LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans

Bu koleksiyon için kalıcı URI

http://hdl.handle.net/11527/19401

Gözat

Alteration of titanium surfaces using hyaluronic acid coated mesoporous silica nanoparticles for local drug release

(Graduate School, 2021-12-17) Erşan, Yeliz ; Karataş Yazgan, Ayten ; Önder, Sakip ; 521181123 ; Biology-Genetics and Biotechnology

Nosocomial infection is still an important problem for developed and developing countries as it decreases the effectiveness of the treatment as well as increases the healthcare expenses due to the prolonged stays in units. Most of these infections are biomaterial-based and caused by biofilm forming bacteria on biomaterial surfaces. Therefore, systemic drug administration is used to prevent biomaterials associated infections and to increase success of the implantation. Otherwise, revision surgery is generally required. Revision surgery means more pain for the patient, and it does not guarantee that osteointegration between the implant and surrounding tissue will be as strong as the first implementation. Local drug release using drug eluting implant materials suggests a great opportunity to prevent implant associated infections. Functional coatings containing therapeutic agents can be deposited on these surfaces by using different surface coating techniques such as layer by layer deposition (LBL), electrophoretic deposition (EPD), physical/chemical vapor deposition (CVD/PVD) etc. Local drug release from these surfaces does not only prevent implant associated infections, it also prevents the systemic toxicity. Moreover, sustained local drug release is possible with drug eluting materials. In the present study, a functional coating based on hyaluronic acid (HA) and mesoporous silica nanoparticles (MSNs) was proposed to prevent implant associated infections. For this purpose, drug loaded HA coated MSNs were prepared and deposited onto the Ti implants using EPD technique. Mesoporous silica nanoparticles (MSNs) were synthesized by the sol-gel/emulsion method and analysed using different characterization techniques such as transmission electron microscopy (TEM), scanning electron microscopy (SEM) and dynamic light scattering (DLS). After that, MSNs was silanized by using APTES (3- Triethoxysilylpropylamine) to form amino groups (-NH2) on the MSNs surfaces. In order to form HA coatings on MSN surfaces, HA was dissolved in MES buffer, NH2 -MSNs were added into the solution, EDC (N-(3-Dimethylaminopropyl)-N'-ethyl- carbodiimid-hydrochloride) and NHS (N-hydroxysuccinimide). Characterization of silanized (-NH2-MSNs) and HA-coated MSNs (HA-MSNs) were done by using SEM, Zeta Sizer and Fourier Transform Infrared Spectroscopy (FT-IR). SEM micrographs showed that almost homogeneous spherical MSNs ranging from 185 nm to 240 nm were synthesized. DLS analysis was showed that their sizes are ca. 8.7 μm. The cause of the difference was due to the environment MSNs exposed during characterization. SEM analysis is performed on dry nanoparticles while watery environment is used for DLS analysis. TEM analysis gave similar results with SEM for the sizes of the MSNs. Porous structure of synthesized MSNs were shown in this analysis. Moreover, characteristic peaks at 696 cm-1 and 1540 cm-1 that are attributed to –NH2 groups after silanization and peaks at 1639 cm-1 that are attributed –CH groups following the HA coating were determined in FTIR analysis. According to these results MSNs, -NH2-MSNs and HA-MSNs are synthesized and modified successfully. Ti plates were treated mechanically and chemically prior to deposit nanoparticles. Deposition onto the Ti surfaces were carried out using two different procedures. In the first procedure, HA-MSNs were dissolved in ethanol (70%) and HA coated MSNs deposited onto the Ti substrates for 1, 3 and 5 min. at 30V. In the second procedure, different ratios of MSNs and HA solution (MSNs:HA (w/w; 1:0.5, 0.5:1, 1:1)) were prepared and electrodes were then placed in this solution. Same coating parameters were applied for both coating procedures. The surfaces were analysed using SEM, FTIR and X-ray spectroscopy (EDS) to examine the surface morphology and chemical composition of the surfaces following the coating process. Characterization studies showed that both procedure 1 and procedure 2 can be used to obtain homogenous coatings on the surfaces. Neverthless, MSNs:HA should be 1:0.5 because coatings that were deposited using higher HA concentrations detached from the surfaces due to the thick film formation. In addition, SEM and EDS analysis showed that coating thickness can be increased with prolonged deposition time. The coating thicknesses were determined using mechanical profilometer and different thicknesses from 0.48 to 1.9 micrometer. were measured on the surfaces for different coating times. According to results of analysis, thickness was increased while increasing deposition time. Drug loading and release studies were carried out in PBS for free (synthesized MSNs, -NH2-MSNs, HA-MSNs) particles and coated particles (HA-MSNs) that were prepared using two different procedures. Ciprofloxacin as a model drug was used in this study, and it was both loaded into free MSNs and coated surfaces by diffusion. In accordance with the drug loading studies, drug loading efficiency was higher for HA-MSNs (ca. 80 %) when compared to HA free MSNs (ca. %40). Moreover, it was shown that drug release was possible using free MSNs for ca. 15 hours at 37 oC. Moreover, synthesized and silanized MSNs did not show sufficient drug loading and release rate like HA- MSNs. Finally, drug release from the coated Ti surfaces were examined. According to the drug release profiles, it was possible to have 10 hours drug release was determined from the Ti surfaces. In sum, HA-coated MSNs can be used as a functional coating to design a drug eluting Ti implant material and to prevent implant associated infections.
Improving bone tissue integration of hard tissue implamants using bioactive materials

(Graduate School, 2022) Kerem, Gizem ; Kılıç, Abdulhalim ; 867049 ; Department of Molecular Biology-Genetics and Biotechnology

Hard tissue implants which made of different materials are used widely in medical applications in bone and tooth deficiencies. Metallic and ceramic materials are preferred as hard tissue implants because of their strength and toughness. Due to low density, high corrosion resistance and biocompatibility properties of titanium and titanium alloys, their usage as an implant has gradually increased. Implant materials should not cause an immunogenic reaction in the body and also in recent years thanks to new technologies in biomaterial field there are some applications to improve interaction in a positive way between implant and implantation tissue. Some morphological changing applications like grooving and acid etching, some physicochemical activation treatments like hydroxyapatite (HA) coating and titanium oxide (TiO2) coating, some biochemical activation treatments like coating with biopolymers and immobilization of bone morphogenic proteins and inducer chemicals are studied on titanium surface to improve bone formation, bone bonding and antibacterial property. At this study, titanium (grade 2) surfaces are chosen to increase its bone tissue integration by designing bioactive surface by using chitosan microspheres as a natural polymer and water-soluble dexamethasone (DEX) as an inducer for SAOS-2 cell differentiation. Chitosan microspheres were produced by using single emulsion-crosslinking method. Different amount of glutaraldehyde with different percentages was used as a crosslinker agent to obtain optimum DEX loading into chitosan microspheres. Crosslinking time parameter was also changed for optimization. Dexamethasone loading was tried in two ways first one is adding drug into dissolved chitosan solution then produce chitosan microspheres, second one is adding produced chitosan microspheres into the drug solution. DEX was loaded into chitosan microspheres via diffusion. After optimization process, chitosan microspheres were produced by using glutaraldehyde as a crosslinker. Chitosan microspheres were placed into DEX solution for loading process. Drug release studies were performed by using DEX-loaded chitosan microspheres, and released amount of DEX was determined. Drug loading efficiency was found as 50.16% and release of DEX was observed for 12 hours and the released amount of the loaded DEX was calculated as ~32.6%. Before chitosan microsphere coating onto titanium surfaces, they were treated chemically (polished, oxidized and silanized to produce amino groups on titanium surfaces). Then the samples were activated by glutaraldehyde and coated with chitosan microspheres.
The computational study of the interaction of POT1 with SSDNA and TPP1

(Graduate School, 2022) Gürbüz Önder ; Balta, Bülent ; 740109 ; Molecular Biology-Genetics and Biotechnology Programme

Linear DNA sequences in eukaryotes have telomere sequences at the chromosome ends. In humans, telomeres consists of repeating TTAGGG sequences. Shelterin complex mainly interacts with telomere sequences. It interacts with both telomeric dsDNA and ssDNA. It protects it from DNA repair mechanism. POT1 is one of the members of shelterin complex. It interacts with telomeric ssDNA. It controls the length of the telomere sequence by forming complex with TPP1. The N-terminal domain of POT1, POT1N, binds to ssDNA while C terminal domain of POT1, POT1C, is known to interact with TPP1. The interaction between POT1 and ssDNA depends on many factors. In the shorter sequences, POT1 prefers to bind 3' end of the sequence by 10 fold. The preference changes as TPP1 binds to POT1. POT1 slides on ssDNA after forming complex with TPP1. There is no information about the mechanism of action in the literature. TPP1 interacts with POT1 and increases its affinity to ssDNA. The affinity of POT1 on ssDNA increases after interacting with POT1 and POT1-TPP1 interaction is regulated by cell cycle as well. It is likely that the POT1-TPP1 complex has at least 2 different conformations and these conformations have different interactions, conformations and geometries. There is no information about these structures in the literature. The preference of POT1 on ssDNA, potential initial binding and sliding mechanisms, POT1-TPP1 complex and the allosteric effect of TPP1 were investigated by standard Molecular Dynamics (MD) simulations and Replica Exchange Molecular Dynamics (REMD) simulations. Free energy of the system was calculated by Molecular Mechanic/Poisson-Boltzmann Surface Area (MM/PBSA) method. Another candidate for the mechanism was investigated by Potemtial of Mean Force (PMF) method. According to our results, the binding preference of POT1 depends on the length of ssDNA. In a longer DNA sequence (22 nucleotides), POT1 has the highest abundance in the middle of sequence, ~27%, followed by 5'end, ~ 17% and 3' end ~ 16%. In a shorter DNA sequence (16 nucleotides), POT1 prefers binding to the 3' end over 5' end (~32% and ~22%). This results shows that the preference of POT1 is influenced by ssDNA length. Our results indicate that extra nucleotide in the longer ssDNA simulations form secondary structure with each other and this might explain 5' end preference over 3' end. In the shorter ssDNA simulations, 3' end is loosely bound to POT1 ~23% of the time and ~8% in the solvent, while 5' end has ~8% protein bound and ~14% in the solvent and this explains 3' preference in the shorter ssDNA simulations. Our results show that there are multiple potential initial binding and sliding mechanisms. Most of the time, POT1 slides only 1 OB fold at a time while other OB fold conserves its position. The sliding happens per nucleotide in this mechanism. The relative abundance of these OB shifted structures were ~ 21%. Here, OB2 fold shifted structures had higher abundance than OB1 fold shifted ones which confirms the literature. Another potential mechanism involves the repetetive nature of telomere sequence. POT1 might prefer to skip a telomere repeat and OB fold can bind to the next telomere sequence. When OB1 fold binds to telomere sequence, OB2 fold moves 6 nucleotides and then OB1 folds shall slide on ssDNA. Furthermore, when ssDNA was interacted with only OB1 fold or OB2 fold, it clearly showed that OB1 fold prefers to bind ssDNA. The relative abundance of these structures were ~4.5% and ~1.5%, respectively. When whole sequence was shifted by 1 nucleotide, which is considered a potential mechanism, the structure has the lowest abundance. In order to test this for 2, 3, 4 and 5 nucleotides, 5 new initial structure was created. In these structures, ssDNA was 11 nucleotide long. The crystal structure and 6 new structures were simulated. G results show that all of these structures are not a candidate for a potential intermediate structures during the sliding. There are 2 different complexes for POT1-TPP1. In the first complex, it should slide easily on ssDNA and it should have less specific interactions. In second complex, it should bind it tightly and have specific interactions. Both structures were constructed based on O. nova. In the simulations, they survived more than 1 µs.
In vitro and in silico investigation of NFIB-SUMO interactions

(Graduate School, 2022) Özkan, Ayberk ; Kumbasar, Aslı ; 718174 ; Molecular Biology-Genetics and Biotechnology Programme

