LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji Lisansüstü Programı

Bu topluluk için Kalıcı Uri

http://hdl.handle.net/11527/19400

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A comparative study on immobilization of Jack Bean urease on different matrices

(Graduate School, 2023) Okuşluk, Abdullah Said ; Kılıç, Abdullahim ; 874308 ; Molecular Biology – Genetics and Biotechnology Programme

Biotechnological applications of urease enzyme prevalently involve biosensor development, wastewater treatment systems, artificial kidney machines, restoration of cultural heritages, beverage industries, bioregenerative long-distance space travel systems and rehabilitation of agricultural fields. However, the integration of free urease to industrial, medical, or agricultural applications results in considerable drawbacks, such as activity loss in process conditions and high cost of isolation and purification for reuse. To overcome these mentioned restrictions and develop new biotechnological products, in this thesis study, we immobilized soluble urease onto the eggshell membrane and inner epidermis of the onion bulb scale. Natural supports for the immobilization of Jack Bean urease are already available as waste products of the food sector, with materials that are biocompatible, biodegradable, non-toxic and low-cost. Further, considering sustainability and feasibility issues, the eggshell and onion membranes were chosen for this approach. The eggshell membrane is essentially made of cross-linked collagens as a flexible protein fiber. Additionally, the inner epidermis of the onion bulb scale is predominantly composed of microfibrillar cellulose. The surface morphology of these natural membranes, as displayed by SEM imaging, provided a suitable support for the adsorption technique. This method is the simplest, undemanding, and economically attractive enzyme immobilization approach, relying on weak electrostatic interactions or physical bonding. For the immobilization of Jack Bean urease, each supportive membrane was initially washed with water, air-dried, and cut into 1 cm2 pieces. Subsequently they were treated with branched polyethyleneimine to generate polycationic surfaces. This adsorbent is generally recognized as an FDA-approved safe substance. The urease activity was detected by measuring the amount of released ammonia, as an indicator of residual urease activity.
A molecular dynamics study of the prion protein

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

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

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

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

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

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

(Graduate School, 2024-06-28) Akaydın, Aslı Nur ; Çakar, Zeynep Petek ; 521211101 ; Molecular Biology - Genetics and Biotechnology

