LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Yüksek Lisans
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ÖgeDADA2 hastalarının periferik kan mononükleer hücrelerinde total ada aktivitesinin analizi(Graduate School, 2022-02-09)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.
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ÖgeControlled release of tetracycline hydrochloride from silica based polycaprolactone nanohybrides(Graduate School, 2022-12-23)Biomaterials can be synthetic or natural materials which is designed for interact with the biological systems. There are various type of biomaterials such as metals, seramics and polymers. Biodegradable biomaterials that naturally degrade or completely dissolve in their physiological environments have gained attention for both invasive and noninvasive health monitoring due to providing an unique opportunity for therapeutic field. Biodegradable polymers are classified into two main categories as natural and synthetic polymers. Polymers offer high adjustability in terms of their chemical structure and morphology and they contain hydrolysable bonds and these bonds makes them prone to chemical degradation via hydrolysis or enzymecatalyzed hydrolysis. Biodegradable polymers are using in controlled drug delivery, anticancer drug delivery, protein and peptide delivery, gene delivery, and enzyme immobilization at industry and researches. Biodegradable polymers can degrade in two main ways; partially or fully degrade to monomeric units. Polymers' degradation rate is heavily affected by various factors. Examples of these factors are the following such as the morphology, molecular weight, its distribution, crystallinity temperature and environmental conditions. Polylactic acid (PLA), polyglycolic acid (PGA), poly(lactic-co-glycolic acid (PLGA) and poly(ε-caprolactone) (PCL) are some of the most used synthetic biodegradable polymers. These are polyester polymers that have chemical structure with ester bond linkages and these polymers most used in industry are aliphatic polyesters. Polycaprolactone (PCL) is a linear aliphatic polyester hydrophobic semi-crystalline. PCL can synthesis with two different methods. These are the condensation of epsilon-hydroxycaproic acid and the ringopening polymerisation (ROP) of e-CL. In the condensation of epsilon-hydroxycaproic acid which is a polycondensation, 6-hydroxycaproic acid is polymerising by using lipase from Candida antarctica. ROP is the most preferred route and it gives a polymer with a higher molecular weight and a lower polydispersity. PCL is mainly synthesized by ring-opening polymerization (ROP) of ε-caprolactone (ε-CL) monomer. There are three different ROP catalytic system and metal-based catalysts are the most used catalysts for ROP of ε-CL. The metal based compounds takes part in the ROP of lactones can be describe as catalysts, initiators, initiating systems or catalytic systems. PCL have wide applicability and advantage such as biocompatibility, controlled degradability, miscibility with other polymers, and if its properties can be controlled and it can be made inexpensively, it can be a very useful polymer. Drug delivery systems (DDSs) are developed to prevent problems such as reducing therapeutic efficacy and causing unwanted side effects for improving drug safety and helping to improve patient compliance and convenience. DDS have many important applications in every field of medicine such as cancer, pain, diabetes and ischemia, myocardial treatment. Release pattern of DDS mainly effect by the delivery vehicle , the drug properties, and the environmental conditions. Drug delivery systems varies based on their route, mechanism and materials used in and there are several approaches of DDS. One of these approaches is polymers that used in a variety of fields in pharmaceutical applications. Polymers have advantages in DDSs as providing controlled release of therapeutic agents in constant doses over long periods and their cyclic dosage, and tunable release of both hydrophilic and hydrophobic drugs. In the first part of this study performed RHA preparation and activation of RHA, immobilization followed by polymerization to obtain nanohybrid polymers. Rice husk ash (RHA) is obtained by burning husks of rice. prepared and then silanized with 3-Glycidoxypropyltrimethoxysilane (3-GPTMS). RHA silanized by using 3-GPTMS as support material to immobilize free enzyme Candida antarctica lipase B. Enzymatic ring-opening polymerization (eROP) of ɛ-caprolactone was provided by immobilized lipase enzyme Candida antarctica lipase B (CALB). eROP reaction started along with ε-caprolactone and immobilized enzyme, then G-PCL/RHA nanohybrid obtained after terminating the reaction with chloroform followed by the precipitation. In the second part of the study microspheres prepared by trying different conditions to find best efficiency ones and followed by Drug release experiment to the highest effiency microspheres. Drug loaded microspheres were prepared by W/O/W double-emulsion-evaporation method which is water-in-oil method. Since the drug used in study is a water-soluble drug active substance, have to find the right ratio for both PVA and drug amount. Because of this, tried to find best PVA percentage by using different PVA percentages. Here found the best ratio is %1 PVA. Then looked at the drug amounts by preparing microspheres in %1 and different drug amounts. G-PCL/RHA drug-loaded microspheres with best drug loading efficiency we can get were obtained. Drug release experiments were carried out at pH 7.4 condition The release profiles of the drug-loaded G-PCL/RHA microspheres were determined and afterwards the drug release percentages are calculated. According to drug release results, the highest cumulative drug release percentage was %56 at pH 7.4 in %0.5 PVA, 10mg drug amount conditions microspheres with 72 hours burst level and %29 drug loading. Meanwhile the highest