Nuclear Factor I family of transcription factors are involved in regulation in diverse physiological processes, including neuronal terminal differentiation, gliogenesis, stem cell quiescence as well as in pathologies such as tumorigenesis and cancer progression. NFI family is encoded by four genes: NFIA, NFIB, NFIC and NFIX. NFI proteins contain a highly conserved N-terminal DNA binding and dimerization domain, while their C-terminal domains diverge. Because of their highly similar DNA binding and dimerization domain, NFIs bind to a palindromic consensus site with similar affinity in vitro, potentially regulating the same set of target genes in vivo. Any functional differences between the family members may arise from the more diverse C-terminal domain provides which can promote either transcriptional activation or repression. NFIs may regulate target gene expression via different mechanisms of action. NFI can bind directly to DNA and regulate the expression of the target gene or interact with another protein to affect gene expression indirectly. Moreover, NFIs can interact with histone proteins and cause alterations in the nucleosome structure, thereby being involved in the formation of the transcription complex. NFIs can directly interact with and facilitate recruitment of basal transcription factors. NFIs can also bind co-activator or co-repressors to control transcriptional activation. In addition, NFIs can, along with other transcription factors, co-regulate target gene expression. Finally, NFIs can promote dissociation of DNA methyltransferase from target gene promoters and activate transcription. Any alterations in the production or action mechanisms of NFI proteins lead to important developmental defects as well as cancer. One member of the NFI family, NFIB, is an essential gene as demonstrated by studies on knockout on mice: silencing of NFIB leads to perinatal death due to lung defects. NFIB controls stem cell differentiation in different cell types such as, adipocytes, megakaryocytes, melanocytes and hippocampal neural progenitors. Interestingly, in humans, mutation of one copy of the NFIB gene can result in intellectual disability and brain malformations. These findings underscore the importance of NFIB as a transcriptional regulator, however, the mechanism by which NFIB acts or regulatory events upstream of NFIB have not been fully elucidated. Indeed, scarce data exists regarding NFI post-translational modifications. Phosphorylation, glycosylation, acetylation, sumoylation may regulate the activity of NFI. Among these modifications, sumoylation is conserved by eukaryotic organisms. Sumoylation regulates many cellular mechanisms such as nuclear transport, chromosome segregation, and transcription activation/repression. SUMO (small ubiquitin like modifiers), which is generally observed as a suppressor in transcriptional regulation, can be conjugated to many transcription factors and affects the activity of these factors. The SUMO gene family has five mammalian isoforms: SUMO1, SUMO2, SUMO3, SUMO4 and SUMO5. SUMO peptides are activated by a series of enzymatic processes. These processes are required to form mature SUMO, which is active and may able to conjugate specifically to the target protein. The sumoylation consensus sites and SIM (SUMO interacting motif) on target proteins enable SUMO to specifically recognize and bind to these proteins and regulate their activities. Sumoylation can affect transcription factors in several ways. SUMO can compete with other modifications, may interact with co-activators, and can control the binding of the transcription factor to its target site on chromatin. In addition, sumoylation can control intracellular localization of transcription factors. Sumoylation of NFI has been shown in vitro. Interestingly, a study on neuroblastoma cells exposed to oxidative stress, identified NFIB among sumoylated proteins modified on sumoylation consensus sites. However, sumoylation of NFIs have not been further explored, in silico and in cell culture. In this study, we set out to investigate the functionality of sumoylation consensus site and SUMO interacting motifs of NFIB, using in silico methods and forced expression in cell culture. Currently, there is no experimental or modeled 3D structure of NFI proteins. Information about NFI protein structure is quite limited. As mentioned above, NFI proteins contain an N-terminal DNA binding and dimerization domain and C-terminal transactivation domain. NFI proteins carry four conserved cysteine residues in their DNA binding and dimerization domains, three of which are required for DNA binding activity. Another piece of evidence regarding NFI structure comes from the homology with the MH1 domain of SMAD3. Both proteins have highly similar Cys-His box motifs consisting of three cysteine residues and one histidine residue. Nevertheless, this homology is below 30%. Here, due to the lack of high homology and also fold similarity, we used ab initio modeling method to predict structure of NFIB DNA binding and dimerization domain. Subsequently, these predictions were compared to each other. For this comparison, we focused on the cysteine residues which are required for DNA binding activity as well as the conserved MH1 Cys-His box motif. Then, selected structure prediction models were assessed by molecular dynamic simulations. Finally, with REMD (Replica Exchange Molecular Dynamics) the model that showed higher stability and quality was verified. We performed molecular docking simulations to investigate NFIB SIM-SUMO1 interactions. We found that SUMO1 would preferentially bind to a specific NFIB SIM. Meanwhile, to investigate NFIB-SUMO1 conjugation in vitro; site-directed mutagenesis was performed for generation of a sumoylation consensus site mutant and a SIM mutant. HEK293T cells were co-transfected with SUMO1 and wild-type NFIB or NFIB mutants and NFIB was immunoprecipitated for analysis of NFIB-SUMO1 conjugation. Future experiments are required to validate the putative NFIB sumoylation consensus site and SIMs in cell culture.
Investigation of the mitochondrial metabolism of Helicobacter-activated B cells

(Graduate School, 2022) Şentürk, Zeynep Nur ; Yazgan Sayı, Ayça ; Molecular Biology-Genetics and Biotechnology Programme

Helicobacter pylori (H. pylori) is a gram-negative, microaerophilic, and spiral-shaped bacterium and a member of the Helicobacteraceae family. H. pylori was discovered in 1982 by Warren and Marshall. Helicobacter infection can lead to multiple gastro pathologies such as chronic gastritis, gastric cancer, peptic ulcer disease, and mucosa-associated lymphoid tissue lymphoma. Whereas more than 50% of the world population has been infected with H. pylori, 80% of them are asymptomatic. Similar to H. pylori; H. felis is a gram-negative, urease-positive, spiral-shaped, and microaerophilic bacteria. Studies have shown that H. felis can induce gastric atrophy, metaplasia, dysplasia, chronic and persistent inflammatory response, and gastric cancer in mice models. Because H. pylori have less capacity to activate an efficient immune response in mice compared to Helicobacter felis (H. felis); H. felis is used to generate mice models for studying this pathogen. B cells play critical roles in adaptive immunity with antigen presentation to T cells and antibody production. Recently, new B cell subsets have been shown to exert anti-inflammatory and immune suppressive properties were discovered. These B cells are termed regulatory B cells (Bregs) by Bhan and colleagues. In mice; mainly CD19+CD21hiCD23hiCD24hi transitional 2 marginal-zone precursor cells (T2-MZP), IL-10 producing CD1dhiCD5+ B10 cells, CD19+CD21hi CD23- marginal-zone (MZ) B cells, Tim-1+ B cells, CD19+CD5+ B1a cells, CD9+ B cells, CD138+ plasma B cells, and CD138+CD44hi plasma blasts are identified as Breg subsets. For maintaining host immune tolerance and balance effector immune responses, these Breg cells secrete IL-10, IL-35, and TGF-ꞵ cytokines. In addition to cytokines, Breg cells also use cell membrane-bound molecules such as CD39, CD73, programmed death-ligand 1(PD-L1), or aryl hydrocarbon receptors for their functions. Stimulation of B cells with H. felis; signals via TLR2 and MyD88 and results with IL-10 producing Bregs. Bregs can induce differentiation of naive CD4+ T cells to IL-10-producing regulatory Tr1 cells by direct B and T cell interactions for suppressing Helicobacter-associated pathologies. Metabolism is the collection of all anabolic and catabolic reactions which are the generation and breakdown of cellular substances respectively. Oxidative phosphorylation (OXPHOS) is one of the major metabolic pathways inside the cell. It occurs in the mitochondria and consists of the tricarboxylic acid cycle (TCA) and electron transport chain reactions for generating ATP. Shortly, in OXPHOS electrons formed from the tricarboxylic acid cycle (TCA); are combined with molecular oxygen (final acceptor of electron transport chain) and this results in many oxidation/reduction reactions where energy is released for the production of ATP from ADP. Mitochondria are double membrane organelles that have both outer and inner mitochondrial membranes (OMM and IMM) and are found in eukaryotic cells. Membrane transporters and electron transport chain (ETC) complexes of mitochondria localize on the inner mitochondrial membrane. IMM encloses a viscous structure called a mitochondrial matrix. Enzymes, mitochondrial DNA (mtDNA), ribosomes, and nucleotides are placed in the mitochondrial matrix. Mitochondrial DNA encodes 37 mitochondrial genes including 22 transfer RNAs, 2 ribosomal RNAs, and 13 important oxidative phosphorylation polypeptides: ND1, ND2, ND3, ND4L, ND4, ND5, ND6 (parts of Complex I); Cytochrome b (parts of Complex III), COI, COII, COIII (parts of Complex IV) and ATP6, ATP8 (parts of Complex V). To provide expression of a mitochondrial gene, mitochondrial DNA needs to be transcribed by mitochondrial transcription factor A (Tfam), mitochondrial RNA polymerase (POLRMT), and mitochondrial transcription factor B1 and B2 (Tfb1m and Tfb2m). Two of the most critical features of multicellular life are metabolism and immunity. These can be explained as the need to distribute nutrients across cells, tissues & organs and protect from injury and inflammation. In recent years, studies have focused on elucidating the metabolism of immune cells in the context of their survival, activation, differentiation, and functions. For the activation of immune cells, signals which are triggered by metabolic intermediates and ATP molecules are required. In order to maintain proper immune cell activation, differentiation, and function, mitochondrial metabolism which generates energy plays a critical role. Studies have demonstrated that B cell activation with B cell receptor (BCR) or different Toll-like receptor (TLR) ligands changes mitochondrial dynamics. LPS-stimulated B cells enhance mitochondrial mass, and co-stimulation of B cells with BCR ligand IgM and TLR9 ligand CpG increases mitochondrial biogenesis. In addition to that, anti-CD-40 and IL-4 stimulated B cells to undergo OXPHOS. However, there is no information in the literature about the mitochondrial metabolism of Helicobacter-activated B cells. The main aim of this study is to elucidate the mitochondrial metabolism of Helicobacter-infected B cells. For this purpose, B cells were magnetically isolated from spleens of C57BL6 mice and treated with H. felis antigen, PAM3CSK4, and LPS for 6h, 24h, and 48h. Afterwards, cells were collected at respective time points for mitochondrial mass and membrane potential staining by using Mitoview Green and Mitoview 633 or TMRE dyes respectively in the flow cytometry. The supernatant of these cells is used for the IL-10 ELISA experiments for checking their IL-10 secretion. H. felis, PAM3CSK4, and LPS-stimulated B cells increased their IL-10 production most noticeably at 24h and 48h indicating the suppressive capacity of that cells. Also, compared to the unstimulated control group, all of the stimulant groups increased both the mass and membrane potential of mitochondria at 24h and 48h time points. The second aim of our study was to investigate whether B cells with high mitochondrial membrane potential (Mitoview 633+ B cells) produce IL-10 or not. For that, after B cells were isolated from IL-10 GFP reporter (VertX IL10 egfp) mice, they were treated with H. felis antigen, PAM3CSK4, and LPS for 6h, 24h, and 48h. Mitochondrial membrane potential were analyzed by flow cytometry. Afterwards, at 6h, 24h and 48h we evaluated mitochondrial membrane potential of the IL-10 producing B cells with quadrant analysis using flow cytometry. At 6h time there were no significant changes on IL-10+ Mitoview 633+ B cells. But at 24h and 48h time points; H.felis, PAM3CKS4, and LPS-stimulated B cells increased IL-10+ Mitoview 633+ B cells. These data show that in all stimulated B cell groups; high portion of the IL-10 producing B cells also have high mitochondrial potential. Our data shows, H. felis, PAM3CSK4, and LPS-stimulated Mitoview 633 + B cells increased their IL-10 GFP signal both at 24h and 48h time points compared to the unstimulated control group. The third aim of our study was to investigate mitochondrial biogenesis markers: mtDNA:nDNA ratio and mitochondrial transcription factor A (Tfam) gene expression. To perform Q-PCR experiments isolated and treated B cells were collected for DNA and RNA isolation at 6h, 24h, and 48h. For mtDNA:nDNA ratio analysis in Q-PCR; respectively cytochrome c oxidase subunit I (COX1) and 18S ribosomal subunit gene (RPS18) were targeted by using gene-specific primers. While H. felis, PAM3CSK4, and LPS-stimulated B cells decreased their mtDNA:nDNA ratio, they increased Tfam expression level compared to the unstimulated control group. This study showed that H. felis-activated B cells have active/functional mitochondria, and increased oxidative phosphorylation for energy production and their IL-10 production can be related to their mitochondrial metabolism.
Comparative whole genome sequencing and bioinformatic analysis of afreeze-thaw stress-resistant, industrial Saccharomyces cerevisiae strain

(Graduate School, 2022) Şimşek, Burcu Tuğba ; Çakar, Zeynep Petek ; 737560 ; Molecular Biology - Genetics and Biotechnology Programme

Yeasts have been around for thousands of years; they have benefited people in many fields such as science, medicine, food and agriculture. In particular, Saccharomyces cerevisiae is used in multi-enzyme pathways for the expression of protein biocatalysts and to synthesize chemicals and small molecular weight compounds important for medicine and nutrition. Due to these advances, S. cerevisiae is currently the primary model organism for the study of eukaryotic biology and human diseases. S. cerevisiae is a unicellular eukaryote. It has 16 chromosomes with subcellular organelles containing and these organelles commonly found in eukaryotes. S. cerevisiae has a classical eukaryotic cell cycle (including G1, S, G2, and M). Different strains of S. cerevisiae have been established to fill the gaps and requirements in genetic, biochemistry and physiology research. The CEN.PK family is frequently used in industrial biotechnology research, while the BY strain family derived from the S288c strain is mainly used in genetic studies. Yeast contains a large number of orthologous genes in the human genome. By examining the expression of some genes in yeast, the mechanism in more complex eukaryotes can be understood. S. cerevisiae has highly developed homologous recombination and contributes to the basic knockout operation of genes. Furthermore, S. cerevisiae is an important model for understanding the role of stress response genes in living organisms. S. cerevisiae cells can experience different environmental stress conditions such as metal toxicity, heat or cold shock during growth, essential nutrient limitations, hyperosmotic or hypoosmotic pressure, and ethanol toxicity. To overcome these stress conditions, S. cerevisiae cells have been developed to detect stress signals and respond to these signals through general or specific stress response and protection programs. Cryopreservation is a long-term storage method of various living cells, and the freeze-thaw tensile strength is important in cryopreservation. However, this method includes freezing and thawing processes that cause fatal damage to cells. Under freeze-thaw stress conditions, cells are exposed to more than one type of stress. These are; cold during freezing, dehydration, osmotic, ice crystal formation and oxidative stress during thawing. Therefore, it is important to obtain freeze-thaw tolerant organisms and to examine all freeze-thaw tolerance mechanisms. Yeasts are organisms that have a high survival rate when rapidly frozen at -80 °C. However, it is usually applied to commercial products at -20 °C and is highly damaging to cells, predominantly lethal to cells. Applications of freeze-thaw stress in S. cerevisiae are concerned with inducing this cross-resistance to overcome the effects of freeze-thaw stress. Additional mechanisms at gene expression levels are thought to be triggered and maintained during freeze-thaw exposure to achieve multiple stress tolerances and freeze-thaw stress tolerances. Metabolic engineering; it is defined as enhanced production of metabolites and cellular activities. It is done with through manipulation of the enzymatic, transport and regulatory functions of the cell by modifications of cellular networks including metabolic, gene regulatory and signaling networks using recombinant DNA technology. Metabolic engineering strategies can be divided into two groups as rational engineering and inverse metabolic engineering. Evolutionary engineering is a common strategy used in biological research to achieve the desired phenotype by improving its properties such as high environmental tolerance and improvement of product yield. Evolutionary engineering differs from metabolic engineering in that it is based on random methods; genetic modifications are not directed. Ploidy is the number of complete sets of chromosomes in a cell, which means the number of possible alleles for autosomal and pseudoautosomal genes. Many eukaryotic creatures have two sets of chromosomes (diploid) or more than two sets of chromosomes (polyploid). During the evolution of plants, animals, and fungi, ancient whole-genome duplication (WGD) or hybridization events frequently result in diploid and polyploid conditions. Increased chromosomal sets, development, cellular stress, disease, and evolution all cause polyploidy. Yeasts, which belong to the kingdom of fungi, can exist in both haploid and diploid forms. Polyploid yeasts, on the other hand, are widespread. Allopolyploid cells are formed when two or more cells from closely related but not identical species fuse together. Euploidy refers to the stance in which cells have a chromosomal number that is an integral multiple of the characteristic circum haploid number. Due to the common occurrence of polyploidy and aneuploidy in yeast, variable chromosome numbers elicit characteristics that may be beneficial in specific circumstances. As a result, the physiology and fitness of cells with different ploidy levels may differ. Bioinformatics is a highly interdisciplinary field that drives knowledge discovery from biological data using computational analysis. Today, bioinformatics is becoming an important part of most life science research. The process by which the DNA sequence of gene expression is copied into a gene product or RNA is explained by the central dogma of molecular biology. Microarray and more recently RNA sequencing; it has been widely used to measure gene expression levels. In this thesis, ploidy and genomic differences between the industrial Saccharomyces cerevisiae strain R625 and the freeze-thaw resistant evolved strain P8 obtained from R625 by evolutionary engineering were analyzed to gain insight into the complex molecular mechanisms of ploidy and freeze-thaw stress resistance.
Expression, purification and characterization of high-fidelity DNA polymerase

