Moleküler Biyoloji-genetik ve Biyoteknoloji Anabilim Dalı (disiplinlerarası)
Istanbul Technical University26
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Kpkt katil toksininin Saccharomyces cerevisiae ve Pichia pastoris hücrelerinde heterolog üretimi için ekspresyon vektörlerinin oluşturulması
In nature, microorganisms process several defense mechanisms to survive under limited nutritional conditions. Killer yeasts have one of the defense mechanisms. They produce toxins called killer toxins (KTs) against sensitive microorganisms. Killer toxins are produced by killer yeasts into extracellular environment and they show their activities by binding to specific compounds called killer toxin receptors (KTRs). Killer toxins can be produced as either proteins or glycoproteins. While killer yeasts have self-immune system to their own KTs, their cytocidal activities usually process in a two-step mechanism on sensitive strains. In the first step, they bind the receptors on cell-wall of the sensitive strains. These receptors are known as primary KTRs. Secondary KTRs found on cell membrane are the second targets of KTs. They bind to these receptors to kill the sensitive strains to enter the cell. Different action mechanisms have been presented for killer toxins and they depend on the genetic determinants as well as physical-chemical properties of KTs. Kpkt is one of the killer toxins produced by Tetrapisispora phaffii (formerly known as Kluyveromyces phaffii). Kpkt killer toxin is active against wine spoilage yeasts such as Kloeckera apiculata and Hanseniaspora uvarum. Therefore, Kpkt has a potential to be used in wine fermentation since it maintains its zymocidal activity for more than 14 days in wine. Kpkt is a glycoprotein and it has a β-glucanase activity. It shows its activity by hydrolyzing β-1,3- and β-1,6- glucans on cell-walls of the sensitive strains. Kpkt killer toxin is encoded by TpBGL2 gene in T. phaffii. The aim of the study was to construct expression vectors for heterologous production in Saccharomyces cerevisiae and Pichia pastoris. For this purpose, pYES2.1/V5-His-TOPO® vector was utilized for both extracellular and intracellular production in S. cerevisiae as well as pPIC9 vector was utilized for extracellular production in P. pastoris. In the first part of the study, suitable yeast strains resistant to Kpkt were selected for heterologous production of Kpkt. S. cerevisiae YPH501 and BY4741 along with P. pastoris GS115 strains were subjected to killer plate assay using YPD plates containing citrate-phosphate buffer (pH 4.6). P. pastoris GS115 appeared as a suitable strain after this plate assay. Then, S. cerevisiae YPH501 and BY4741 were grown in galactose containing media to observe their growth profiles in galactose containing media since pYES2.1/V5-His-TOPO® vector harbors GAL1 promoter which is active only in the presence of galactose. Finally, BY4741 was selected as a suitable strain for transformation. In this study, three different vectors were constructed: i) TpBGL2 gene in pYES2.1/V5-His-TOPO® under the control of GAL1 promoter; ii) TpBGL2 gene in pYES2.1/V5-His-TOPO® under the control of GAL1 promoter and downstream the sequence; iii) TpBGL2 gene in pPIC9 under the control of AOX1 promoter and downstream the sequence. To construct them, first DNA extraction was carried out from T. phaffii. Next, DNA quality was checked by PCR amplifying with ITS1 and ITS4 universal primers. First, TpBGL2 gene was amplified using BGL2F and BGL2R primers from T. phaffii total DNA in order to ligate it directly to pYES2.1/V5-His-TOPO® vector to construct the first vector (TpBGL2 gene in pYES2.1/V5-His-TOPO® under the control of GAL1 promoter) for intracellular expression in S. cerevisiae BY4741. After ligation, vectors were transformed into Escherichia coli cells and vectors were extracted from E. coli cells through minipreparation. The resulting vectors were further analyzed to investigate both the presence and the orientation of TpBGL2 gene in the vectors by constructing restriction patterns