children between age of 2-17. It is characterized by aseptic,symmetric and recurrent bone lesionsthat tend to cluster around metaphysis of long bones such as femur, tibia, vertebral column and pelvic floor. Although there is no definitive data on prevalence of CRMO, it is estimated to be 1-2 cases per million. Though having an unclarified etiology and pathogenesis, families with multiple affected members suggest genetic background on disease development. Identification of disease-causing genes - LPIN2 and IL1RA in Majeed syndrome and deficiency of interleukin-1 receptor antagonist (DIRA) which are defined as chronic nonbacterial osteomyelitis (CNO) further strengthen the involvement of genetic features. Takayasu arteritis (TA) also known as pulseless disease is a rare, systemic large vessel vasculitis that predominantly affects women younger than 40 years old. Its major clinical symptoms are occlusion and stenosis of aorta and its main branches accompanied with loss of radial pulse whereas involvement of pulmonary, coronary, renal arteries and aneurysmal formations are also encountered. Severity of the clinical progression depends both on the vessels afflicted and ethnicity of the patient. Highest prevalence of TA is reported in East Asia, especially Japan with up to 40 cases per million. In Turkey, it is postulated to be the second mostly encountered vasculitis with a prevalence up to 12.8/million accompanied with 1.11/million incidence. In spite of undefined etiology, several factors including infections are suggested to be involved in TA pathogenesis. Monozygotic twins diagnosed with TA and familial aggregation observed in some cases indicate the role of genetic factors in disease phenotype. Various genome-wide association studies (GWAS) in cohorts of different size and ethnicity replicated significant association of HLA-B*52 and IL12B to TA. Susceptibility loci FCGR2A/FCGR3A, MLX, HLA-B/MICA and HLA-DQB1/HLA-DRB1 also show significant association with TA. In this study we present three Turkish consanguineous families with multiple members diagnosed with TA and one consanguineous family with two brothers diagnosed with CRMO. Clinical diagnosis and follow up of TA families were done at Cerrahpaşa Medical Faculty whereas CRMO patients were diagnosed and followed in Pamukkale University Medical Faculty. Individuals' were informed and their written consent for participating in the study was obtained accordingly to the ethics committee approval (MBG.22/2014). The aim of the study is to identify disease causing genes by using gene mapping techniques as linkage and exome analyses. Linkage analysis and homozygosity mapping were used to identify non-recombinant homozygous genomic regions that were shared between affected individuals but not with healthy members. By exome analysis, possible candidate genes were investigated. Linkage analysis and homozygosity mapping of CRMO1 family revealed 54 regions that are larger than 200 kb with LOD score > 1.50 that were shared among the affected siblings. Exome analysis on these regions did not reveal any homozygous, non-synonymous and rare variants. Therefore, exome analysis was conducted without basis on linkage regions revealing 10 variants out of which only CARNS:c.1145C>T had a closer association with CRMO phenotype when compared to the rest. However, the region it lied was homozygous in all family members eliminating the variant from being a candidate. Two sisters diagnosed with TA are present in all three families under investigation. TA1 and TA2 families indicate autosomal recessive inheritance model. Affected sisters of TA3 family are half siblings (sibs) who share the same mother but different fathers. CRMO1 family two brothers were diagnosed with CRMO. Genomic DNA samples obtained from peripheral blood were used for genotyping via genome-wide single nucleotide polymorphism (SNP) array where ~710,00 fixed markers were genotyped using Illumina Infinium OmniExpress microarray. By performing multipoint parametric linkage analyses under recessive inheritance model and homozygosity mapping using the SNP data, non-recombinant regions that were shared among the affected individuals but not with the healthy members were detected. Exome sequencing data were used to fine map these regions to variant resolution and investigate the presence of a potential candidate variants involved in the TA or CRMO phenotype. Both linkage and exome analyses were conducted under the recessive inheritance model. Variants in the functional regions (exonic and splicing) with minor allele frequency (MAF) < 0.01 were further examined with bioinformatics tools. Expression sites and levels, pathways, interactions, evolutionary conservation of the genes harboring candidate variants were studied to unravel any potential involvement in the immune system or inflammatory response that could lead to the TA or CRMO phenotype. Linkage analysis of TA1 family revealed 59 regions larger than 200 kilobases (kb) with maximal LOD score 3 1.50. Homozygosity mapping confirmed 41 of these to be shared between affected individuals. Co-joint multipoint parametric linkage analysis of TA1 and TA2 families indicated 123 linkage regions larger than 200 kb with LOD score 3 1.50. Nine of these were shared between all affected members of both families. Exome analysis of these sites revealed 9 variants in total of which 5 were in TA1-04 which are RELN: c.103301976_103301984dup, ANXA8L1: c.449C>T, EHBP1L1: c.1321C>T, MYH14: c.565C>T and SHANK1: c.3947G>A. The remaining 4 variants in TA2-01 are AP4B1: c.767C>T, MST1L: c.811dupG, AGAP: c.G8A and ZNF829: c.1173A>T. Variant filtration and prioritization of the exomes of TA3 family members, revealed no common variant that are found in affected sibs. There was no gene that got hits that were present in both TA patients. Although our results were not indicative of a gene involved in the TA development, this is the first study integrating linkage analysis, homozygosity mapping and exome analysis where three consanguineous families with multiple affected members were included to identify possible disease-causing variants. Variants detected in TA patients should be further studied to unravel their possible roles in TA pathogenesis.
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ÖgeExploring the conformational transition between closed and open states of the sars-CoV-2 spike glycoprotein using molecular dynamics simulations(Institute of Science And Technology, 2020-06-15)Coronaviruses (CoVs) are classified as a genus under the Coronaviridae family within the order Nidovirales which includes Mesoniviridae, Arteriviridae, Roniviridae, and Coronaviridae families. The Coronaviridae family includes the Coronavirinae subfamily, which is subdivided into four genera; alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses. Coronaviruses are responsible for a wide variety of diseases in humans and animals; alpha- and betacoronaviruses infect mammals, gammacoronaviruses infect mostly birds and few mammals and deltacoronaviruses infect both mammals and birds. Although coronaviruses have been associated with many diseases, historically, coronaviruses had been associated with 15-30% of self-limiting respiratory infections each year in humans. This situation was accepted as such until a member of betacoronavirus named the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic occurred in 2002. Subsequently, another outbreak caused by another virus belonging to the betacoronavirus genus named the Middle East respiratory syndrome (MERS-CoV) following the SARS-CoV outbreak occurred between 2002-2003 and caused the coronaviruses to be seen as possible pandemic agents. Coronaviruses are able to adapt to new conditions through recombinations and mutations uncomplicatedly, thus, they can alter their host targets efficiently. As a result of these adaptation abilities, a new type of betacoronavirus, which can spread much faster and bind better than SARS-CoV and MERS-CoV, was detected in 2019. A novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the infectious coronavirus disease 2019 (COVID-19) has resulted in a pandemic crisis since its first registration in December 2019. This virus belongs to the same large betacoronavirus family including SARS-CoV and MERS-CoV that caused other epidemic diseases in the past years. Similar to other coronaviruses, SARS-CoV-2 consists of a viral envelope that has a bilayer lipid structure and three structural proteins embedded in this viral envelope: envelope, membrane, and spike (S). Among these structural proteins, S proteins have a critical role on host cell infections since they are involved in the recognition of the host cells and fusion between viral and host cell membranes. S proteins are large trimeric glycoproteins and contain two functional subunits: the S1 subunit responsible for binding to host cell receptors and the S2 subunit responsible for fusion of the host cell and viral membrane. The S1 subunit contains the N-terminal domain (NTD) and the receptor-binding domain (RBD) which can bind directly to the receptor of the host cell. RBD undergoes rigid-body motion to hide or expose the receptor-binding surface. Depending on this movement, S protein can be in receptor inaccessible or receptor accessible states. In the receptor inaccessible state (closed state), all RBDs are in the down position whereas, in the receptor accessible state (open state), at least one RBD is in the up position to engage with the host cell receptor. In order to initiate the binding and fusion mechanism, the S protein switches from the closed state to the open state and bind to the host cell receptor. SARS-CoV-2 S proteins target the angiotensin-converting enzyme 2 (ACE2) receptors located on the membranes of human respiratory epithelial cells and S proteins can bind to ACE2 receptors due to the RBD in their structure. As a result of receptor binding, a series of structural changes