(Graduate School, 2022) Türk, Kübra ; Doğanay Dinler, Gizem ; 783309 ; Molecular Biology-Genetics and Biotechnology Programme

DNA polymerases found in all living cells discovered to date are the enzymes that synthesizes a new DNA strand complementary to template single stranded DNA. These enzymes do not only play a major role in the transmission of genetic information across generations during cell division, they also form the basis of Polymerase Chain Reaction (PCR), which is one of the most important in-vitro diagnostic techniques today. In addition to synthesis ability, DNA Polymerases may also have other properties including processivity, which is known as the ability of continuous polymerization, fidelity, which is known as the synthesis accuracy, and nucleotide selectivity. Thermostable DNA polymerase enzymes are mostly preferred in PCR-based studies because it is high importance that the stability of the enzymes used do not decrease depending on temperature. Taq DNA polymerase is the first discovered polymerase, which is a well-known enzyme used in a wide range of applications. Following the discovery of Taq DNA polymerase, the high-fidelity DNA polymerase was discovered in 1991 as a highly thermophilic DNA polymerase. Due to its high thermostability and proofreading properties, high-fidelity DNA polymerase is widely used in the applications that require high accuracy such as molecular cloning. High-fidelity DNA polymerase is an enzyme with a length of 775 amino acids and a molecular weight of about 90 kDa. This enzyme can perform 3'-5' exonuclease (proofreading) activity, which allows the addition of the correct nucleotides by removing the wrong nucleotides added to the structure during DNA synthesis. Due to this feature, it reduces the error rate during synthesis (1.3×10-6 mutations/base pairs/duplications), resulting in about 8 times less errors compared to Taq DNA polymerase. Many researchers have produced this protein by cloning it from Pyrococcus furiosus, a hyperthemophilic archaea, into different strains of Escherichia coli. The purification step is simplified by adding an affinity tag to the N- or C-terminus during the cloning. Based on these tags and various biophysical properties of the protein, purification protocols were created by affinity chromatography or ion exchange chromatography. In this study, we aimed to purify and characterize the high-fidelity DNA polymerase enzyme by taking the advantage of its thermal stability and 10X Polyhistidine-tag after bacterial production with high efficiency and low cost. For this purpose, commercially purchased pET16B. High-fidelity polymerase's plasmid DNA with a 10X Polyhistidine-tag at the N-terminus was used. The plasmid pET16B. High-fidelity polymerase was transformed into competent E. coli BL21(DE3) cells containing GroEL/GroES chaperonins to ensure soluble expression of the protein. In the first step of purification of High-fidelity DNA polymerase, which is a thermostable protein, all the folded proteins obtained from bacterial cells were heated and centrifuged to separate impurities with less thermal stability. High-fidelity DNA polymerase in soluble form was purified using IMAC affinity chromatography. The pure product was taken into a storage buffer containing 50% glycerol by filtration. The GroEL/GroES chaperonin system is a system that enables unfolded proteins with a molecular weight of 2-100 kDa to be folded in vitro and in vivo. Given that GroEL/GroES system can increase the folding of co-expressed recombinant proteins of different sizes by up to 70%, this system was employed in the production of High-fidelity DNA polymerase. However, while this system increases the amount of target protein, it can also increase the amount of impurities. Therefore, the purification of High-fidelity DNA polymerase was highly challenging. So that, various buffer compositions were used in order to optimize one step IMAC purification. Co-expression system was induced using IPTG for expression of pET16B. Expression of the polymerase regulated by the Lac operon. Since the growth temperature was chosen in the range of 12-20°C, where the metabolic rate of the cell and thus the growth rate was selected, the amount of protein folded by the chaperones was increased. Most of the impurities that increased with the target protein were eliminated with the 90°C heat treatment step. While heat sensitive proteins are eliminated from the environment, thermostable high-fidelity DNA polymerase enzyme, GroEL, and GroES proteins are still present. The separation of GroEL and GroES was achieved by applying IMAC affinity chromatography to increase the purity of the high-fidelity DNA polymerase enzyme. Purification results were analyzed by SDS-PAGE and immunoblotting methods. At the end of 500 ml bacterial production and purification process with three biological repetitions, high-fidelity DNA polymerase enzyme of similar quality with its commercial counterparts was produced, which can be used for a total of 60 000 PCR reactions with ~90% purity. The protein band on the SDS-PAGE gel was excised and analysed by peptide mapping using Liquid Chromatography-Mass Spectrometry (LC-MS) system to confirm that the produced protein is the target protein. According to the analysis, it was concluded that the purified protein was the target DNA polymerase. In order to determine if the purified protein is correctly folded or not, the secondary structure analysis of the protein with a purity over 90% was performed using Circular Dichroism (CD) in the far-UV (<260 nm) range. As a result of the study, the protein showed an apparent α-helix secondary structure with two minima at 208 and 222 nm wavelengths and a maximum at 190 nm wavelengths. Functional analysis of the protein on its folded state was completed by performing the Polymerase Chain Reaction (PCR). The 825 bp DNA region with 49.6% G-C content, and 1947 bp DNA region with 61% G-C content was amplified. These regions were selected considering the processivity of the enzyme. No-template control was used as negative control. The amplified regions were analyzed comparatively with commercial enzymes and it was observed that the target regions were successfully amplified. Commercially available high-fidelity DNA polymerase enzymes do not have endonuclease contamination and exonuclease contamination. Within the scope of quality control experiments, both endonuclease and exonuclease contamination of three biological replicates of our high-fidelity DNA polymerases were compared with commertial ones. λ DNA and λ DNA digested with HindIII were used as positive control. As a result, it has been shown that commercial enzymes and our high-fidelity DNA polymerase enzyme do not have endonuclease and exonuclease contamination. Another important test for demonstrating the quality of commercial enzymes is testing whether the protein remains stable under different conditions. For testing the stability of the polymerase, the effect of freeze-thaw stress repeated 20 times and also the effect of incubation of the enzyme for 5 days at room temperature were evaluated. As a result of these experiments, it has been shown that the freeze-thaw process during the general use of purified enzyme does not cause a negative effect on the activity of the protein, and that if the purified enzyme is forgotten at room temperature for up to 5 days during use, they can polymerize without reducing their activity. In conclusion, with this study, we have produced high-fidelity DNA polymerase with the same stability and processivity as commercial polymerases produced by large biotechnology companies with high efficiency and purity.
Investigation of protonation state dependent conformational dynamics of the nucleotide binding domain of Hsp70 protein homolog DnaK via computational methods

(Graduate School, 2022) Uçar, Umut Çağan ; Balta, Bülent ; 771654 ; Moleküler Biyoloji-Genetik ve Biyoteknoloji Bilim Dalı

70 kDa heat shock proteins (Hsp70) are a ubiquitious and well-conserved protein family with chaperone functions. They supervise protein folding process and assist in proper protein folding and renaturation by preventing partially folded or misfolded proteins from forming amorphous aggregates and amyloid fibrils. In terms of structure, Hsp70s are composed of approximately 45 kDa N-terminal nucleotide binding domain (NBD) with ATPase activity, 25 kDa C-terminal Substrate Binding Domain (SBD) with substrate peptide binding pocket. Also, there is a conserved hydrophobic linker segment connecting the 2 domains. Hsp70 proteins do not work alone. Rather, they need aid of some other proteins called "cochaperones" to work optimally. The two auxillary cochaperones required by the Hsp70s are the 40 kDa Hsp40s(J domain proteins) and nucleotide exchange factors (NEFs). Together with the cochaperones, Hsp70 proteins form 3-membered Hsp70 chaperone machinery. For Hsp70 chaperone system to carry out all of its functions, the NBD and SBD domains must communicate with each other during the functional cycle. This mutual signal transfer and crosstalk between the domains is an intricate example of "allosteric regulation". Basically, nucleotide status of the NBD exerts an effect on substrate affinity. Reciprocally, binding of a substrate protein to the SBD stimulates ATPase activity of the NBD. In ATP bound state of the NBD, the two domains are in docked conformation. On the other hand, in ADP bound or nucleotide free state of the NBD, these two domains are undocked and become independent of each other. In this undocked conformation, the two domains are connected to each other with the hydrophobic linker. The ATP-bound state of the NBD with docked NBD-SBD conformation binds and releases substrates at much higher rates than nucleotide free or ADP-bound state; thus, ATP-bound state of the Hsp70 is known as "low-affinity state" state with substrate binding pocket open. In contrast, when the NBD lacks any nucleotide or is bound by ADP, rate of substrate binding and release occur at way slower rates. This state of the Hsp70 proteins is called as "high-affinity state" with closed substrate binding pocket. Hence, Hsp70s shuttle between open and closed states. The hydrophobic linker is an undispensable component of the Hsp70 chaperone system. By functioning as a dynamic signal switch, the linker conveys messages in either direction, from NBD to SBD and from SBD to NBD, keeping both domains in touch. In literature, the linker, particularly the hydrophobic 388 VLLL 392 sequence, have been demostrated to be vital for both interdomain communication and dynamics of the ATPase domain. Likewise, there are many residues of the NBD playing key roles in the Hsp70 cycle, particularly in the mechanism of ATP hydrolysis. Especially, D8, K70, E171, D194, T199, and D201 residues of E.coli Hsp70 homolog DnaK and correspondants of these residues in other Hsp70 homologs have been given attention. In this study it was aimed to elucidate the conformational dynamics of the nucleotide binding ATPase domain (NBD) of E.coli DnaK in nucleotide free state by means of computational simulation techniques. First of all, so as to understand if protonation states of critical residues of interest have any effect on the opening-closure dynamics of the NBD in nucleotide free state, initially open and closed structures with different protonation states were prepared. By changing the protonation states of residues D194, D201, and H226 in distinct combinations, charge states +12, +13, and +14 were attained. Four protonation states, namely D194 protonated (194pr), D201 protonated (201pr), both D194 and D201 protonated (2pr), and both D194 and D201 unprotonated (2dep) were tested for both initially open and initially closed conformations. In each case, another residue of interest H226 was taken in protonated form. To broaden conformational space scanned and explore contribution of linker position to the dynamics of the NBD, positions of the linker were changed manually multiple times for each protonation state. According to our standard molecular dynamics (MD) simulation results (each 500 ns) of DnaK 1-392 construct of initially open conformations with these 4 protonation states, it was seen that protonation state can dramatically influence the tendency of initially open conformation towards closure. The cases in which D194 were unprotonated (2dep and 201pr) exhibited a tendency to close. Especially, 2dep became totally closed after 200 ns simulation period. On the other hand, 194pr had no tendency towards closure at all. On the other hand, all of the initially closed conformations for each of the 4 protonation states of the DnaK 1-392 retained their closed forms. Based on these simulation results, it can be deduced that the energy barrier between open and closed conformations was highest for 194pr and lowest for 2dep. In order to both decide on relative abundance of open - closed structures and circumvent sampling problems encountered during classical MD simulations, we carried out temperature replica exchange molecular dynamics (T-REMD) simulations for each protonation state. In addition to the 4 protonation states with protonated H226 residue investigated during MD simulations, 2 additional states, namely D194 protonated-H226 deprotonated (194prHID226) and D201 protonated-H226 deprotonated (201prHID226), were prepared by only removing the epsilon H atom of H226 from 194pr and 201pr cases, respectively. To elucidate whether the linker favors closed conformations, apart from these 6 different protonation cases with residues 1-392 of NBD, 6 further constructs were prepared by stripping the last 4 residues 389VLLL392 of the linker away from each of 1-392 construct. In total, 12 T-REMD simulations, 6 with 1-392 and 6 with 1-388 residues, and each with 300 ns-long were performed. Based on our T-REMD results, the most essential point to be understood is that closed conformations are much more favorable compared to open ones. Considering the ratios of open conformations, even the highest fractions of open conformations obtained in the case of 194pr388 did not exceed 25%. Looking at other cases, the percentage of open conformations can be as low as 2-3% for 201pr cases. If we look at 194pr cases, irrespective of the presence of last 4 residues of linker and protonation state of H226, open structures are most abundant in the cases of 194pr. Among 194pr and thereby all other protonation states-structures, 194pr388 (226 protonated) promotes, by far, the open structures most. Additionally, the role of the length of linker in favoring closed structures over open ones becomes more prominent when H226 is protonated with nearly 7% higher closed frames in 194pr392 than 194pr388. As opposed to 194pr, regardless of linker or protonation state of H226, 201pr cases are out and away the protonation states that exhibit lowest tendency towards opening with fraction of open structures no more than 5%. Unlike 194pr, there is no evident impact of linker on opening-closure behavior. Protonation state of H226 seemed not that important in either protonation state. In agreement with the standard MD simulations, 2dep was monitored to tend to have high number of closed frames. Indeed, 2dep comes after 201pr in terms of closed structure fractions. The contribution of the linker to the closure of the NBD was minor and seen only the last 200ns. As to 2pr states, the 2nd highest fraction of open frames, both in the presence and absence of 4 terminal residues of the linker, after 194pr were obtained. Another point of interest in this study was to scrutinize the pH dependence of DnaK ATPase domain. The active site of the ATPase domain comprises multiple charged amino acid residues; therefore, it can be expected from Hsp70 proteins to be susceptible to changes in pH conditions. To the best of our knowledge, only two experimental studies in early 2000's underlining pH dependent behavior of isolated DnaK NBD have been conducted thus far. On the other hand, no computational study has paid attention to change in the dynamics and activity of either full-length protein or NBD alone with respect to pH. Hence, which of the candidate residues in the active site are in protonated or deprotonated form around physiological pH remained elusive. To solve this mystery, thermodynamic integration (TI) simulations were performed, again for both DnaK 1-392 and DnaK 1-388. For TI simulations, for each of 12 protonation states used in the T-REMD, 2 initially closed structures alongside one open structure were picked from cluster analysis performed on the T-REMD trajectories. Additionally, pKa values of E171 and epsilon position of H226 were calculated in various protonation/deprotonation scenarios of D194 and D201. Regarding pKa values of epsilon H226, we inferred that pKa values were more or less the same around 9, indicating the fact that neither protonation states of active site aspartate and glutamate residues nor the lenght of the linker altered pKa values of H226. Protonation state of E171 is likely to depend on the protonation states of D194 and D201. When these aspartates were both deprotonated, E171 was protonated. D194, D201, and H226, on the other hand, must be in protonated states around physiological pH conditions. Consequently, according to our pKa values, we got +12 charge state, not +13 or +14. All in all, combining all atom MD simulations with REMD simulations and free energy TI simulations, the following questions were tried to be clarified in this thesis study: 1) Effect of protonation state on opening-closure abundance and dynamics of NBD 2)Whether the linker induces a change in the ratio of open to closed conformations. 3) pH dependence of the NBD through demonstration of the possible protonation/deprotonation status of critical residues 4) Whether linker promotes change in pKa of any of critical active site residues. At the end, we came to these conclusions. Dynamics of the DnaK NBD opening-closure were protonation state dependent. Intriguingly, protonation of 2 active site aspartate residues have opposite effect in such a way that protonation of D194 increases the abundance of open conformations, whereas D201 promotes more closed form of the NBD. In addition, at different charge states, there must be multiple protonation states in equilibrium. Regarding the linker, there was no direct indication of the linker being involved in opening/closure or pH dependence of the isolated DnaK NBD. Likewise, no apparent role of the linker in shifting the pKa of any critical residue in the active site.
Expression, purification, and characterization of recombinant human IL-2