drug loading, efficiency, was in %1 PVA and 10mg drug amount conditions with %54 drug loading. In the last part of study, experiments has been finalized with charactization of microspheres to analyse microspheres by using Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). FTIR spectra of G-PCL/RHA nanohybrid and drug-loaded G-PCL/RHA microspheres were compared along with literature tetracycline hydrochloride FT-IR spectra and the presence of tetracycline hydrochloride in the micropheres demonstrated. FT-IR results showed both G-PCL/RHA nanohybride and tetracycline FT-IR spectra on G-PCL/RHA microspheres FT-IR results. This proved the tetracycline hydrochloride presence on G-PCL/RHA nanohybrid microspheres. TGA results showed the decomposition temperatures and organic weight losses of drug-loaded G-PCL/RHA microspheres and tetracycline hydrochloride. According to DSC analysis, showed the melting and crystallization points of PCL polymer. Microsphere structures were observed by SEM analysis. Particles of the G-PCL/RHA microspheres were seen to be spherical and sphere-like structures with various size but they were more disadvantage cause of not being as highly porous compare to the literature microspheres. For better understanding drug-polymer interactions and higher how to get higher drug efficiency of microspheres on drug release mechanism, analysis methods can be developed in further studies. Beside drug release of hydrophylic drugs loaded microspheres can be carried out in vivo. Such studies exist in the literature.
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ÖgeThe synthesis, SLIC based labeling, and characterization of microbial rhodopsins by using custom build spectroscopic methods(Graduate School, 2023-01-26)Type I opsins (also known as microbial opsins) are seven transmembrane-domain proteins with retinal chromophore absorbing incoming light. Most of them are ion channels or pumps although they do not directly bind to G protein complexes. They are found in all three domains of life. Numerous homologous forms of rhodopsins have been identified in the microorganisms, including light sensors (sensory rhodopsins), transmembrane chloride pumps (halorhodopsins), and energy saving transmembrane proton pumps (bacteriorhodopsin or proteorhodopsin). Rhodopsin proteins are widely used in the field of biotechnology. For example, it is used to determine the membrane voltage level in neurons. The use of rhodopsins as tools to control membrane potential with light is another technique for transformative optogenetics technology. Membrane voltage is present in all cells, and it creates an electric signal to carry the signal across the cell membrane and provides cell-cell communication. Since the absorption methods are not sufficient to measure voltage signal due to low signal to noise ratio in cells, more sensitive fluorescent methods based on rhodopsin are strongly preferred for a wide spectrum of applications. An understanding of the dynamics of the microbial rhodopsin proteins is essential to tune the photophysical properties of rhodopsins. It is also necessary to label them with fluorescent proteins and characterize their localization in detail. For the fluorescent signal to vary with the amount of light absorption in the membrane protein, the linker peptide between the proteins has to be optimized for various applications. For electrochromic fluorescent energy transfer, it is necessary to select the appropriate fluorescent protein. The emission signal of the fluorescent protein must overlap with the absorption signal of the membrane protein. After the most suitable fluorescent proteins are selected by calculating the amount of overlap, the structure of the peptide that binds the membrane and the fluorescent protein should be determined. Since both the length and the bending ratio of the selected peptide are important, it is necessary to optimize by testing different constructs. Here we have synthesized, purified, and studied the rhodopsin by using molecular biology and various spectroscopy methods. After the synthesis of rhodospins in BL21 cells, it was purified with his-tag affinity column chromatography and reconstituted with a detergent solution. The color tuning of rhodopsin as a function of pH was investigated by using absorption spectroscopy. We found that BPR undergoes a large red shift under acidic conditions. A pH value was increased the color turned from orange to red at the basic solution. We concluded that the deprotonation of the retinal at the rhodopsin center results in a significant change in the color of BPR. We have also measured the transient absorption changes of BPR by using a custom home built spectrometer that was equipped with two laser lines and an op amp light detector. The data acquisition and the control of lasers were performed an by arduino and field programmable gate array device programmed with arduino and labview respectively. Our results indicate that BPR underwent an absorption change after stimulated with a 532 nm diode laser. Finally, the fluorescent proteins were also cloned into the SRII gene by using SLIC cloning method and expressed in BL21 cells to determine the changes in fluorescent emission. Sensory rhodopsin was similarly characterized by using absorption spectroscopy. As conclusion, BPR undergoes a large spectral shift due to deprotonation upon decreasing pH and alters the color of the protein. SLIC method provides a cost-efficient method to prepare fluorescently labeled rhodopsin proteins. Contrary to the standard cloning techniques used in molecular biology, the SLIC method, which is convenient in terms of time and cost, has been studied and the method has been optimized. The optimized SLIC method can be used as an alternative to other molecular cloning techniques. The custom build pump-probe system can also be used for the characterization of fluorescently labeled other rhodopsin proteins in future studies.