Saccharomyces cerevisiae has an important place in human life with its wide usage in various processes like fermentation, brewing, bread and wine production from the oldest times in history. Since its genome sequencing in 1996, it has become one of the most well-known and studied model organisms in many different areas of biology such as cell biology, biotechnology, cancer and aging research. Compared to other model organisms, its ease in genetic manipulation and cultivation conditions made it a convenient host for the production of heterologous proteins and economically valuable products. Yeast shares 30% of homology with many human genes, thus it is a convenient platform to study eukaryotic cell metabolism along with disease models. Aging is a common process all living organisms share and involves numerous metabolic and physiological changes that usually result from accumulated damage and deterioration. Despite the developed technology and improved living conditions through every passage of human life, it is estimated that the aging population will cover one-fifth of the whole population of the world by end of the century and age related diseases will cause a socioeconomic burden to governments. Studying aging in humans is complicated because of the long lifespan and economic and ethical concerns. Although a wide range of organisms share similar aging patterns, using yeast provides a convenient and accurate eukaryotic model with a shorter lifespan and easier growth capability. There are two main approaches to studying aging in yeast: chronological lifespan (CLS) and replicative lifespan (RLS). CLS seeks to analyze the lifespan of undivided yeast cells after they enter the stationary phase, mostly caused by decreased nutrients or toxic metabolite accumulation. RLS defines the number of cell divisions a cell undergoes before its death. CLS analysis is particularly useful for analyzing the response and survival of the cell against certain stress factors and modeling G0 cells that arrest their cell cycle. Metabolic engineering is an effective biotechnological approach for improving metabolic processes and product formation of the organism by altering the existing mechanisms or introducing new ones through the usage of recombinant DNA technology. In classical metabolic engineering, information on metabolic, genetic and physiological data of the strain is gathered, then the manipulations on relevant factors are employed to obtain the desired phenotype. Yet in the inverse metabolic engineering approach, for example when evolutionary engineering is employed, the desired phenotype is achieved using laboratory-based evolutionary settings. In this strategy, yeast strains can become resistant to certain stress factors or produce desired molecules throughout the increased stress treatment during culture. After obtaining the desired phenotype, yeast strains are examined by genomic or transcriptomic analyses to further determine the molecular changes in the genome or transcriptome. With more advanced xix technologies such as CRISPR-Cas9, altered genetic traits can be transferred to wild type strains to evoke the same resistant phenotype. In this study, previously obtained stress-resistant S. cerevisiae strains were analyzed for their CLS and viability performances. Stress factors selected for this purpose were antimycin, boron and freeze-thaw stresses which can affect the production efficiency or viability of yeast strains. In parallel with the general evolutionary engineering strategy, strains were obtained by increasing the stress levels gradually, which is the concentration of the compound in the case of antimycin and boron, and repeat numbers in the case of freeze-thaw stress, in selection cultures until the resistant population is achieved. It was shown previously that the evolved strains could become cross resistant to other stress types or their longevity can be affected by the process. The aim of this study was to determine which type of stress resistances can extend or shorten the CLS of the yeast thus affecting the lifespan of the industrial and laboratory yeast strains. For this aim, both quantitative and semi-quantitative CLS analyses were carried out. In the semi-quantitative CLS experiment, OD600 values of the yeast strains were set to 6 before they were spotted onto agar plates every 2nd day with serial dilutions. The longevity of the resistant strains was compared with their control strains visually, based on the growth on the plates. According to the results, P8 which is the freeze-thaw stress-resistant, industrial polyploid strain had a longer CLS than its industrial reference strain, whereas the antimycin and boron-resistant strains did not have a longer CLS than their reference strain. In the second part of the study, quantitative CLS analysis was done by spreading the long-lived industrial P8 strain along with its industrial reference strain R625 and the laboratory reference strain 905 to agar plates. The longevity was measured by counting colony-forming units (CFUs). The experiment was repeated until the viability of the cultures was reduced to 0.0001% from day 0 of the experiment where the viability was accepted as 100%. In the second part of the study, further validating the results from the semi-quantitative analysis, P8 exhibited longer CLS compared to its industrial reference strain and could live until the 10th day of the experiment. Among the various stress-resistant strains tested in this study, only the freeze-thaw stress-resistant, industrial P8 strain was found to have a longer CLS. However, the antimycin and boron-resistant yeast strains did not show a longer CLS compared to their laboratory reference strain. Since the freeze-thaw response was previously associated with oxidative stress response and nutrient metabolism alterations, the longer CLS of the freeze-thaw stress-resistant industrial strain can be related to changes in respective pathways that originated from the evolutionary engineering process. In the scope of the research done for this study, despite being studied in other organisms, the effect of boron resistance on longevity was studied in yeast for the first time. Similarly, antimycin resistance was examined for its effect on longevity in yeast for the first time, as well. Further studies to analyze genomic and transcriptomic changes that occurred by the acquired resistance can be performed and these changes can be transferred to wild-type or reference strains to assess the viability and CLS profiles.
Clinical and molecular collaboration studies: i)comparison of CNV by NGS&MLPA in hereditary breast and ovarian cancer ii)effective covid-19 sampling and storage