(Graduate School, 2022-01-18) Akgün, Buse ; Doğanay Dinler, Gizem ; 521181103 ; Molecular Biology – Genetics and Biotechnology

Cytokines, which are small proteins secreted by the immune system, are in charge of directing the immune system. Through their formation, differentiation, and activation functions, cytokines govern the maintenance of innate and adaptive immune responses. They are primarily formed by mononuclear phagocytes, dendritic cells, and antigen-presenting cells. Interleukin (IL) is a kind of cytokine that acts as an immunomodulatory protein. It induces a variety of cell and tissue responses. Interleukins mediate the interaction of leukocytes (white blood cells) and initiate a response by attaching to high-affinity receptors on the surface of the cells. They play a critical role in the regulation of cellular formation, differentiation, and activation that occurs over the course of inflammatory and immunological responses. Each family is assigned an IL based on sequence homology, receptor chain similarity, and functional qualities. Interleukin-2 (IL-2) was the first cytokine discovered to stimulate the growth of T lymphocytes. T cells, B cells, natural killer (NK) cells, lymphokine-activated killer cells, and macrophages all require IL-2 to regulate their proliferation and differentiation. Mier et al. discovered the molecule and named it "IL-2" since it was produced by and acted on leukocytes. Its discovery is regarded as a milestone in immunology. However, there is one issue that is common to all lymphokines when it comes to the molecular and functional characterization of IL-2, and it is due to their production in small quantities. The cloning of cDNA for IL-2 was a significant turning point in 1983, precipitated by the discovery of IL-2. The Jurkat T cell leukemia cell line was employed for the IL-2 cDNA clone development. IL-2 is a 15.5 kDa glycoprotein that belongs to the cytokine family four α-helical bundles. There are 153 amino acid residues in a single polypeptide chain of IL-2. IL-2 binds to and communicates with a receptor complex composed of three different subunits known as IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2R (CD132). Different combinations of these three components bind to IL-2 with varying degrees of affinity. The αβγ heterotrimer, βγ dimer, and α chain monomer all bind to IL-2 with "high," "intermediate," and "low" affinity, respectively. Binding of IL-2 to the IL-2R heterodimer complex activates several pathways. In response to an interaction between interleukin-2 and its receptor, kinases connect to cytoplasmic areas of the receptor subunits, resulting in the tyrosine phosphorylation of many proteins and the activation of a number of signaling pathways, including JAK/STAT, PI-3K/AKT, and Ras/MAPK. IL-2 activity promotes cell survival, proliferation, cell cycle progression, and targeted gene transcription. Due to its ability to activate both T and NK cells, IL-2 was the first cytokine to be successfully used in cancer treatment. The US Food and Drug Administration authorized high-dose IL2 for the treatment of melanoma and renal cell carcinoma in xxii 1992 and 1998, respectively. Moreover, the use of recombinant IL-2 therapy may help researchers understand better the coronavirus disease 2019 (COVID-19), which is caused by a virus that leads to severe acute respiratory illnesses and has rapidly spread throughout the world. As a prospective treatment for this condition, the use of rIL2 may be beneficial for patients since it has the potential to accelerate disease recovery by increasing the number of lymphocytes in the body. A major difficulty is figuring out how to direct IL-2 activity toward Teffs and away from Tregs, which inhibit the immune system. IL-2 is available in two recombinant forms derived from E. coli, but only aldesleukin is FDA-approved. Recombinant IL-2 differs structurally from its natural version. IL-2 recombinant is not glycosylated and lacks N-terminal alanine. To avoid the formation of an incorrect disulfide bond, serine has been substituted with cysteine at amino acid position 125. The pharmacological actions of endogenous and recombinant human IL-2 are similar. In this study, E. coli Rosetta (DE3) was used as the host cell. Induction of protein expression was accomplished by the use of IPTG. Following that, inclusion bodies, which develop in the cell as a result of excessive protein expression, were separated and solubilized from cell lysates and refolded by step-wise dialysis. Anion exchange chromatography was used to separate the target protein from the rest of the protein mixture. After purification, the yield was determined to be 0.114 mg per liter of cell culture. SDS-PAGE and immunoblotting methods were used to validate the effectiveness of the purification. The molecular weight is estimated using intact mass analysis through LC/MS. The CE-SDS analysis revealed that rIL-2 has a purity of around 80%. In addition, the pI value of the protein was determined as 7.31 using the capillary isoelectric focusing method. The peptide mapping on LC-MS/MS is used to figure out the main structure of the protein that has been purified. The secondary structure of pure human interleukin-2 (hIL-2) was investigated using circular dichroism (CD), and the results revealed that it included a high concentration of alpha helices. The biological action of our IL-2 is determined by phosphorylation of one of the MAPK pathway proteins, extracellular signal-regulated kinase 1/2 (ERK), on human monocytic cells, THP-1. An active protein has been produced as a result of this work. The experimental results indicate that the procedures established for generating and purifying the rIL-2 protein may be employed to create a pure product that maintains its bioactivity.
DADA2 hastalarının periferik kan mononükleer hücrelerinde total ada aktivitesinin analizi

(Graduate School, 2022-02-09) Demirci, Turna ; Turanlı Tahir, Eda ; 521171122 ; Molecular Biology, Genetics, and Biotechnology

Deficiency of Adenosine Deaminase Type 2 is an autosomal recessive disease caused by biallelic mutations in the ADA2 gene. It was first defined as monogenic vasculitis syndrome in 2014 as a result of studies conducted by two different groups independently. Although it has been shown that the prevalence of ADA2 Deficiency maybe 4 in 100,000, the prevalence of the disease may differ between ethnic groups, depending on the degree of consanguinity and the presence of founding variants. Adenosine deaminase is an enzyme involved in the regulation of adenosine homeostasis and purine metabolism by converting adenosine to inosine and 2'-deoxyadenosine to 2'-deoxyinosine. There are two isoforms of adenosine deaminase in humans, and one of them, the 57-kDa homodimer ADA2 protein, is produced by the Adenosine Deaminase 2 (ADA2) gene. The N-terminal portion of the ADA2 protein is responsible for growth factor activity, while the C-terminal portion is responsible for adenosine deaminase activity. In addition to the catalytic domain, the ADA2 protein also has a protein dimerization domain and a cell surface binding domain. ADA2 proteins bind to different cell surfaces via glycosaminoglycan chains and to T cells via adenosine receptors. In this way, it shows both cytokine-like and autocrine-type growth factor properties. Although the ADA2 protein is involved in macrophage polarization, it also has an important regulatory function for neutrophil activation. In addition, it significantly reduces the formation of neutrophil extracellular traps, which are caused by extracellular adenosine and can lead to the activation of proinflammatory cytokines. Despite the clinical manifestations of DADA2 being very diverse, episodic clinical findings are usually observed in patients with fever and systemic inflammation. The most common type is vasculitis findings. In addition to dermatological and neurological symptoms, it is also rarely defined by renal involvement and gastrointestinal system findings. More than half of patients have attacks of non-infectious fever. Symptoms include recurrent oral and genital ulcers, musculoskeletal symptoms, recurrent abdominal pain, inflammatory bowel disease, and immunodeficiency. Hematologic findings include cytopenia, anemia, and rare bone marrow failure. The diagnosis of the disease is made based on the detection of pathogenic variants on the ADA2 gene or the measurement of ADA2 activity in serum/plasma. Treatment methods are selected depending on the symptoms and the severity of the disease. Currently, anti-TNF-α is the most common treatment modality, especially for patients with signs of vasculitis. Hematopoietic stem cell transplantation can be used in the treatment of hematological diseases. In addition, although it is not a suitable choice for long-term treatments, fresh frozen plasma infusions are also among the treatments applied. Enzyme-linked immunosorbent assay (ELISA) is a method used to detect and quantify protein in soluble substances, based on antigen-antibody interaction and measuring enzyme activity by colorimetric analysis. The purpose of this study was to compare the total adenosine deaminase (ADA) activity in peripheral blood mononuclear cells of patients diagnosed with DADA2 with the control group. 8 patients diagnosed with ADA2 deficiency and 5 healthy individuals were studied. Two of the patients are Syrian and have a G47R/G321E heterozygous mutation. 3 of the patients have G47R homozygous. Total ADA activity was measured in lysates prepared from subjects' peripheral blood mononuclear cells using a colorimetric ADA Activity Assay kit that is a commercial kit. ADA activity was calculated by following the protocol written in the kit, and then the statistical comparison of the results was analyzed by performing the t-test. The disease-causing variant p.G47R, which occurs in the dimerization domain, affects the stability of the homodimer required for enzyme activity of the ADA2 protein. Therefore, due to the decrease in ADA2 catalytic activity in patients with p.G47R mutation, it is expected that the total ADA activity will be lower than in the healthy group. As a result of statistical analysis, a significant difference was observed in ADA activity (p=0.0008). As expected, ADA activity was lower in the patient group compared to the healthy group. In addition, when patients with heterozygous mutations were compared with patients with homozygous mutations, lower ADA activity was observed in patients with heterozygous mutations. In this case, it can be said that the G321E mutation plays an important role in catalytic activity.
Preparation and characterization of PCL/lignin sponges for bone tissue engineering applications

(Graduate School, 2022-05-09) Elmalı, Gizem ; Kök, Fatma Neşe ; 521181108 ; Molecular Biology-Genetics and Biotechnology

Organs and tissues may be damaged by accident, congenital anomalies, diseases and similar reasons. Although living tissues have the capacity to heal these damages, large defects or severe tissue losses can only be repaired to a certain extent. Tissue engineering techniques have been developed to trigger, assist and accelerate the healing process, or directly replace the targeted tissue. In tissue engineering, the most suitable three-dimensional scaffold structure is produced to match the characteristics of the targeted tissue and provide the initial structural integrity and organizational backbone for cells to replace the damaged tissues and organs. Especially bone and its related diseases can cause a significant impact on a person's health and quality of life if they are not treated and healed properly. Millions of bone injuries have been occurring each year and they are not only affecting people's life but also cause to a serious burden on country's economies. Bone tissue engineering was presented as a solution to address these problems. In this thesis, a synthetic and natural polymer blend was used to create a scaffold that is suitable for bone tissue engineering. Polycaprolactone (PCL) was used as synthetic polymer, while alkaline lignin was used as natural polymer. Solvent casting, particulate leaching and freeze-drying methods were used to create porous sponge-like scaffolds. PCL and lignin samples were prepared at different lignin concentrations (0, 10, 15, and 20 mg/ml) while keeping the PCL concentration constant at 200 mg/ml, PCL200, PCL200/Lignin10, PCL200/Lignin15 and PCL200/Lignin20, respectively. A sample with PCL concentration of 100 mg/ml and lignin concentration of 20 mg/ml was also prepared to see the effect of PCL concentration (PCL100/Lignin20). For all scaffolds, except for the control sample, NaCl crystals were used as porogen. Lignin containing scaffolds were appeared more brownish than pure PCL scaffolds. Since the structural integrity of PCL100/Lignin20 sample was very poor, and PCL200/Lignin20 without the salt addition cannot be removed from the mold surface, they were not used in further experiments. Even though, mechanical properties of the scaffolds have not been studied in this thesis, based on preliminary visual and manual analysis rest of the PCL/Lignin type scaffolds were observed to be more flexible than PCL constructs. Water uptake analysis showed that there is no significant difference between the samples with different lignin concentrations. The water uptake behavior of the scaffold, however, was dramatically increased with the lignin addition. While the water uptake for PCL200 was 44%, it was 380-400% for the samples with lignin after 24 h. In Fourier-Transform Infrared Spectroscopy (FTIR) analysis, typical peaks for PCL are identified for all scaffold types. Even though special peaks for lignin could not be observed, a reduction in the intensity of a peak, which represent –OH groups, was determined in all PCL/Lignin scaffolds compare to the PCL200. This can be evidence of reactions and new bonds between PCL and lignin polymers. Since water uptake and FTIR analysis showed that there is no significant difference between different lignin concentrations, it was decided to continue with only one sample type, PCL200/Lignin10, for further experiments. The porosities of pure PCL200 and PCL200/Lignin10 determined as 73% and 76%, respectively using liquid displacement technique. Pore sizes were investigated with ImageJ software on SEM images. For PCL200 scaffold, 31% and 58% of the pores on the surface were in the range of 100-300 µm and 300-600 µm, respectively. For PCL200/Lignin10, 30% and 43% of the pores were in the range of 100-300 µm and 300-600 µm, respectively. In addition, many pores that are smaller than 20 µm were observed for both sponges. The melting temperature of PCL200/Lignin10 scaffold was %5.4 higher than PCL200 scaffold as determined by Differential Scanning Calorimetry. The increased melting point might be the result of a new bond between PCL and lignin. Hydrolytic degradation was not seen within 7 days for both samples, but enzymatic degradation in lipase presence was observed for both sponges. After 7 days, only 43% weight loss was recorded for PCL200/Lignin10, while PCL200 sponge was completely degraded after the fifth day. Lignin's natural resistance to enzymatic degradation or the modifications to form an alkaline lignin might be the reason of this unexpected decrease in degradation. Biomineralization process was triggered by soaking scaffolds to modified-simulated body fluid solution. The mineralization rate was analyzed by SEM. SEM micrographs revealed significant mineral accumulations for both scaffold types at the end of the 7th day. When the 7th day samples were compared, it was seen that the hydroxyapatite-like structures were mostly deposited on top of each other in the PCL200 sample, while the aggregations were more evenly spread over the surface on the PCL200/Lignin10 sample. Furthermore, even though an intense accumulation was observed on the scaffold surface of the PCL200, the inside of the pores as appeared to be smooth. Lignin containing samples, on the other hand, had more mineral deposition within the pores. The elemental analysis of the formed hydroxyapatite-like structures was analyzed by EDX. It was observed that the Ca/P ratio of the accumulations on the PCL200/Lignin10 samples at all determined times was closer to the ideal hydroxyapatite Ca/P ratio (1.67). Finally, cell proliferation assay was performed with hFOB cells to examine the effects of lignin addition on cell adherence and proliferation on the surface. The proliferation of hFOB was significantly higher on PCL200/Lignin10 scaffolds compared to PCL200. The proliferation on PCL200/Lignin10 sponge was found to be 212% and 50% higher after day 3 and 7, respectively. Further studies are necessary in order to evaluate the usability of PCL200/Lignin10 sponge scaffolds in bone tissue engineering. However, the findings obtained within this thesis suggest that this blend is promising candidate for bone tissue engineering.
The study of colorimetric pH probe and optical detection of heme attachment to cyt C by using genetically encoded indicators in living cells