with MlyI and XbaI enzymes as well as by performing sequence analysis. In addition, PCR amplification was carried out using GAL1F and BGL2R primers. All results confirmed that the insert was in frame with GAL1 promoter. To construct the second and the third vectors, first TpBGL2 was amplified from T. phaffii total DNA with FW2 and RV2 primers having EcoRI and NotI restriction sites, respectively. TpBGL2 gene flanked by EcoRI and NotI restriction sites was ligated into pGEM-T Easy Vector by TA cloning. After transformation and cloning in E. coli cells, extracted primers were digested by EcoRI to confirm the presence of the insert. Next, both empty pPIC9 vectors and pGEM-T Easy vectors having TpBGL2 gene flanked by EcoRI and NotI restriction sites were digested by EcoRI and NotI enzymes. Then, they were ligated to construct the third vector (TpBGL2 gene in pPIC9 under the control of AOX1 promoter and downstream the sequence). Both sequence analysis and PCR amplification with AOX1-BGL2R primer set confirmed that the insert was in frame with AOX1 promoter and sequence. To construct the second vector (TpBGL2 gene in pYES2.1/V5-His-TOPO® under the control of GAL1 promoter and downstream the sequence), TpBGL2 gene downstream the sequence was amplified using ALFA and BGL2R primers and the amplicon was ligated into pYES2.1/V5-His-TOPO® vector. However, sequence analysis showed that the insert was in wrong orientation and no further studies were carried out with this vector. pYES2.1/V5-His-TOPO® + TpBGL2 vector was transformed into S. cerevisiae BY4741 strain for intracellular production of Kpkt killer toxin and the presence of the insert was confirmed by PCR analysis in S. cerevisiae transformants. The selection was carried out on YNB selective medium without uracil plates. One of the six transformants called T1 was cultivated in 2% (w/v) galactose and 2% (w/v) raffinose containing buffered yeast minimal medium. After 24 h and 48 h, sampling was carried out for crude extraction and for taking supernatant from the culture. Next, well plate assay was performed to investigate killer activity on buffered YPD plates spread by sensitive S. cerevisiae DBVPG 6500 strain to Kpkt killer toxin. T1 was also cultivated in 0.2% (w/v) galactose and 2% (w/v) raffinose containing buffered yeast minimal medium for 48 h and it was transferred into 2% (w/v) galactose containing minimal medium. After 24 h incubation, well plate assay was performed again for both crude extract and the supernatant of the culture. In addition, T1 was cultivated in 2% sucrose (w/v) containing buffered selective minimal medium and it was transferred into 2% (w/v) galactose containing buffered selective medium after 48 h of growth. Well plate assay was performed again after 24 h of growth in galactose containing medium. All studies showed that Kpkt killer toxin did not perform its killing activity when it was expressed intracellularly. Finally, the third vector (PIC9 + TpBGL2) was transformed into P. pastoris GS115 cells for extracellular expression of Kpkt in P. pastoris. To do that, P. pastoris cells were prepared to obtain competent cells for transformation which was carried out through electroporation. For transformation, linearized plasmids obtained by digesting with either SacI or BglII enzyme were utilized along with linearized empty pPIC9 vectors with the same enzymes. Selection was carried on minimal dextrose plates. Next, the transformants were grown on both minimal dextrose and minimal methanol plates to determine putative Mut+ (Methanol utilization plus) or MutS (Methanol utilization slow) phenotypes of the transformants since only Mut+ phenotypes can grow on methanol containing medium if the insertion results in functional AOX1 gene encoding alcohol oxidase, which is the main enzyme in methanol utilization pathway. Finally, transformants were subjected to PCR amplification using AOX1 and AOXR primers to investigate whether they have inserts. The evaluation of the killer activity of Kpkt killer toxin in P. pastoris is in progress.