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

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

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

Oxidative stress occurs as the oxidant and antioxidant balance in an organism is disrupted in the direction of oxidants, and oxidants come to a position that can potentially cause damage. All aerobic organisms use oxygen for respiration and oxidation of nutrients for producing energy. For this reason, while oxygen is necessary for aerobic organisms to live, it is also harmful because of the reactive oxygen species (ROS) that emerge during these processes that are necessary for life. Lipid, protein and DNA molecules, which form the basis of life, can be damaged directly or indirectly due to oxidative stress caused by ROS. Aerobic organisms have developed various mechanisms to protect themselves from the harmful effects of oxidative stress. These mechanisms involve enzymatic and non-enzymatic (antioxidants) systems. In addition to these mechanisms, organisms use various repair mechanisms, especially to repair damaged DNA. Oxidative stress from ROS is biomedically important and widely researched, as it causes many neurodegenerative and autoimmune diseases, as well as cardiovascular diseases, cancer, and aging in mammals. The yeast Saccharomyces cerevisiae is used as a model organism, because of its beneficial characteristics. S. cerevisiae, which is a eukaryotic organism, has a short-life span. It is a single cell organism and can be found as haploid, diploid or polyploid in nature. Thus, S. cerevisiae is an important model organism for elucidating the processes in eukaryotic complex organisms. The aim of this study was to characterize an oxidative stress-resistant mutant S. cerevisiae strain at genetic and transcriptomic levels. The main idea, however, was to comprehensively examine the complex molecular infrastructure of oxidative stress resistance. In addition, as a physiological analysis, the cell wall properties of the mutant strain was also tested and information was obtained about the effect of stress on the cell wall structure. In this thesis study, transcriptomic analysis of the mutant S. cerevisiae was performed by comparing the transcriptomic profiles of the oxidative stress-resistant, evolved S. cerevisiae and the reference strain. Transcriptomic data of the strains were obtained by microarray analysis. As a genomic analysis, the entire genomes of the oxidative stress-resistant strain and the reference strain were sequenced and the differences in the genome were determined to find the mutations in the evolved srain that are related to oxidative stress resistance. In addition, cell wall analyses of the reference strain and the mutant strain were performed, using lyticase susceptibility test. As a result of transcriptomic analysis, it was observed that the expression level of 869 genes changed by at least 2-fold, 349 genes changed by at least 3-fold, 144 genes changed by at least 4-fold, and 67 genes changed by at least 5-fold in the oxidative stress-resistant mutant. Among these 869 genes that were differentially regulated by 2 times or more, 459 genes were upregulated and 410 genes were downregulated. The genes whose expression were decreased are generally related with ribosomal RNA, nuclear transport, organelle integration, tRNA cell cycle, mitosis and RNA polymerase. Expression levels of stress response genes, carbohydrates, lipid, protein, precursor metabolites and ion/metabolite transport-related genes were generally increased in the mutant strain. According to the ESR analysis results, a positive correlation was observed between the ESR-induced genes and the genes of the oxidative stress- resistant mutant with increased expression, according to the database. In addition, a positive correlation was observed between ESR-suppressed genes and genes of the mutant with decreased expression. The expression levels of two genes related to oxidative stress decreased and twenty three of them increased in the oxidative stress-resistant, evolved strain. The expression levels of six autophagy-related genes in the oxidative stress-resistant mutant decreased and 30 of them increased. According to whole genome sequencing results, 34 missense, 2 nonsense, 1 deletion and 12 silent mutations were found in the genes of the mutant strain, and 13 mutations were detected in chromosomal regions outside the coding regions. A nonsense mutation in the NRG1 gene, which is a transcriptomic regulator, results in the formation of a truncated protein. Further genomic and proteomic studies would be necessary to clarify the role of these genes and mutations in the oxidative stress resistance.
Computational investigation of reaction mechanism of FET3 protein in yeast

(Graduate School, 2023-02-17) Ahıshalı, Büşra ; Balta, Bülent ; 521191104 ; Molecular Biology-Genetics and Biotechnology