occur in the S protein required for fusion mechanism. There are two main cleavage sites in the structure of S protein that are cleaved by the host cell enzymes: the S1/S2 and S2' sites. Upon cleavage of the S1/S2 site, S1 and S2 subunits remain non-covalently bound with each other in the pre-fusion conformation. Cleavage of the S2' site is a prerequisite for the separation of S1 subunit and viral anchored S2 subunit, and for the fusion of the viral and host cell membranes since fusion peptide (FP) at S2 subunit is exposed to the solvent environment after cleavage on this site. Thus, conformational changes that are essential for the fusion mechanism occur in critical fusion mechanism structures heptad repeat 1 (HR1) and heptad repeat 2 (HR2) structures. Conformational change of HR1 to an extended alpha-helix promotes FP insertion in the host cell membrane. This interaction triggers the rearrangement of HR2 to fold over HR1 and form a six-helix bundle. Consequently, viral and host cell membranes are pulled into proximity, allowing the fusion of these membranes and the release of the viral genome into the host cell. This pre-fusion to the post-fusion transition of the S protein can be blocked at any point during the process to prevent the release of the genome into the host cell. Since the recognition of the host cell and release of the viral genome into the host cell are the most crucial steps for pathogenesis and viral infectivity, exploration of the binding and fusion mechanism of S proteins as a potential therapeutic target for developing antiviral drugs has become prominent. In the literature, the binding and fusion process using the complete S protein structures has not been modeled at an all-atom level using molecular dynamics (MD) simulations. MD simulations provide effective insights into structural, dynamic, and energetic information at the atomic level which are difficult to access by experimental techniques. The main aim of the thesis is the modeling of the transition between closed and open states of the SARS-CoV-2 S protein RBD by performing MD simulations. In this thesis, the switching mechanism of the RBD from its closed state to open state is modeled and analyzed using MD simulations and statistical thermodynamic methods. Initial conformations that are used in MD simulations were obtained from crystal structures having PDB ID 6VXX and 6VYB where the RBD of the SARS-CoV-2 S protein in closed and open states, respectively. These structures cover 76.4% of the protein sequence. The remaining parts of the protein sequence were completed using the homology modeling method. Glycan molecules attached to S protein in the crystal structures were conserved throughout the MD simulations. For both open and closed state structures, MD simulations were performed. Based on the results, S protein in the open state was found to be more mobile than the closed state. Salt bridge and hydrogen bond analyses between RBD structures in up and down positions showed that there were a different number of interactions between two positions; and the difference of these interactions between up and down positions might be the reason for the mobility difference between them. In addition, a steric clash between S protein and ACE2 was found in the closed state. This steric clash between ACE2 and the closed state S protein prevents ACE2 binding in the closed state. Therefore, an inhibitor that binds to the S protein in the closed state does not need to compete for the binding interface with the ACE2 receptor. This finding indicates that inhibitory molecules targeting the S protein in the closed state might be developed to prevent RBD-ACE2 binding. In silico pulling experiments were performed to obtain the transition between the two states using steered MD (SMD) simulations. SMD simulations were initiated from closed and open state structures that are obtained with MD simulations and performed for two directions by pulling the RBD structure from down to up conformation to switch between closed to open state and up to down conformation to switch between open to the closed state. In this way, the switching path between closed and open states is obtained. Results of MD and SMD simulations were used for principal component analysis (PCA) to determine the most important and dominant movements taking part in the transition. PCA is an effective and proven method used to determine the most prominent movements of a protein. The covariance matrix of alpha carbon atom coordinates in the protein structure was constructed using the MD simulations data. The diagonal elements of this matrix give the variance value for each amino acid. Based on the simulation results, the first two principal components (PCs) correspond to 96.1% of the total variance, thereby identifying the two most prominent PCs of the protein. Using the determined PCs, the free energy surface was created, and based on the generated energy landscapes, the minimum free energy