(Graduate School, 2022-06-17) Genceroğlu, Mehmet Yunus ; Bayraktar, Halil ; 521191118 ; Molecular Biology-Genetics and Biotechnology

The cytochrome c (Cyt c) is a metalloprotein that has a heme as a cofactor. The heme has a reduced iron atom (Fe2+) on the center of its structure. The Fe2+ atom is important for formation of thioether bonds. The thioether bonds are formed between the thiol groups of Cys18 and Cys15 residues of apocytochrome c (apocyt c) that is unfolded forms of Cyt c and the vinyl groups of heme by catalyzing of holocytochrome c synthase (HCCS) or cytochrome c heme lyase (CCHL). Met81 coordination change also affects the folding of Cyt c. The Cyt c has two main function in human cells. One of them is to transfer the electron from Cyt bc1 to Cyt c oxidase in electron transfer system (ETS) which is an important step to reduce oxygen to water in respiration. Another function is to bind apoptotic protease activating factor-1 (Apaf-1) complex to activate caspases in intrinsic pathway of apoptosis.The Cyt c is encoded in CYCS gene which is located on 7p15.3 region of chromosome 7. The substitution mutation of G41S on CYCS gene in megakaryocytes results in an autosomal dominant genetic disorder called thrombocytopenia. In this case, the platelet cannot form their mature forms due to enter apoptosis in their premature forms and does not have a function. The CCHL is encoded on HCCS gene which is located on Xpter region of X chromosome and is an evolutionary conserved gene. The rearrangement of Xpter to Xp22 results in a neurodevelopment genetic disorder called as microphthalmia with linear skin defects syndrome (MLS). The MLS is lethal for males since they have only one X chromosome, whereas the females have two X chromosome and they show the disease in phenotype due to random X inactivation. The Cyt c is detected and studied by various biological methods. The Western blot, enzyme linked immunosorbent assay (ELISA) and flow cytometry are extensively used to understand the role of Cyt c in cells. These methods use specific antibodies to detect Cyt c. Circular dichroism (CD), the hydrogen/deuterium (H/D) exchange, the Fourier transform infrared (FTIR), gel filtration and the isothermal titration calorimetry (IATC) are spectroscopic techniques that were used to study the structural changes of Cyt c. Although all of these techniques are sensitive to detect Cyt c in vitro, they are not useful to study conformational changes Cyt c in vivo especially in living cells at spatial and temporal resolution.The genetically encoded fluorescence probes enable to visualize cellular functions in living cells and organisms in real time. The green fluorescence protein (GFP) is the first isolated protein from Aequorea victoria. The fluorescence protein gene is cloned into a vector and the desired protein gene is also cloned upstream or downstream of fluorescence protein gene. Then, the vector is transformed into the cell such as a bacteria or cell line and expressed in the cell recombinantly. Then, the fluorescence probe characterized by spectroscopic techniques and used to follow cellular functions in vivo in real time by light microscope. The fluorescence resonance energy transfer (FRET) system is a kind of genetically encoded fluorescence probe that uses two different fluorescence proteins. One of them is the acceptor that is placed to upstream of the desired protein. Another is the donor that is placed to downstream of the desired protein. There are actually different types of FRET such as ligand-dependent FRET probes and catalytic probes. For example, there is a sensor domain between acceptor (CFP) and donor (YFP) proteins. If a ligand binds to sensor domain, there is a conformational change occurs and results in the approaching of acceptor and donor with each other. In this case, the acceptor absorbs the signal from laser microscope and the donor quenches the signal from acceptor, emits a FRET signal to detector. In this study, we firstly characterized a FRET construct called Cyt c-Venus as a colorimetric and ratiometric pH probe and compared to the other control constructs which are Cyt c and Apocyt c-Venus. These constructs were designed by recombinant cloning methods in our previous study, but their pH sensitivity were not characterized in detail. We expressed these constructs in BL21 DE3 E.coli bacterial cells and treated with pH 4.5, 5.5, 6.5, 7.5 and 8 PBS buffers. We observed that there are changes in the colors of Cyt c-Venus and Apocyt c-Venus, but there is no change in the color of Cyt c under visible light, as the pH declines from 8 to 4.5. We observed fluorescences of Cyt c-Venus and Cyt c in pH 8 and 7.5, but the fluorescence of Cyt c-Venus and Apocyt c-Venus were diminished in pH 6.5, 5.5 and 4.5, whereas there is no fluorescence in Cyt c in all pH values since it does not have fluorescence protein under UV light. The absorbances of these proteins were measured by UV-Vis spectrophotometer. We observed that there were two signal peaks in Cyt c-Venus in pH 8 and 7.5. One of them is at 408 nm which belongs to Cyt c, another is at 515 nm which belongs to Venus. The signal peak of Venus in Cyt c-Venus started to decrease, as the pH reduced and it was disappeared in pH 4.5. On the other hand, we did not observe any change in the signal peak of Cyt c in all pH values. To get smooth peaks in absorbance graphs and prevent background signals due to cellular wastes, we lysed the cells and purified the proteins by ion-exchange chromatography from other cellular waste components. Then, we run the proteins in SDS-PAGE. We successfully purified Cyt c and Cyt c-Venus. We titrated the pure Cyt c-Venus protein by dropping mild HCI. We had observed that there was a color change in Cyt c-Venus compared to Cyt c and non-titrated Cyt c-Venus. Indeed, we had expected that the color of Cyt c-Venus turned from orange to red at acidic pH under visible light due to sensitivity of Venus to pH. Although it did not turn to red, orange color was reduced. We also observed a decline in its fluorescence compared to non-titrated Cyt c-Venus that had a brilliant fluorescence under UV light and compared to Cyt c that did not have any fluorescence. We measured the absorbance or excitation of Cyt c-Venus during titration and observed that there was a decline of signal peak of Venus at 515 nm and it finally disappeared at low pH values. There was no change at the signal peak of Cyt c at 408 nm. We also titrated pure Cyt c by mild HCI to use as a control and observed no change in the color, fluorescence and absorption signal of Cyt c.Secondly, we also investigated the heme binding properties and conformational changes of Cyt c by design a FRET probe. We placed a CFP on upstream of Cyt c as acceptor and a YFP on downstream of Cyt c as donor. Then, we expressed the construct in neuronal RPE cells.We calculated the netFRET values to exclude background signals and we normalized the netFRET values to normFRET values which are between 0 and 1. We also used C17V as positive control which has a 17 amino acid linker between Cerulean and Venus. We observed that there was a FRET signal in Cerulean-Cyt c-Venus construct in the presence of heme and heme lyase compared to C17V which has also FRET signal. We observed that there was an increase in FRET signal in Cerulean-Apocytc-Venus in the presence of only heme. On the other hand, there was no FRET signal in Cerulean-Apocytc-Venus construct in the absence of heme and heme lyase or in the presence of only heme lyase. As the concentration of heme increased, we observed that the FRET signal increased in Cerulean-Apocytc-Venus. The expression of heme lyase in the absence of heme did not change the FRET signal indicating that heme is important for binding of heme lyase and Cyt c. As a control, we separately expressed Cerulean and Venus and compared the FRET signal to C17V in the presence of heme. We observed that the heme did not bind to C17V.As a conclusion, we have characterized that the Cyt c-Venus is a sensitive protein construct to pH changes. It can be used to visualize pH changes in real time. We have used FRET method to determine the intermediate state of Cyt c in RPE cells. We have also found that the heme can bind to Cyt c non-covalently in the absence of heme lyase and causes conformational changes in Cyt c by using FRET method.
Inverse metabolic engineering of KCl-resistant Saccharomyces cerevisiae

(Graduate School, 2022-06-20) Morkoç, Ogün ; Çakar, Zeynep Petek ; 521181116 ; Molecular Biology - Genetics and Biotechnology

Bu çalışmada, KCl tuzu kaynaklı hiperosmotik strese dayanıklı S. cerevisiae suşları elde etmek için, tersine metabolik mühendislik yaklaşımı olan evrimsel mühendislik kullanılmıştır. Evrimsel mühendislik ile elde edilen suşların fizyolojik ve metabolik analizleri yapılmıştır. Evrimsel mühendislik için seçilim deneyi, kademeli olarak artan KCl stresi altında gerçekleştirilmiştir.
Biochemical characterisation of truncated amylopullulanase from Thermoanaerobacter brockii brockii

(Graduate School, 2022-06-24) Kayrav, Aycan ; Karagüler Gül, Nevin ; 521201105 ; Molecular Biology-Genetics and Biotechnology