In Saccharomyces cerevisiae, a yeast species, iron uptake into the cell takes place with the Reducing Iron Uptake Model. Ferric chelates (Fe3C-L) are degraded on the cell surface by being reduced from Fe3+ to Fe2+ by the cell surface reductases Fre1p and Fre2p. The free reduced Fe2+ ions are taken up by Fet3p-Ftr1p, a high-affinity oxidase-permease complex, or by Fet4, another metal carrier. In this study, the reaction mechanism and the role of Fet3p in reducing iron uptake are examined. Fet3p is a membrane-bound protein and a member of the multicopper oxidase protein family. It metabolizes iron uptake with a high affinity for Fe2+ and plays a role in iron uptake together with iron-permease Ftr1p. Since Ftr1p can only transport the oxidized form of iron, Fe2+ needs to be oxidized before entering the cell. Fet3p couples the four-electron reduction of O2 to H2O with the one-electron oxidation of four Fe2+. The oxidized iron leaves the iron-binding site in Fet3p and is transferred to Ftr1p. Thus, Fe3+ ions are transported into the cytoplasm by a permease, Ftr1p. The understanding of the mechanism of Fet3p is of great importance to shed light on other multicopper oxidase members such as laccases and human ceruloplasmin, some having wide industrial applications. When the active site structure of Fet3p is examined, it has 4 copper as a cofactor in the active site. These coppers are divided into 3 types according to their characteristics: Type 1 (T1), Type 2 (T2), and binuclear Type 3 (T3a and T3b). T2 and T3 coppers form the trinuclear cluster (TNC). Iron as a substrate is not observed in any of the crystal structures of Fet3p. However, according to the information obtained from mutation studies and comparing them with the crystal structures of other MCOs, especially copper efflux oxidase (CueO), the amino acids in the iron-binding region of Fet3p and the location of iron were determined. Fet3p couples four one-electron oxidations of 4 Fe2+ as a substrate to the four-electron reduction of dioxygen to water by taking four protons from the environment. This process is mediated by oxidation-reduction reactions of copper ions as cofactors and consists of two stages. In the first stage, the O2 molecule, which will be reduced to H2O during the reaction, enters the TNC through the solvent channel and binds to the TNC. The O-O bond is cleaved by taking two electrons from two coppers (T1 and one of T3 coppers). Proton donation of E481 to one of the oxygens bridging T3 coppers facilitates this cleavage. Finally, all coppers are oxidized to Cu2+, and one O2- and two OH- ions are formed. In the second stage, the four reductions from Cu2+ to Cu+ with oxidation of four Fe2+ to Fe3+, and four protonations occur, and OH- and O2- ions are converted to two water molecules. In the literature, most of the first stage of the reaction mechanism of MCOs, especially dioxygen-cleavage and peroxy intermediate structure are known. However, the exact mechanism of the second stage, the order of electron and proton transfer reactions is not known because this part occurs fast. Due to the rapidity of these reactions, they have not been studied before and the order of the reaction is unknown due to the difficulty of following the protonation order experimentally. In addition to examining the reaction scheme, it is known that D283 plays an important role in iron binding to substrate-binding site, and electron transfer (ET) is enhanced by D283. However, in the crystal structure, the loop containing D283 is oriented away from the active site, suggesting that it closes only after the binding of Fe2+. Thus, to find out the role of D283 on ET and reaction pathways, the geometries are separately examined when the loop containing D283 is open and closed. In order to elucidate the unknown parts, computational methods were used in this present study, so the possible reaction mechanism will be determined. Thus, it is aimed to understand the mechanisms of other multicopper oxidase members through Fet3p. The calculations and geometry optimizations were carried out using the Quantum Mechanics/Molecular Mechanics QM/MM approach. The M06-2X method, a Density Functional Theory (DFT) method, was used for quantum mechanical (QM) calculations. B3LYP, TPSS, and M06 methods were also used to investigate whether M06-2X is the most suitable method for energy calculations and geometry optimizations of Fet3p containing copper and iron metals. Although M06-2X is not recommended to be used on metals in the literature, all necessary electronic states and spin densities could be obtained only with M06-2X in this study. For this reason, the results were interpreted over the energies obtained with M06-2X. The determination of the QM region to be calculated during the QM/MM calculations is of great importance for the calculations to obtain more accurate results. While choosing the most ideal QM region, residues that have the potential to affect the reaction, electron transfer, and proton exchange, especially close to the region where the reaction took place, were determined. The proximity of amino acids that will contribute to electron transfer around copper and Fe was investigated; therefore, calculations were made accordingly by choosing different QM regions. Considering the computational costs, the most ideal QM region was determined. In the structure where the loop containing D283 is closed, the first Fe2+ oxidation occurs exothermically without protonation while T1 is reduced. Protonation of OH- or O2- ions are not needed due to the cost of protonation. It is examined whether the first electron transfers from T1 to TNC before the second iron binds; nevertheless, the structure could not be obtained without protonation. With the protonation of the TNC region, electron transfer to the TNC has yielded a stable structure. After the oxidized iron leaves, the second Fe2+ binds. Meanwhile, the electron already transferred from the first iron remains in the protonated TNC. Considering the necessity of a second proton transfer before oxidation, the proton taken from D94 returns back to D94 during the optimization, thus the second proton transfer is not necessary. The electron from Fe2+ transfers to T1 copper, and oxidation of the second iron takes place. The second oxidation, which was endothermic when D283 was open, is exothermic in the structure where the loop is closed. The results draw attention to the importance of the loop containing D283. After the second oxidation, the oxidized Fe3+ is replaced with the third Fe2+. For the third iron, structures with two protons, three protons, and four protons are examined. For the third and fourth Fe2+ the geometries when the loop with D283 is open are also examined. According to the results, even three protonations are not enough for third oxidation, and a fourth protonation is needed. When the loop containing D283 is open, the oxidation of the fourth Fe2+ is endothermic even in the presence of four protons in TNC, which is the maximum number of protons TNC can take. In the oxidation reactions protonation of TNC-O2- decrease the negativity of TNC; thus, electron transfer to TNC is more favorable. The protonation of TNC is important to reduce coppers at TNC (T2 and T3 coppers) and for transferring an electron from the substrate to TNC. Similarly, the transfer of an electron from the substrate to TNC and the reduction of TNC coppers force the TNC-O2- or T3-OH- to take proton.
Controlled release of tetracycline hydrochloride from silica based polycaprolactone nanohybrides