Evrimsel mühendislik yöntemi ile elde edilen kobalta dirençli Rhodobacter sphaeroides bakterisinin fizyolojik analizi
Rhodobacter sphaeroides is a gram negative purple non-sulfur α-proteobacterium found in soil, mud, deep lakes and stagnate waters. It has metabolic diversity, as it is able to grow under photoheterotrophic, photoautotrophic, chemoheterotrophic and chemoautotrophic conditions. It can alter its metabolism according to the changing environmental conditions and nutrient deficiency. Due to its metabolic versality, sequenced genome and no requirement of unusual conditions for growth, it is commonly used in biotechnological applications. R. sphaeroides is capable of synthesizing and storing biodegradable and biocompatibile polyester polyhydroxybutyrate (PHB) as insoluble inclusion bodies in cytoplasm. Some of the other byproducts of R.sphaeroides in industry are 5-aminolevulinic acid, Coenzyme Q10, carotenoids, vitamin B12, indole terpenoids and plant hormones. Large scale H2 production in a photobioreactor, ZnS nanoparticle production for medical purposes and potential usage in bioremediation are also reasons for biotechnological importance of R. sphaeroides. R. sphaeroides has the ability to survive under changing stress conditions by modifying its cellular and genetic composition. For instance, oxidative stress causes down-regulation of the carotenoid genes, while heavy metal stress increases membrane integrity. In another study, cobalt-resistant R. sphaeroides population was obtained under gradually increasing cobalt stress conditions, using evolutionary engineering approach. The last (64th) population was able to survive 15 mM cobalt stress, with a survival rate of 21%. The last population was spread onto solid Luria Broth plate and eight colonies (called G1-G8) were selected from there. In this study, the selected individual mutants were phenotypically characterized.
Donma-eri̇me stresi̇ne di̇rençli̇ Saccharomyces cerevisiae laboratuvar ve endüstri̇yel suşlarinin gen anlatim düzeyleri̇ndeki̇ farkliliklarinin qRT-PCR i̇le anali̇zi̇
The aim of this thesis study was to analyze gene expression level differences between freeze-tolerant laboratory and industrial strains of Saccharomyces cerevisiae, to gain insight into the molecular mechanisms of freeze tolerance in this yeast. For this purpose, S. cerevisiae laboratory strain '905', industrial strain 'R625' and their freeze-thaw resistant mutant individuals 'F1' and 'P8' were used. Freeze-thaw stress-related genes were chosen according to whole-genome transcriptomic analysis results of 905 and F1, which have been obtained previously. S.cerevisiae is a small, unicellular fungus that has been used as a model eukaryotic organism in biological research for a long time. Also, it has been commonly used in industrial processes since ancient times. The ability to ferment sugars makes the budding yeast is commercially important in bread-making and wine industries. The organism is also being used in bioethanol and fuel ethanol production, recently. S. cerevisiae is also named as baker's yeast because of its ability to leaven dough. It is also known that the yeast cells are exposed to freeze-thaw stress during long term storage conditions. Thus, cryopreservation process is a stressful condition for yeast cells. The freeze-thaw resistant mutants F1 and P8 were obtained in a previous study by an inverse metabolic engineering approach, based on random mutation and selection under freeze-thaw stress conditions. Whole-genome transcriptomic analyses of 905 and F1 were also done previously. To verify the transcriptomic analysis results obtained by DNA microarray technique, gene expression levels of selected genes were determined by Quantitative RT- PCR. The genes were selected from microarray data according to their high up- or downregulation levels. Highy upregulated genes HSP12, FMP45, HSP26, HXK1 and highly downregulated genes PHO84, NSR1, ZRT1 were chosen for gene expression analysis by qRT-PCR. The qRT-PCR experiments were performed under control and stress conditions for '905', 'F1' 'R625' and 'P8' strains. The expression profile of 'F1' was normalized to that of the wild type laboratory strain '905', and the expression profile of 'P8' was normalized to that of the wild type industrial strain 'R625'. In conclusion, it was found that freeze-thaw stress causes significant changes in gene expression patterns of both laboratory and industrial strains. In addition, some differences were also found between gene expression level results of microarray and qRT-PCR analyses. This indicates the importance of verifying DNA microarray results by qRT-PCR, as qRT-PCR is a more sensitive method.