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ÖgeEvolutionary engineering of freeze-thaw stress-resistant yeasts without using chemical mutagenesis(Graduate School, 2023-06-20)Saccharomyces cerevisiae, also known as budding yeast or baker's yeast is a unicellular microorganism from the fungi kingdom. It has been consistently used in winemaking, brewing and baking bread throughout human history. After 1930s, laboratory studies were conducted to obtain strains with increased product quality. Today, S. cerevisiae is the most popular yeast strain due to its good fermentative abilities. S. cerevisiae with high fermentation performance and tolerance to environmental stresses is preferred for industrial applications. During bread production, yeast cells are exposed to a variety of environmental stresses including freeze–thaw, high sugar concentrations, air-drying and oxidative stress. Stress conditions cause a decline in cell growth rate, product yield and quality. Cells give responses to stress conditions, as environmental stress response (ESR) and stress-specific response. ESR mechanism is not specific to the stress factor and it can be used to explain the cross-resistance of the yeast cells against various stress types. One of the reasons for cross-resistance is the use of the same transcription factors as a response to a variety of different stresses. S. cerevisiae is exposed to freeze-thaw stress during the cryopreservation and frozen dough process. Freeze-thaw stress causes physiological injuries to cells. At high freezing rates, formation of intracellular ice crystals causes cellular damages; while at low freezing rates formation of extracellular ice crystals causes cellular dehydration. The thawing process causes oxidative stress which leads to oxidative damage on proteins, nucleic acids and other biomolecules inside the cell. Studies conducted in S. cerevisiaes' stress-specific response against freeze-thaw stress revealed cells focus on regulating the contents of the cell membrane, protecting cell wall integrity, increasing degradation of damaged proteins from stress and increasing overall protein synthesis under stress conditions. Cryoprotective agents can be added to decrease ice crystal formation under freezing conditions. Alternatively, yeast levels in the product can be increased to increase product yield. However, these methods can decrease product quality and increase cost. Thus, stress-resistant S. cerevisiae strains are preferred for industrial applications. Stress-resistant strains can be obtained by metabolic engineering. Evolutionary engineering is an inverse metabolic engineering method that mimics the natural evolution process. In this approach, the desired phenotype is selected first and the genes responsible for the phenotype are determined later by reverse engineering methods. In this study, freeze-thaw resistant yeast strains were obtained with the evolutionary engineering method. A reference yeast strain was exposed to freeze-thaw stress in the form of pulse stress selection. The evolved strains obtained under stress conditions generally show mutations mainly in their stress-induced genes. This allows ease in reverse engineering studies to determine genes related to the applied stress. Freeze-thaw stress was applied in the form of pulse stress selection to maintain the survival rate of cells with increasing stress levels and to induce selective pressure. In this study, a S. cerevisiae CEN.PK113-7D reference strain was exposed to gradually increasing freeze-thaw stress until the final population was obtained. The final population was obtained after 10 cycles of freeze-thaw stress application. Ten mutant individuals were randomly selected from the final population and their resistance to freeze-thaw stress was tested with the spot assay method. Four evolved strains labeled as FT-1, FT-5, FT-6 and FT-9 that showed the highest freeze-thaw resistance were selected for detailed analysis. Further physiological characterizations of the evolved strains were made by cross resistance analysis. FT-1, FT-6 and FT-9 showed cross-resistance to potassium chloride (KCl) and iron stress. KCl, at high concentrations, causes hyperosmotic stress to the cell. This cross-resistance could be the result of a similar response mechanism activated by the cell to protect itself from dehydration caused by freezing stress. Metals such as iron increase generation of ROS in cell and cause oxidative stress. The cross resistance to iron stress could be the result of activation of similar pathways used by the cell as a response to oxidative stress caused by thawing process. All evolved strains tested showed resistance to boric acid. Boric acid disrupts cell wall synthesis in S. cerevisiae. The freezing process also causes cell wall damage in S. cerevisiae. Inducing cell wall synthesis due to freezing stress may also result with increased resistance to boric acid. The aim of this study was to obtain freeze-thaw stress-resistant S. cerevisiae strains from a reference laboratory strain, without using chemical mutagenesis, by evolutionary engineering. Physiological characterization of the evolved strains was also performed by determining their cross-resistance to selected stress factors. Further genomic, transcriptomic and proteomic analyses could be performed on the selected FT-9 strain to identify the genes, pathways and molecular mechanisms responsible for resistance against freeze-thaw stress and the pathways that cause cross-resistance to selected stress factors.
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ÖgeExpression, purification, and characterization of recombinant human IL-2(Graduate School, 2022-01-18)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.