(Graduate School, 2022-12-23) Cengiz, Aybüke ; Güvenilir, Yüksel ; 521191130 ; Molecular Biology - Genetics and Biotechnology

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.
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.
Detecting binding activity of a therapeutic monoclonal antibody targeting vascular endothelial growth factor using surface plasmon resonance

(Graduate School, 2024-07-25) Erdem, Serim ; Kılıç, Abdulhalim ; 521191137 ; Molecular Biology – Genetics and Biotechnology

The use of biopharmaceuticals, particularly monoclonal antibodies (mAbs), has become a major application following advancements in recombinant DNA technology. The treatment of a wide range of diseases, among them cancer, asthma, cardiovascular conditions, infections, inflammatory and autoimmune disorders, including allergies, has become possible with the development of mAbs. Surface plasmon resonance (SPR) is a well-known and reliable method that can evaluate biomolecular interactions in real-time in terms of binding affinity and kinetics without requiring labeling procedures. Using SPR technology to determine the kinetic parameters of a mAb molecule resulting from its binding with its target receptor is a common approach to characterize the binding activity of the final product. To characterize the binding kinetics between two molecules, one molecule is first stabilized on the sensor chip surface by immobilization or capturing steps. Then, the other molecule is flowed over the sensor chip surface at variety concentrations in a running buffer to observe the interaction between the two molecules and to determine the resulting kinetic parameters. In this study, after an IgG monoclonal antibody was captured on a Protein A chip surface, the target VEGF121 growth factor was flowed over the surface at five different concentrations. By employing single-cycle kinetic analysis, the kinetic parameters were measured, providing information about the interaction between the two molecules. To develop a robust kinetic assay, it was necessary to optimize several parameters, including the capture molecule (mAb) concentration, the concentration range of the VEGF121 molecule, dissociation time, regeneration time, and the established method sequence. While evaluating the robustness and reliability of the method, important factors considered included quality control parameters from the evaluation software, plotted residuals, and the consistency of Rmax and KD values between replicates. As a result of the method development studies, a capture concentration of 13.4 nM, a dissociation time of 240 seconds and a regeneration time of 30 seconds were decided upon, ensuring that the deviation in KD values between repeated studies was less than 20 %. After the KD value was obtained with low deviation between replicate studies, qualification studies including specificity, system precision, repeatability and intermediate precision parameters were initiated. As a result of 6 replicate studies conducted by two different analysts, the percentage difference between the analysts was determined to be 0.14%. Additionally, kinetic binding studies were performed with mAb molecules from 12 different production batches to demonstrate the effectiveness of the developed method in determining the binding activity of mAb molecules in different lots. These studies showed quite similar kinetic parameters for different production batches.
Development of novel BCL-2 inhibitors for glial tumors by using in vitro and in vivo systems

(Graduate School, 2022-05-31) Çalış, Şeyma ; Turanlı Tahir, Eda ; Avşar, Timuçin ; 521162117 ; Molecular Biology – Genetics and Biotechnology

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

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

Thermophilic microorganisms that survive and grow in extreme environments, above temperatures of 50 °C, have been well studied over the last decade allowing us to increase our knowledge of the compositional and functional potential of these microbial communities. These microorganisms are of great importance for industrial processes since they express heat-resistive enzymes with the potential to serve as a biocatalyst in the future. Developing proteomic and metaproteomic approaches to discover novel enzymes from environmental samples is growing research of interest owing to the advanced mass spectrometry (MS) based techniques. In this study, proteomics and metaproteomics approaches were applied to discover novel enzymes from harsh environmental conditions. Geothermal sources are among the habitats of thermophilic bacteria. In Turkey, there are many spas that have the potential habitat for numerous thermophilic bacteria, and this offers a good opportunity for the discovery of new thermophilic microorganisms. In this study, a thermophilic bacterial consortium of the Armutlu Hot Spring in the Yalova region of Turkey was investigated in a culture-dependent manner using proteomic approaches.
Evolutionary engineering of freeze-thaw stress-resistant yeasts without using chemical mutagenesis

(Graduate School, 2023-06-20) Balaban, İrem ; Çakar, Zeynep Petek ; 521211107 ; Molecular Biology - Genetics and Biotechnology

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.
Evolutionary engineering of rapamycin-resistant yeast

(Graduate School, 2022-12-02) Esen, Ömer ; Çakar, Zeynep Petek ; 521191120 ; Molecular Biology-Genetics and Biotechnology