The last point reached as a result of the chemical transformation carried out by nature is life. Enzymes are responsible biomolecules for the chemical transformation that makes life possible. People had benefited from the reactions carried out by enzymes to obtain various products in many fields throughout history, from the times when they did not know the existence of these catalysts. Over time, people had understood how great potential these biocatalysts have with the structure and working principles. Since then the characterisation of novel enzymes and the usage area of these enzymes have been expanded day by day. Today, more than 3.000 enzymes are used in biotechnological and industrial fields and it is reported that the size of this enzyme market has reached 6 billion USD in 2021. The enzymes that dominate this market with a share of 30 % are those used in the food and beverage industry and the ones that have the upper hand in this group are the enzymes used in starch processing applications. Starch, the main component of many agricultural products and the primary source of carbohydrates for most people, is the main source of carbon in the world. In addition to starch, oligosaccharides, disaccharides and monosaccharides obtained as a result of hydrolysis are also used in various fields such as food, pharmaceuticals and biofuels. To obtain these hydrolysis products, starch is subjected to a three-step process: gelatinisation, liquefaction and saccharification. The viscous solution which is obtained by hydrolysis of α-1,4- and α-1,6- glycosidic bonds in the starch structure and gelatinisation is achieved by using α-amylase, β-amylase, glucoamylase and pullulanase enzymes during the liquefaction. During all these processes to remain the enzymes stable for a long time and maintain their activity, pH adjustment and the addition of Ca2+ ionsare required . Remove both the formed salt as a result of pH adjustments and excess Ca2+ ions causes additional steps and all of these lead to an increase in the cost of production. To eliminate these steps and perform a one-step liquefaction-saccharification process can be possible with an alternative enzyme. Amylopullulanases (E.C. 3.2.1.1/41) are enzymes capable of hydrolysing both α-1,4- and α-1,6- glycosidic bonds, while those obtained from extremophilic organisms can maintain their activities under harsh industrial conditions. In this study, the amylopullulanase (TbbApu) enzyme belonging to the Thermoanaerobium brockii brockii organism, is one of the strong candidates for starch hydrolysis processes, is examined. Previously, for the recombinant production of TbbApu, the whole apu gene had been obtained by using the primary walking method by our group and the optimisation of expression studies was in progress. As a result of the studies, pET-28 a (+) vector and E. coli BL21 (DE3) were chosen as the appropriate expression vector and and E. coli BL21 (DE3) the appropriate host, respectively. Additionally, the variants TbbApuΔSH3 without SH3 domain and TbbApuΔCBM20 without CBM20 domain were constructed to investigate the effect of SH3 and CBM20 domains. In the scope of this thesis, apart from TbbApuΔSH3 and TbbApuΔCBM20 variants, four additional truncated constructs namely TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1 and TbbApuΔX25-2-CBM20 were also obtained to disclose the effects of X25 domain. The biochemical characterisation, raw starch binding ability and kinetic studies of TbbApu and its six variants have been accomplished. The function of the X25 domain on substrate binding, activity and stability of the enzyme has been revealed for the first time with this study. The genes belonging to each construct were amplified by PCR with specific forward and reverse primers that have SacI and NotI restriction sites at their 5' ends and using the whole apu gene as a template. As a result of the amplification, tbbApuΔX25-1-SH3, tbbApuΔX25-2-SH3, tbbApuΔX25-1-CBM20 and tbbApuΔX25-2-CBM20 genes with a length of 4065, 3750, 3375 and 3060 base pairs, respectively, were obtained. Then, the obtained genes and the pET-28 a (+) expression vector were cut with SacI and NotI enzymes to form sticky ends and the ligation reactions were set up for the insertion of the genes into the pET-28 a (+) expression vector. Then, transformation was performed using competent E. coli BL21 (DE3) host cells. For the determination of positive colonies from the colonies obtained as a result of the transformation, half of the colonies were inoculated on agar plates containing red pullulan. 1 µM IPTG and 40 µg/mL kanamycin were also included in the agar plate for induction and selection respectively. For the control of detected positive colonies from the PRR plate, the other half of the colonies were inoculated into Luria-Bertani (LB) medium and plasmid isolation was performed from these cells. Then, the isolated plasmids were cut with FastDigest SacI enzyme and linearised to determine the length of the plasmids. By linearisation, tbbApuΔX25-1-SH3 - pET-28 a (+) vector with a length of 9434 base pairs; 9119 base pairs long tbbApuΔX25-2-SH3 -pET-28 a (+) vector, 8744 base pairs tbbApuΔX25-1-CBM20- pET-28 a (+) vector and 8429 base pairs tbbApuΔX25-2-CBM20- pET-28 a (+) vector were obtained from all selected colonies. After the cloning, TbbApu, TbbApuΔSH3, TbbApuΔCBM20, TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20 and TbbApuΔX25-2-CBM20 were over-expressed using magic media. To purify these recombinant enzymes, purification was achieved in three steps by using metal affinity chromatography, ion exchange chromatography and heat purification respectively. Then, the pullulanase and α-amylase activities of TbbApu and its six variants were checked by the red pullulan and starch-containing polyacrylamide gel. The biochemical characterisation of the enzymes was completed. As a result of the studies, the optimum reaction temperature for pullulanase activities was determined as 70 °C for TbbApu, TbbApuΔSH3 and TbbApuΔCBM20, 75 °C for TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3 and TbbApuΔX25-1-CBM20 and also 80 °C for TbbApuΔX25-2-CBM20 domain. The optimum reaction temperature for α-amylase activities was specified as 75 °C for TbbApu, TbbApuΔSH3 and TbbApuΔCBM20, and 80 °C for TbbApuΔX25-1-SH3, TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20 and TbbApuΔX25-2-CBM20. The pure variants were incubated for 24 hours at variable temperatures to examine the effect of temperature on the stability of enzymes. It was observed that the most stable temperature of 60 °C for TbbApu, TbbApuΔSH3, TbbApuΔCBM20, TbbApuΔX25-1-SH3 and TbbApuΔX25-2-SH3, 50 °C for TbbApuΔX25-1-CBM20 and 40 °C for TbbApuΔX25-2-CBM20. Optimum pH values for both activities were found to be 6.5 for TbbApu, TbbApuΔX25-1-SH3 and TbbApuΔX25-1-CBM20, 6 for TbbApuΔSH3, TbbApuΔCBM20 and TbbApuΔX25-2-SH3, and 7 for TbbApuΔX25-2-CBM20. It has been determined that the enzymes reached 80 % of their α-amylase activities with pH 5 and protected it up to pH 8. When the effects of different pH values on the stability of the enzymes were examined, the most stable pH according to pullulanase activities was found as pH 4 for TbbApu, TbbApuΔSH3, TbbApuΔCBM20 and TbbApuΔX25-2-CBM20, pH 6 for TbbApuΔX25-2-SH3, pH 6.5 for TbbApuΔX25-1-SH3 and TbbApuΔX25-1-CBM20 after 24 hours of incubation. According to their α-amylase activities, the most stable pH was found as pH 3 for TbbApu, TbbApuΔSH3, TbbApuΔCBM20, pH 6 for TbbApuΔX25-2-SH3, TbbApuΔX25-1-CBM20 and TbbApuΔX25-2-CBM20, pH 7 for TbbApuΔX25-1-SH3. In addition, it is represented that TbbApu and its variants can maintain their stability between pH 3 and 8 and TbbApu can be used in starch hydrolysis processes without pH adjustment. When the effect of metal ions was examined, while Mn2+ and Co2+ ions increased both pullulanase and α-amylase activities of TbbApu and its variants whereas, Mg2+, Zn2+ and Al3+ ions decreased both activities of all enzymes except pullulanase activity of TbbApuΔCBM20 variant. Although α-amylase enzymes used in starch processing are Ca2+ ion-dependent, TbbApu and its variants are not dependent on Ca2+ ion for activity and stability, making the enzyme and its variants a strong candidate for starch hydrolsis process. Also, in the presence of 20 % and 50 % hexane and acetone, with some exceptions, both activities of TbbApu and its variants were increased and 20 % and 50 % butanol, DMF and DMSO presence strongly inhibited both activities of the enzymes. When the effects of organic solvents on the stability of enzymes were examined, it was observed that, with some exceptions, the stability of the enzymes increased in the presence of 20 % and 50 % acetone and hexane, compared to both pullulanase and α-amylase activities, after 24 hours of incubation. In the case of inhibitors and detergents, it was observed that inhibitors moderately inhibited pullulanase and α-amylase activities of TbbApu and its variants with some exceptions, whereas all inhibitors increased the pullulanase activity of TbbApuΔCBM20 and nonionic and anionic enzymes inhibited both activities moderately with. However, the activities of all enzymes were strongly inhibited in the presence of CTAB, which is a cationic detergent. Then, the kinetic parameters of the enzymes were elucidated. The results imply that, truncation SH3 domain improves both α-amylase and pullanase activities of the enzyme. However, truncation of CBM20 and X25 domains from TbbApu caused to loss of affinity and specificity of the enzyme to soluble starch and to shift in the specificity of the enzyme to pullulan. The removal of the SH3 domain and also CBM20 domain, the carbohydrate-binding module in the enzymes did not have any effect on the raw starch binding capacity of TbbApu. The removal of the SH3 domain and also CBM20 domain, the carbohydrate binding module in the enzymes did not have any effect on the raw starch binding capacity of TbbApu. However, sequential removal of X25 and SH3 domains resulted in 27.3 % and 58.4 % reduction in raw starch binding ability, while removal of CBM20 and X25 domains resulted in a 90 % decrease in raw starch binding ability. Thus, it was revealed that the X25 domain is involved in binding raw starch. The results of TLC analysis represented that TbbApu and its variants released maltotriose and maltose as a result of pullulan hydrolysis and maltotriose starch hydrolysis, respectively. The results of TLC analysis represented that TbbApu and its variants released maltotriose and maltose in pullulan hydrolysis and maltotriose in starch hydrolysis, respectively.
Recombinant production and characterization of chitinase enzyme from Pseudomonas mandelii KGI_MA19

(Graduate School, 2022-06-24) Saraç Cebeci, Emine Tuğçe ; Karagüler Gül, Nevin ; 521201111 ; Molecular Biology-Genetics and Biotechnology

Biological catalysts, generally found in protein structure, that catalyze the biochemical reactions necessary for life are called enzymes. They increase the reaction rate by lowering the activation energy of the reaction catalysed. In recent years, enzymes have gained a large share in different industrial areas, like food, agriculture, pharmacy, cosmetics, waste removal, leather processing, detergent, and medical applications. Enzymes can minimize the formation of unwanted by-products, they are environmentally friendly and cheap, and have biodegradable properties. They are also considered safe for the cleaning, health, and food industries. In addition to their preferred properties, enzymes that remain active at low/high temperatures, in the presence of organic solvents, at different salt concentrations and pH values, and with an affinity for different substrates are attracting the attention of the industry. Meeting the increasing demand for the use of biomolecules with different properties in the industry is possible with the development of the physical and biochemical properties of the biocatalysts defined today with the help of protein engineering, metagenomics, advanced DNA technologies, nanotechnology, and finally, the discovery of new biocatalysts. Scientific research has been increasing in this direction, especially because enzymes from living things that live in extreme conditions can be used in a wide range of industries. Chitin (C8H13O5N)n is an inelastic, hard, white, non-elastic biopolymer formed by the bonding of N-Acetyl-D-glucosamine monomers (Glc-NAc) with β-1,4 glycosidic bonds, which ranks second after cellulose among the most abundant biopolymers in nature. It is a nitrogen-containing polysaccharide. Chitin is found in the cell wall of fungi, the outer shells of insects, the shells of sea creatures such as lobster, crab, shrimp, and the mouth areas of cephalopods such as cuttlefish and octopus which have a high annual production in the world. According to the Food and Agriculture Organization (FAO, 2019) data, 10.5 million chitin-rich shellfish (12.3%) are grown in aquaculture. The need for converting chitin wastes, which are rising in quantity by the day, into biologically useful products develops. In recent years, the employment of biocatalysts in the removal of chitin wastes instead of chemical methods, which are poisonous to the environment and expensive, has allowed for a safe conversion for the environment. Chitinase enzymes (EC 3.2.1.14), in the hydrolase class, catalyse the destruction of β-1-4 glycosidic bonds in chitin (C8H13O5N)n and separate N-acetyl-D-glucosamine molecules from the polysaccharide chain. The chitinase enzyme, which is one of the most common hydrolase enzymes in nature, is found in insects, plants, fungi and viruses. It is also commonly found in different bacterial genera such as Aeromonas, Arthrobacter, Bacillus, Chromobacterium, Flavobacterium, Pseudomonas, Sanguibacter, Serratia, and Streptomyces. With the increase in green and environmentally-friendly technology in recent years, the interest in chitinases is increasing day by day. Chitinase enzymes, which have agricultural importance, replace chemicals and pesticides used in agriculture with their ability to be used as biocontrol agents. It creates a positive effect on the marine ecosystem by recycling marine waste. Due to their pharmaceutically antifungal and antibacterial properties, they are used in the production of anti-inflammatory and anti-fungal drugs, anti-cancer and immune-enhancing agents, wound dressings, contact lenses, and surgical sutures. It is a bioprotective additive used to increase shelf life in the food industry. Antarctica with the highest elevation and lowest temperature has recently become a popular research area. 70% of the freshwater resources on the Antarctic continent, which has not been touched by human hands until lately, are in the form of ice. It also features powerful winds, extremely cold temperatures, and is exposed to low temperatures in the winter and high UV rays in the summer. It has a natural and distinct habitat as a result of these factors. Because of the harsh circumstances, it is unavoidable to uncover new species and enzymes and genes. Extremophiles are living systems that can thrive in harsh environments. Biocatalysts derived from extremophilic organisms and demonstrating catalytic activity even under adverse circumstances have been dubbed extremozymes. Psychrophiles and psychrotolerants are extremophilic microorganisms that can live at 0-20 °C and 0-30 °C, respectively. Because of their low energy needs, flexibility, and high catalytic activity, cold-compatible enzymes derived from psychrophilic and psychrotolerant organisms are crucial for commercial applications. Biotechnological approaches help the identification of novel and diverse extremophiles and extremozymes from Antarctica that has not been described in the literature yet. In the study, which was carried out using Antarctic sediment samples collected within the scope of the 2nd National Antarctic Science Expedition (TAE-2), 12 sediment samples taken from 8 different regions were cultured and enriched. Subsequently, freeze-thaw stress was applied to each cultured sample. After the applied freeze-thaw stress, it was observed that five cultures were resistant to stress. Two morphologically different colonies were selected from each stress-resistant sample, DNA isolations were made and 16S rRNA analyzes were carried out. The sample with the lowest similarity rate according to 16S rRNA analysis was sent for whole-genome analysis and according to the results of the analysis, a new strain of Pseudomonas mandelii, Pseudomonas mandelii KGI_MA19, was identified. Phenotypic and biochemical characterizations of the identified KGI_MA19 strain were performed. The next step was the recombinant production and the characterization of the chitinase enzyme of the psychrotolerant Pseudomonas mandelii KGI_MA19. The gene region of the chitinase enzyme of Pseudomonas mandelii KGI_MA19 was amplified by polymerase chain reaction (PCR) by designing gene-specific primers containing SacI and NotI restriction sites. The amplified gene region of interest and the pET-28a (+) vector were cut with SacI and NotI restriction enzymes. T4 DNA ligase enzyme was used for ligation of the cut PCR product and expression vector. Plasmid pET-28a (+) containing the PCR product was transformed into Escherichia coli BL21 (DE3) competent cells. The colony PCR method was applied to determine the colonies containing the relevant gene as a result of the transformation, and plasmid isolation of two colonies selected from among the colonies thought to be positive was performed. Then, to understand whether the colonies contain the gene product or not, the plasmid was cut with the EcoRV endonuclease enzyme, which has a fast-cutting feature. The DNA sequencing results showed that the chitinase gene-specific PCR product was correctly inserted into the pET-28a (+) plasmid. The Magic MediaTM (Invitrogen), which is commercially available and used in the expression of E.coli cells, was used to monitor the expression level of the chitinase gene in E.coli BL21 cells. At the end of Magic Media incubation, cells were treated with lysis (0.1M Tris-HCl, pH 8.0, 0.3M NaCl) buffer, and homogenization was performed by ultrasonication using the M73 probe. A high amount of produced chitinase enzyme was purified by the His-Tag affinity chromatography method using the His-Trap column. The activity of the chitinase enzyme obtained in high purity was determined by the 3,5-Dinitrosalicylic acid (DNS) method which allows one to determine the amount of reducing sugars expected to be released as a result of the reaction performed by the usage of colloidal chitin as a substrate. Biochemical characterization including optimum pH, pH stability, and optimum temperature, thermal stability has been completed. A new, psychrotolerant strain, Pseudomonas mandelii KGI_MA19, was identified from sediment samples from the Antarctic King George Island, and its molecular, phenotypic, morphological, and biochemical characterization was completed. In addition, the chitinase enzyme which has an increasing impact in the industrial field has been successfully produced and biochemically characterized by using recombinant DNA methods. The cold-adaptive chitinase enzyme of the Pseudomonas mandelii KGI_MA19 strain, which was obtained for the first time within the scope of this thesis, is thought to be a promising potential biocatalyst for industrial applications.
Expression, purification, and characterization of soluble recombinant TNFR1

(Graduate School, 2022-06-28) Hatipoğlu, Derya ; Dinler Doğanay, Gizem ; 521191107 ; Molecular Biology – Genetics and Biotechnology