pathway representing the transition between the down and up state of the S protein protomers was constructed. Energy landscapes suggest the existence of a semi-open state between down and up states of the S protein protomers and several additional substates at various locations were determined. While RBD is in the semi-open state, RBD of one protomer is halfway between its down and up positions while the RBDs of the remaining two protomers are in the down position. The semi-open state shows a different network of interactions than the down and up states and does not show any steric clash to binding with the ACE2 receptor. These findings show the possibility of RBD-ACE2 binding while the S protein in the semi-open state. In the thesis, investigations have been made to discover structural characteristics and transition pathway of the SARS-CoV-2 S protein RBD. This thesis provides an extensive insight into the interdomain interactions, dynamics, and solvent accessibility of the SARS-CoV-2 S protein RBD in its open and closed states and its transition pathway between the closed and open states.
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ÖgeSurface modification of titanium substrates with nano hydroxyapatite coated chitosan microspheres(Institute of Science And Technology, 2020-06-15)Today, implants are increasingly used to replace or support the damaged structure or function in human body. Titanium and Ti based materials are commonly preferred as implant materials due to their superior mechanical features and biocompatibility. Although implants are biocompatible, well designed, and functional, they carry the risk of infection. Implant associated infections are serious problem after surgical operations because they generally lead to revision surgery and thus increase morbidity, length of hospitalization and health care cost. These infections are generally caused by the attachment of microorganisms to the implant surface and subsequently biofilm formation. In order to address this problem, surface modification of implant materials and drug delivery strategies have been studied. Releasing antibiotics from the implant surfaces is an effective approach to increase the implant success by local antimicrobial delivery. For further improvement, the hydroxyapatite coating is one of the most preferred methods to fasten osseointegration. In this study, Ti surfaces were modified with antibiotic loaded chitosan microspheres and coated with nano hydroxyapatite (nHA) to prevent implant related infections and increase the osseointegration. Firstly, antibiotic (ciprofloxacin) loaded chitosan microspheres were prepared via emulsion/cross-linking method using different stirring rates (300, 400 and 500 rpm) and analyzed under light and electron microscopes. It was seen that more homogenous size distribution and smaller microspheres were obtained with increasing stirring rate and 500 rpm was preferred for microsphere production. Before the immobilization of microspheres, Ti plates were oxidized and then silanized with APTES (3-Triethoxysilylpropylamine) to form amino groups (- NH2) on the surfaces. Ti plates were characterized with Fourier transform infrared spectroscopy (FT-IR) to analyze the chemical composition of surfaces and scanning electron microscopy (SEM) to examine surface morphology. Characterization studies proved that chemical groups required for cross-linking were formed on the surfaces. After that, the chitosan solution was used for immobilization of the microspheres. Microsphere - chitosan solution (2%) was prepared and spread on Ti surfaces activated with glutaraldehyde GA (8%) for cross-linking, then the samples were freeze-dried and analyzed by SEM. The amount of chitosan solution and microspheres were optimized, and the chitosan to microsphere ratio was chosen as 50:5 (µl:mg) for 1.5 cm2 Ti plate. Secondly, microsphere – chitosan modified Ti samples were coated with nHA using CaCl2 (1.25 mM) and Na2HPO4 (0.75 mM) solutions. The presence of nHA was analyzed with FT-IR and X-ray diffraction spectroscopy (EDS), and the crystalline structure of nHA was analyzed with an X-Ray diffractometer (XRD) then nHA structures on the surfaces were viewed by SEM. Characteristic functional groups (-OH and -PO4 -3 ) of nHA were detected in FT-IR analysis and Ca and P minerals were observed in EDS analysis. Before the drug release study, the whole system was tested xx in PBS for 30 day and flaking or fractures were not observed showing the stability of the system. Finally, drug release studies were carried out with free and immobilized microspheres (uncoated and nHA coated) and it was seen that sufficient amount of drug can be released from Ti surfaces although drug release profiles were affected by immobilization and nHA coating. Thus, a dual-functional system with antibacterial activity and tissue integration ability could be made. This system will be used in animal experiments after the antibacterial and bioactivity studies.