The budding yeast Saccharomyces cerevisiae, a unicellular eukaryotic microorganism, is widely used in many industrial processes such as baking, alcohol fermentation, biofuel and recombinant protein production as well as in basic research to understand the complex biological processes of advanced eukaryotic organisms, including humans due to its well-characterized genome and proteome, ease of growth and manipulation, as well as the similarity of its genes and pathways to higher organisms. Rapamycin is a macrolide compound that is produced by the bacterium Streptomyces hygroscopicus. In the medical field, rapamycin and its analogs are used to prevent organ transplant rejection, coat coronary stents and treat tumor cells. Many key metabolic pathways including cell growth and lifespan, protein synthesis and ribosome biogenesis, regulation of cell cycle and size, environmental stress response, nutrient uptake, starvation control, and autophagy in S. cerevisiae and also other higher eukaryotes are affected by rapamycin due to its inhibition of the target of rapamycin (TOR). Fkbp12 forms a complex with rapamycin that interacts with TOR resulting in its inhibition where the exact mechanism is still unknown. Fkbp12, immunophilin, is conserved in its structure and function among eukaryotes from yeasts to mammalians. Fpr1 found in S. cerevisiae is an orthologue of Fkbp12 found in humans. In this study, an inverse metabolic engineering strategy, evolutionary engineering was used to obtain rapamycin stress-resistant S. cerevisiae. Thus, serial batch cultivation of the S. cerevisiae CEN.PK113-7D reference strain under gradually increasing rapamycin stress was carried out. Before selection, a screening experiment was performed and 3 ng/ml rapamycin stress was determined as the initial stress level. During the selection process, the concentration of rapamycin in the medium was gradually increased from 3 ng/ml to 200 ng/ml over 61 daily passages or populations. From the final population, fifteen individual colonies were randomly selected which were named R1 to R15. Every individual (R1 to R15) was highly resistant to rapamycin stress in comparison to the reference strain (905). Afterward, R1, R3, R7, R12 and R14 were selected to continue for genetic stability test in which they were found to be genetically stable and their resistance to rapamycin was shown to be permanent. Genetically stable strains were tested by spot assay for their cross-resistance or sensitivity against various stress types, including 0.5 mM NiCl2, 2.5 mM CrCl3, 3 mM CoCl2, 17.5 mM MnCl2, 50 mM NH4Fe(SO4)2, 10 mM AlCl3, 20 mM CuCl2, 15 mM LiCl, 50 mM H3BO4, 100 µM AgNO3, 0.5 M NaCl, 15 mM caffeine, 4 mM vanillin, 200 ng/ml propolis, 1mM coniferyl aldehyde and 200 ng/ml cycloheximide. R12 was found to be the mutant with the highest number of cross-resistance and sensitivities: R12 strain was cross-resistant to CuCl2, NH4Fe(SO4)2, NaCl, coniferyl aldehyde, vanillin, cycloheximide, propolis; and sensitive to AlCl3, CoCl2, H3BO4 and AgNO3 in comparison to the reference strain (905). In order to determine the cell wall integrity of the rapamycin-resistant mutant (R12), lyticase susceptibility assay was performed. The result of this experiment showed that R12 resisted lyticase more than 905, under nonstress condition. On the other hand, under rapamycin stress, 905 resisted lyticase more than R12. Furthermore, the presence of rapamycin stress did not change the lyticase resistance of the evolved strain R12, in comparison to the nonstress condition. The chronological lifespan of rapamycin resistant strain (R12) was determined by using semi-quantitative and also quantitative chronological lifespan analysis. The result of the experiments correlated with each other in which R12 was found to have a shorter CLS, in comparison to 905. Comparative whole genome sequencing analysis of the rapamycin-resistant mutant (R12) revealed four single nucleotide variations (SNVs). These SNVs were located in four different genes. The precise functions of these genes in rapamycin response and resistance in yeast should be examined in greater detail in future studies. In conclusion, a rapamycin-stress-resistant and genetically stable S. cerevisiae strain (R12) was successfully obtained by using evolutionary engineering in this thesis study, and characterized at genomic and physiological levels. These results indicate that TOR pathway-related changes occurred in rapamycin-resistant mutant in order to overcome the high levels of rapamycin stress. However, in order to fully comprehend the molecular basis of rapamycin-resistance of R12, its comparative transcriptomic and metabolic analyses would be necessary as future studies.
Exploration of novel serine protease do-like HtrA from acigöl

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

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

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