Tumor Necrosis Factor Alpha (TNF-α) is a trimeric cytokine secreted by macrophages and monocytes. It belongs to type II transmembrane protein family and is involved in both innate and adaptive immune system. TNF-α exists in two forms: transmembrane TNF (tmTNF), which is synthesized as a precursor form, and solubilized TNF (sTNF), which is created after further processing. TNF-α functions in a variety of biological processes by interacting with specific receptors namely as TNFR1 and TNFR2. TNFR1 consists of intracellular, transmembrane, and extracellular domains. The extracellular part consists of four cysteine-rich parts. When interacting to TNFR1, TNF-α can activate various signaling pathways for instance inflammation and apoptosis. TNF-α level is undetectable in normal individuals, yet under inflammation conditions the protein concentration in serum increases proportionally to the inflammation level in the body, thus making the protein detectable. Controlled production of TNF-α is crucial for tissue healing and fight against infection, but a constantly high level of TNF-α can cause various diseases including rheumatoid arthritis (RA), ankylosing spondylitis (AS), psoriasis/psoriatic arthritis, and Chron's disease. TNF-α inhibitors are designed and used in treatment of diseases caused by overexpression of TNF-α. An alternative pathway includes employment of TNFR1 extracellular domain as a biotherapeutic tool to inhibit the effects of overexpressed TNF-α. The latter approach seems to be way more preferred than other therapeutics due to its targetselectivity and high affinity. Production of proteins like TNFR1 are characterized by cysteine-rich domains by using bacterial systems is quite advantageous in terms of cost, yield, and time. However, lack of an effective translation system in Escherichia coli (E. coli) may hinder production of such disulfide bond containing proteins mainly in terms of generating mismatched cysteine residues or failing in acquiring a sulfide bridge formation thus leading to protein aggregation and inclusion body formation. Recovering proteins from inclusion bodies is time-consuming and expensive while considering the protein loss and incorrectly folded proteins due to the wrongly formed disulfide bonds. Dsb protein family including DsbA, DsbB, DsbC, DsbD, and DsbG, is responsible for the formation of disulfide bonds in bacteria. The DsbA-DsbB complex plays role in the oxidative pathways, while DsbC-DsbD complex functions in the isomerization pathway. Among all Dsb proteins, DsbA and DsbC are the most extensively studied and widely used in the field of biotechnology. Our previous studies showed that the extracellular portion of TNFR1 forms inclusion bodies in E. coli, and in vitro refolding steps are needed to obtain the correct xxvi conformation. Protein aggregations during the refolding process also caused a decrease in yield. The purpose of this study is to obtain the extracellular part of TNFR1 as a soluble protein in E. coli. Initially, a SHuffle T7 strain with a cytoplasmic DsbC copy was selected as the host cell and expression experiments were carried out accordingly. However, the target protein formed an inclusion body. To assess the effect of DsbC in the correctly folded-soluble TNFR1 production, a expression vector containing fusion protein DsbC-TNFR1 was constructed. Formation of DsbC-TNFR1 fusion protein was held on the basis of plasmid design conferring the order of a 6x histidine tag, DsbC, TEV cleavage site, and TNFR1 from N to C terminus respectively. It was assured that periplasm target sequence was removed from DsbC gene, allowing its production within the bacterial cytoplasm. DsbC-TNFR1 production was verified by SDS-PAGE and immunoblotting analyses, which revealed that some conditions led to the production of the fusion protein in soluble form. In order to detect the optimum conditions to produce DsbC-TNFR1, four different E. coli strains as host cells were employed, namely as Rosetta (DE3), Rosetta-gami 2, SHuffle T7, and BL21 (DE3). However, since production of the Rosetta (DE3) strain was higher than others, the research was conducted on that particular strain. Induction conditions including IPTG induction and autoinduction medium were also assessed to get higher yield from the selected cells. As a result of induction trials, the target protein was obtained in soluble form when autoinduction was utilized. Following sonication and centrifugation of the cells, the affinity chromatography was performed for separation of the recombinant protein from other host cell proteins. Anion exchange chromatography (AEX) was used as a second purification step to remove remaining impurities. The efficiency of purification was evaluated by using SDS-PAGE and immunoblotting analyses. Then, protein characterization studies were performed. The isoelectric point of the pure protein was calculated by isoelectric focusing via capillary electrophoresis device, and the purity of the protein was checked using the purity determination method. The secondary structure of the fusion protein was analyzed by circular dichroism spectroscopy and it was determined to be in the α-helix structure. Intact mass analysis through LC/MS was also performed to calculate the molecular weight of the protein. In addition, the peptide mapping was used to identify the amino acid sequence. The native structure of the pure protein was investigated by using blue native polyacrylamide gel electrophoresis (BN-PAGE), which revealed that the protein exists in both dimers and different oligomer structures. The functionality of the fusion protein was assessed by performing a pull-down assay, which showed that DsbC-TNFR1 is functional as it was capable of binding to TNFR1 ligand TNF-α. As a consequence of the research, it was revealed that the implementation of such a fusion protein is a successful tool for expressing TNFR1 in its soluble form in bacterial cells. After analysis of the pure protein generated as the outcome of downstream steps, it was shown that the TNFR1 produced by using the implemented fusion method is functional.
Investigation of the energy metabolism of helicobacter-activated B cells

(Graduate School, 2022-07-07) Çalçı, Mehmet ; Yazgan Sayı, Ayça ; 521181128 ; Molecular Biology-Genetics and Biotechnology

Helicobacter pylori is a gram-negative, spiral-shaped and microaerophilic bacterium. It has been declared as type 1 carcinogen by the World Health Organization since it causes stomach cancer. Although more than 50 % of the world's population is infected with this bacterium, only 10-20 % of them have serious gastrointestinal diseases. For the development of infection-disease murine models, H.felis which is instead of H.pylori can be used. The immune system is the system of defense and protection against any infectious disease. It is categorized into two groups as innate and adaptive. An early line of defense against microbes is formed by innate immunity. Innate immunity includes both cellular and biochemical defense mechanisms that are present even before infection and are ready to respond immediately to infections. On the other hand, adaptive immunity becomes active when the organism encounters an infectious agent. Memory response generation is one of the most basic features of adaptive immunity. B cells play a significant role in the regulation of immune response due to the key functions including antibody production and antigen presentation. The regulatory role of B cells was shown with the use of mice lacking B cells which could not overcome autoimmune encephalitis. Helicobacter felis stimulates B cells via TLR2 and Myd88 and suppress immune responses. This suppression brings about persistency of bacteria in gastric mucosa Therefore, a balance between regulatory and effector response could determine the fate of infection. As demonstrated by our previous laboratory studies, H. felis-mediated activation of B cells induces two different B cell subgroups: IL-10- producing IL-10+ B cells and TGF-β-producing IL- 10- B cells. When a resting immune cell encounters an antigen, it undergoes metabolic reprogramming. The metabolic profile of immune cells for energy production plays a significant role in the regulation of the immune system. The energy (ATP) production mechanism required by immune cells during proliferation and differentiation into an effector cell differs from cell to cell. Glucose which is one of the key biomolecules used as the basis for ATP production is taken into the cell via glucose transporters and metabolized during glycolysis. The pyruvate formed at the end of glycolysis can pass into the mitochondria and enter the TCA cycle, increasing oxidative phosphorylation, or it can remain in the cytoplasm and produce lactate. The oxidative phosphorylation pathway can also utilize fatty acids and amino acids. Glycolysis and oxidative phosphorylation are the two main pathways involved in energy production in the cell. Studies have shown that there is a decrease in the proliferation of LPS-stimulated B cells treated with 2-DG, a glycolysis inhibitor. In addition, it has been reported that treatment of B cells with oligomycin, an ATPsynthase inhibitor, during stimulation with anti-CD-40 and IL-4, decreases the activation marker CD86 and cell viability. However, there is no information in the literature about how B cells undergo metabolic reprogramming when stimulated by Helicobacter species. B cells that do not receive stimulation in cell culture can not maintain their viability for a long time. In a study, a low dose of recombinant B cell-activating factor (rBAFF) was added to B cell culture and increased the viability of the cells. Based on this information, it was aimed to investigate the effect of rBAFF on Helicobacter-activated B cell viability and function since cell viability was very critical in the metabolic analysis studies that would follow. B cells isolated by magnetic separation from C57BL/6 mouse spleen were stimulated with H.felis sonicate, PAM3CSK4, and LPS for 6 and 24 hours. The main purpose of choosing 6h time point is to investigate how cells respond only in the presence of stimuli before they proliferate, and the main reason for choosing the 24h time point is that B cells begin to secrete the anti- inflammatory cytokine IL-10. To increase the viability of B cells in the culture, the effect of rBAFF added at a low dose on the viability is determined by the flow cytometry, its effect on the function with the expression levels of CD86 and CD9 surface markers in the flow cytometry again, and the effect on the IL-10 secretion level by the IL-10 ELISA method. researched with. Although rBAFF added to the B cell culture medium significantly increased the viability of the control group, which did not receive stimulation, it was excluded from the experimental plan because it would affect the results of metabolism studies since it changed the IL-10 levels of the cells. Because rBAFF was intended to be given to the control group, which was not stimulated in metabolism studies, in case it did not show any effect on the function while increasing B cell viability, rBAFF was not given to the experimental groups since it slightly changed the IL-10 production levels of B cells. Another aim of the study is to investigate how the metabolic profile of splenic B cells stimulated with H.felis is characterized. The metabolic flux analysis method was used to determine which pathways are activated for the required energy production while becoming an effector by being exposed to stimuli from the resting state. B cells were cultured with H.felis sonicate, PAM3CSK4, and LPS for 6 and 24 hours, and then the change in energy metabolism was measured in XFe96 device by extracellular flux analysis method. Since the number of dead cells exceeding 20 % would adversely affect the results of metabolism studies, dead cell removal was performed by the magnetic separation method, especially in the control groups. Only cells with high viability were included in the metabolism studies. The proportion of viable cells in the non-stimulated groups increased from 50% to 80% after the magnetic removal of dead cells. Since the viability data of the stimulus groups were suitable for metabolism experiments, no dead cell separation was made in these groups. According to the results obtained from our study, as a result of stimulation of B cells with H.felis sonicate, PAM3CSK4 and LPS for 24 hours, glycolysis and glycolytic capacity tend to increase significantly as a result of the measured ECAR data. Although the groups that received stimulation at 6 hours increased both glycolysis and glycolytic capacity compared to the control groups that did not receive stimulation, this rate is much less when compared to 24-hour stimulation. Basal and maximum OCR values of cells are directly proportional to the amount of oxygen they consume during OXPHOS. As a result of stimulation of B cells with H.felis sonicate, PAM3CSK4 and LPS for 24 hours, both basal and maximum OCR values increased significantly compared to the non-stimulated groups. This increased rate is much lessin the 6-hour warning groups. The OCR data also gives information about the mitochondrial ATP production of the cells. B cells stimulated with H.felis sonicate, PAM3CSK4 and LPS also significantly increased their mitochondrial ATP production after 24 hours. As a result of this study, the effect of rBAFF on cell viability and function was investigated for the first time in mouse splenic B cells stimulated with H.felis for both short (6 hours) and long (24 hours) periods. At the same time, the specificity of the study is high since it was investigated for the first time how the metabolic profile required for energy production is shaped in mouse splenic B cells stimulated with H.felis for both short (6 hours) and long (24 hours) periods.
Characterization of BAG-1S/C-raf interaction targeting peptide

(Graduate School, 2022-07-25) Çebi, Ecenur ; Doğanay Dinler, Gizem ; 521201110 ; Molecular Biology-Genetics and Biotechnology

ERK/MAPK cascade is one of the most important cellular pathways, which regulates many distinct physiological functions such as cell proliferation, migration, differentiation, and apoptosis. Since it has vital roles in many cellular functions, dysregulation of its members usually results in uncontrolled proliferation and development of cancer cells such as breast cancer. Ras and Raf serine/threonine protein kinases are mostly deregulated components of this pathway and mutations in both proteins have been identified approximately one-third of all human cancer types. Although there are several inhibitors against oncoproteins, because of the improving drug resistance, there is a need for the development of new drugs. One of the emerging interest is to target protein-protein interactions (PPIs) related with cancers. Human Raf kinase family has three isoforms: A-Raf, B-Raf, and C-Raf. C-Raf and B-Raf are the important members of ERK pathway. Although B-Raf is dysregulated mostly, C-Raf has ability to suppress apoptosis and hence, both of the Raf kinase is vital for the cancer progression. The Raf kinases are regulated by several phosphorylation events and also, interactions with some proteins like BAG-1. Bcl-2 associated athanogene 1 (BAG-1) which is an anti-apoptotic co-chaperone protein is usually overexpressed in several cancer types making it possible oncoprotein. Moreover, its known that the interaction between BAG-1 and C-Raf promotes the C-Raf activation and stabilization. Therefore, targeting this interaction with small molecules and/or peptides would be effective therapeutics against different cancer types. In our previous studies, the interaction surface between BAG-1S and C-Raf was determined and a peptidomimetic which coded as Pep3 was designed against the BAG-1S from natural sequence of C-Raf. The aim of this study was to characterize the interaction between BAG-1S and Pep3 and also, the interaction between BAG-1S and TPep3 which is cell penetrating form of Pep3. Firstly, His-tagged BAG-1S proteins were produced in mammalian and bacterial cells. Then, BAG-1S proteins were purified by Ni-NTA affinity purification in two steps. After first step, His-tag was cleaved with TEV protease and tagless proteins were eluted as flow-through in second step while the impurities and TEV proteases were remained bound to resin. Then, the purities of both proteins (produced in mammalian and bacterial) were calculated as >80% by SDS-PAGE. Secondary structure analysis of proteins was performed with circular dichroism and both of the proteins has been folded and showed primarily ɑ-helix characteristics. After BAG-1S proteins were characterized. Pep3 and TPep3 characterization were performed by mass spectrometry and circular dichroism analysis. Pep3 and TPep3 has been synthesized by Fmoc-based solid phase peptide synthesis and they were dissolved in 20 mM AMBIC and water respectively since the Pep3 shows hydrophobic characteristics. Molecular weights of peptides were measured as 2.066 kDa for Pep3 and 3.653 kDa for TPep3 which were compatible with the theoretical calculations. Both of the peptides showed β-sheet characteristics approximately 30% combined with random coils. The interaction between BAG-1S and peptides were confirmed by crosslinking reaction with medium length crosslinker DSS (disuccinimidyl suberate). BAG-1S proteins were incubated with peptides separately to form interaction. After they formed interaction, the crosslink reaction was performed with 50-fold molar excess of DSS. The reactions were analyzed by immunoblotting. BAG-1S was shifted 2 kDa when interacts with Pep3 and 4 kDa with TPep3 comparing to the BAG-1S only reaction. These results were confirmed the interaction of BAG-1S with two peptides. The binding kinetics of BAG-1S and Pep3 was measured by Surface Plasmon Resonance (SPR) with multi-cycle kinetics method. BAG-1S protein was captured on Protein G chip with Anti-BAG-1 and different concentrations of Pep3 was injected. Binding kinetics was calculated as 68.56 nM by 1:1 binding model showing that Pep3 binds BAG-1S with high affinity. The effects of TPep3 on MAPK pathway and cell viability of MCF-7 breast cancer cells were analysed. MCF-7 cells were treated with different concentrations (0 uM to 50 uM) of TPep3 and total proteins were analysed with immunoblotting. C-Raf and p-C-RAf (S338) levels were decreased with the increasing concentration of TPep3. As a result of p-C-Raf inactivation, p-MEK levels were also decreased which showed the decrease in ERK pathway. Moreover, B-Raf and p-B-Raf (S446) levels were decreased. So, it can be said that peptide does not only affect the activitation of C-Raf but also B-Raf. To see the effects of peptide on other cell survival pathways, Akt and p-Akt (S473) levels were analysed and it was seen that their levels remained unchanged even with the highest peptide concentration. Lastly, the IC50 value was calculated as approximately 18 uM from the cell viability MTT assay. Together with all the results, TPep3 and Pep3 peptides have potential to be a promising therapeutics for cancer types relying on ERK pathway since the activity of pathway was decreased with the peptide treatment.
Investigation of structural differences between wild-type and mutant forms of mutsα by molecular dynamics simulations

(Graduate School, 2022-08-03) Buran, Clara Xazal ; Gür, Mert ; 521201107 ; Molecular Biology-Genetics and Biotechnology

Cancer is the most common cause of death after cardiovascular diseases. Among the cancer types, colon cancer, which is very common in our country and the world, endangers human health. The aim of the studies in this thesis is to determine the changes in the structure and dynamics of the MutSα protein due to mutations in the protein complex associated with colon cancer. Molecular Dynamics (MD) Simulations provide important information about protein dynamics by providing the opportunity to observe changes in the structures and functions of proteins. Computational methods have been a remarkable field in recent years because they provide energetic, structural and dynamic information at the atomic level that cannot be obtained using experimental techniques. In this study, the effects of mutations in MutSα on protein structure and dynamics were analyzed using computational and theoretical methods. MutSα complex, formed by the MSH2-MSH6 heterodimer is responsible for the recognition and repair of mismatches. Some mutations in MutSα have been associated with cancer studies. Certain mutations that occur on protein complex cause the accumulation of mutations in the cell and eventually causing cancer. Only some of the mutations in protein complex have been associated with cancer, but the effect of some mutations, defined as pathogenic variation, on protein activity is not yet known. The MSH2 and MSH6 proteins that make up the MutSα complex are divided into five major regions: the mismatch binding domain, the connector domain, the lever domain, the clamp region, and ATPase domain. The mismatch binding domain recognizes mismatched bases and binds to these bases. The connector domain is responsible for allosteric signaling, while the lever region is thought to be responsible for signaling between the mismatch binding domain and the ATPase domain. The clamp domain stabilizes the DNA by interacting and bonding with the DNA backbone. The ATPase domain is the region where the energy required for the protein to move along the DNA is obtained by hydrolysis of ATP. MutSα complex detects mismatches by scanning the DNA and binds to mismatch region to start repair mechanisms. In a healthy cell, DNA repair mechanisms work smoothly, DNA damages are repaired, and apoptosis occurs in case of irreparable damage. However, mutations in the proteins in the DNA repair mechanism prevent the correct functioning of the DNA repair mechanism, causing the accumulation of damaged/mutant DNA in the cell and causing tumor cells. Studies have revealed that specific mutations in MutSα, one of the DNA mismatch repair proteins, can be hereditary and cause cancer. Some mutations obtained as a result of genetic screening tests by Prof. Dr. Levent Doğanay and his research group, with samples taken from individuals with a family history of colon cancer have been reported to our study group. Ala733Thr, Arg577Cys and Ser127Asn mutations were detected, and the effects of these mutations on MutSα without DNA were investigated by performing all-atom MD simulations. In order to look at the changes that these mutations may cause in protein function, first of all, systems were prepared for the MD simulations of the MutSα protein using structures with PDB ID: 2O8B. Two replica systems were created for each construct to compare the simulation results for wild-type and mutant proteins. While simulating the proteins, the required ion concentration was provided by considering the cell's physiological conditions. After the structures were dissolved in water, short minimization and equilibrium simulations were performed. The equilibrated structures were simulated for 200ns. Two replicate simulations were carried out for wild-type and three mutant proteins, and totaling 1600ns MD simulations were performed. Root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), heat capacity, interaction analysis and principal components analysis (PCA) were performed to investigate the effect of the mutations on the MutSα complex. The RMSD analysis gives information about whether the simulation has reached equilibrium and whether there has been a conformational change. The average RMSD values of mutant proteins were higher than the wild-type protein. Therefore, these mutations resulted in changes in protein conformation and dynamics. RMSF analysis is used to provide insight about structural flexibility, mobility and thermal stability. In this thesis, fluctuations were investigated for five domains separately and it was observed that the mutant proteins had similar or higher fluctuations than the wild-type protein in general. For the wild-type, the region with the highest fluctuations was observed to be the clamp region. Heat capacity can provide information about the thermodynamic properties of the protein. The findings obtained as a result of the analysis showed higher heat capacities for all mutants compared to that of the WT protein and show that the thermodynamic properties of the protein may have been differentiated. As a result of the interaction analysis, it was observed that the mutants had changes in hydrogen bond and salt bridge interactions compared to the WT protein. With PCA analysis, the two most dominant movements in the protein were obtained. Conformations were projected onto principal components and based on the conformational space sampled by the proteins during the MD simulations heat map surfaces were constructed. In this study, the effects of mutations in the DNA-free MutSα protein structure and dynamics were investigated and it was concluded that mutant structures showed different thermodynamic and structural properties compared to wild-type.
Immunoreceptors modulate eosinophilic functions in viral immunity

(Graduate School, 2022-11-09) Durmuş, Lübeyne ; Muğan Çıracı, Ceren ; 521201116 ; Molecular Biology-Genetics and Biotechnology

Immunity a term used as resistance to pathogens, also referred to reactions of the body to noninfectious compounds such as tumors, harmless environmental substances and even sometimes host's components, each of which are called as tumor immunity, allergy and autoimmunity, respectively. The whole components like cells, tissues and molecules that generate this immunity is defined as the immune system and the immune response is coordinated by these components to foreign substances. Providing protection against infections or eradication of the infectious compounds from the body is the major physiological function of the immune system. Besides, growth of some tumors and cancers can be inhibited by stimulating immune reactions against cancer cells. The human defense system against pathogens can be divided into 3 levels: physical and chemical barriers, innate immunity and adaptive immunity. The innate immunity relies on a finite number of receptors for detecting the invading pathogens. These limited number of receptors target large groups of pathogens by recognizing conserved microbial patterns. Furthermore, activation of adaptive immune response is achieved by innate immune response. Innate immune cells include both hematopoietic and nonhematopoietic origin which make them different from adaptive immunity which relies on T and B lymphocytes. Innate immune response development involves hematopoietic cells that differentiate into monocytes, macrophages, mast cells, dendritic cells, natural killer cells, natural killer T cells, basophils, neutrophils and eosinophils. Moreover, hematopoietic cells include skin and epithelial cells reside in respiratory, genitourinary and gastrointestinal tracts. Innate immunity depends a few germline encodedreceptors which sense pathogen specific structural motifs. These receptors are collectively called pattern recognition receptors (PRRs) and microbial components recognized by PRRs are called pathogen associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). PRRs can exist in various cellular compartments like cell membrane and endosomal membranes, or cytosol. Moreover, they can be found in extracellular environment such as bloodstream or interstitial fluids. They are mainly divided into four major types: 1) Toll-like receptors (TLRs), 2) Nucleotide binding and oligomerization domain-like receptors (NLRs), 3) Retinoic acid-inducible gene 1-like receptors (RLRs), 4) C-type lectin receptors (CLRs). Activation of these receptors of the innate immune cells result with induction of adaptive immune cells. Hence, PRRs help to generate immune response to eradicate infectious agents, for example they induce the death of the infected cells. Humans are constantly under the threat ofinvasive opportunistic microorganisms including viruses, fungi, bacteria and parasites. The detection and development of an appropriate immune response against invading viruses are crucial for the outcome of virus infections. Recognizing viral nucleic acids is the first step for sensing virus infection. This recognition mainly depends on the genetically predetermined and germline encoded PRRs. There are TLR protein family members recognize viral genetic material such as TLR3, TLR7, TLR8 and TLR9. Upon recognition of viral nucleic acids or proteins by PRRs, production of type I IFN is induced resulting in the activation of target cells in both autocrine and paracrine manners. TLR7 and TLR8 have the ability to detect the existence of various single stranded RNA (ssRNA) viruses. These viruses include influenza, vesicular stomatitis virus (VSV), HIV, Sendai virus (SV) and a number of coronaviruses and flaviviruses. TLR3 is an endosomal nucleotide sensor which is located on endosomes and activated by double stranded RNA (dsRNA). As dsRNA is the genome for many viruses or they synthesize dsRNA during their life cycles, TLR3 also detects the existence of dsRNA and DNA viruses. Unlike TLRs, RLRs are RNA detectors locolized in the cytosol. RLR protein family includes three members: RIG-I, MDA5 and LGP2. Upon viral RNA association and oligomerization, RLRs bind to mitochondrial antiviral signaling protein (MAVS) through its CARD. MAVS has an essential role in RLR signal transduction as an adaptor protein whichinduces TBK1 and IKKε leading to the activation of IRF3 and IRF7. These transcription factors and NF-κB, together, activate the production of type 1 IFNs. Moreover, NLRs are important part of the cytosolic innate immunity and have a role in various key pathways such as inflammasome, MAPK, NF-κB and type 1 IFN signaling. Activation of inflammasome complexes is essential because it induces inflammation by leading to the production and secretion of IL1β and IL18 also known as inflammasome dependent cytokines. Eosinophils attract considerable attention with their identified roles in many pathological processes such as acute and chronic infections, cancer and thrombosis. Although their accepted roles in parasitic infections, involvement of eosinophils in fungal, bacterial and viral infections is an on-going research topic in the field of immunology. These granulocytes contain and generate substances with antiviral functions such as RNases and they may also involved in adaptive immunity with their potential antigen-presenting ability. Together, findings about eosinophils indicate potential antiviral role for eosinophils which need to be explored further. Despite the studies on animal models and primary human eosinophils demonstrating the importance of eosinophils against viral infections, the question of how eosinophilic functions are regulated following he viral infections is still ambiguous. Thus, the aim of the study is to investigate the changes in eosinophilic functions upon activation of TLR3, TLR7 and TLR8 with proper ligands, poly(I:C), R848, and ssRNA40, respectively. low number of eosinophils in blood (1-6%) makes them difficult to study in vitro. Therefore, we utilized EoL-1 human eosinophil line as a model in our study which was previously shown to serve an ideal model due to the expression of eosinophilic markers In this study we initially determined the activation of Eol-1 cells upon treatment with viral ligands, eosinophilic PRRs and immune receptors. Secondly, surface markers of eosinophils were determined to further understand the eosinophilic functions. Also, cytokines that they released were analyzed to understand the potential involvement in the induction of adaptive immunity. Moreover, production of matrix metalloproteinases was measured. We observed significant increase in IL-6 and IFN-γ secretion upon TLR8 activation. Also, the number of IL5Rα and PDL1 expressing Eol-1 cells were augmented with TLR3, TLR7/8 and TLR8 inductions. Our data suggested roles for TLR3, TLR7 and TLR8 in eosinophilic functions. We then investigated the changes in granule content of eosinophils during viral infections. We showed that ECP mRNA levels were upregulated upon the ssRNA40 treatment in Eol-1 cells. This data also, demonstrate the possible roles of ECP on the regulation of antiviral eosinophilic functions. MMPs are matrix-degrading enzymes which have roles in leukocyte recruitment to chemokines during microbial infections. In addition to their functions in immune regulation, MMPs can also lead to tissue damage as a result of persistant pathogen infections or spread. Furthermore, CD147 is a matrix metalloproteinase inducer that plays critical roles in various viral infections. Recently, CD147 was shown as an alternative receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Therefore, we also investigated the MMPs and MMP inducer CD147to better understand their roles in eosinophilic antiviral immune responses. The results from RT-qPCR showed a relationship between both CD147 and TLR activation as well as the data obtained from gelatin zymography clearly indicated the relationship between MMP-9 and TLR activation in Eol-1 cells. All stimulants tested in this study elevatedthe CD147 at mRNA level in Eol-1 cells. Additionally, TLR3 and TLR8 ligands significantly increased the Pro-MMP9 production at 24 hours post-induction in EoL-1 cells. Upregulation of TLR8 led us investigate the potential adaptor molecules which may have roles in downstream signaling pathways through TLRs in eosinophils. CARD9 is a multifunctional adaptor protein which participates in different phases of the immune system including fungal, bacterial and viral infections. Thus, we investigated the CARD9 expression in Eol-1 cells upon TLR3, TLR7/8 and TLR8 inductions. Despite the decreased mRNA expressions of CARD9, there was a significant increase in CARD9 expression at protein level in Eol-1 cells. Next, we measured the expression levels of cytosolic PRR RIG-I at protein level because of its well-known roles in antiviral immunity. We observed 1.49-fold increase in the cells stimulated with TLR8 agonist (ssRNA40, 2 μg/ml). Then, we measured NOD2 as it is already known as the interaction partner for CARD9. Numerous studies formerly showed the NOD2 involvement in response to many RNA virus infections such as parainfluenza virus, VSV, RSV and IAV. Here we showed that NOD2 protein expression was significantly increased after induction with TLR7/8 and TLR8 ligands in Eol-1 cells. Overall, of all the stimuli we tested, ssRNA40 (TLR8 signaling) was the most potent in inducing the mRNA expressions of ECP and CD147, and protein expression of immune receptors, surface markers of eosinophils and cytokines. Interestingly, CARD9 which is a critical adaptor against fungi infections was significantly increased after induction with TLR7/8 ligands, suggesting an important role for TLR7 and TLR8 rather than TLR3 in antiviral immune response generated by eosinophils. Our findings will pave the way for future studies focusing on eosinophil related infectious diseases.

Gözat

Çıkarma tarihi ile LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans'a göz atma

Sayfa başına sonuç

Sıralama Seçenekleri