Evrimsel mühendislik yöntemi ile Rhodobacter sphaeroides'in CoCl 2 stresine dirençli suşlarının eldesi
Rhodobacter sphaeroides is a metabolically diverse organism which is able to fix molecular nitrogen and produce H 2 as side product. It can grow under both oxic and anoxic conditions heterotrophically or photosynthetically. This wide repertoire of metabolism puts this organism in the centre of metabolic regulation and bioremediation studies. R. sphaeroides is used in the production of vitamin B 12 , coenzyme Q10, 5-aminolevulinic acid (ALA), porphyrin, as well as in bioremediation of heavy metals. R. sphaeroides follows different metabolic strategies when exposed to different stress factors. It is able to regulate its photosynthetic machinery sensing O 2 tensions. When partial O 2 pressure is reduced, R. sphaeroides develops invagination in its cellular membrane. Biosynthesis of chromatophores, which are crucial for photosynthesis, takes place in these invaginations. There are three systems involved in regulation of photosynthetic genes: the PrrBA two subunit activation system, the PpsR repressor and the FnrL, anaerobic regulator. The genes are regulated mainly by O 2 levels. R. sphaeroides has two chromosomes and five naturally occurring plasmids. Many of its genes are duplicated between the two chromosomes. These gene duplications generate the genetic diversity which allows the bacteria widen its spectrum of metabolic capabilities. Chromosome I contains 3106 open reading frames whereas chromosome 2 contains 874. It possesses a flagellum similar to that of E. coli which is able to rotate only in clockwise direction. When exposed to heavy metal stress, R. sphaeroides alters fatty acid composition to increase its membrane integrity. The accumulation of heavy metals in the environments imposes new strategies for restoring the original site conditions. Bioremediation, providing high specificity and versatility has received a great attention in this area. In this study, R. sphaeroides was used to obtain a cobalt-resistant strain. Evolutionary engineering approach of metabolic engineering was used to increase the cobalt tolerance of the bacterium, by growing it under gradually increasing stress conditions. This approach being a 'bottom-up' approach does not require extensive information about the metabolism of the organism. The R. sphaeroides strain R-26 was used in this study. Initial cobalt concentration to be used for selection was determined after screening analysis of the reference strain at 11 different cobalt concentrations. According to the screening results, the selection of the populations was started with 0.1 mM cobalt concentration. Cobalt concentrations were increased gradually from 0.1 mM to 15 mM through 64 generations. Survival rates were monitored every 24 hours by measuring OD 535 values of control and stress cultures. At the same time points, cultures were spread on LB plates to confirm that no contamination has occurred. Cultures which were grown on solid medium were examined morphologically. Cultures that produced carotenoids were identified by their characteristic red colour. When the survival rate decreased to 0.2, the selection process was terminated for further analysis. Growth behaviour of the reference strain and the last population obtained by evolutionary engineering were xixcompared in media with and without cobalt. The cultures showed similar growth characteristics when there was no cobalt in the medium. However, there was a significant difference, when cultures were grown in a medium containing 4 mM cobalt. The 64 th population cells were able to maintain their growth rate nearly the same when exposed to 4 mM cobalt, while the reference strain's growth was inhibited. These results indicate the improved resistance of 64 th population towards cobalt.
Nano-ölcek metal yüzeylerde moleküler tanıma esaslı kendiliğinden montaj olabilen protein tasarımı
In recent years, a wealth of novel, nature-inspired materials have been explored across a wide range application in medicine and technology. Hierarchical organization is one of the key components that are reflected in the diverse structure and function in biological materials. In mimicking these functions in our engineering approaches have been challenging due to the complexity of these organization. Biological self-assembly principle based upon precise molecularscale recognition is the key in Nature`s design. Biological machinery facilitates self-organization and self-regulation that we observe in biological materials. Proteins are perceived as crucial interaction components in this machinery owing to their versatile characteristics. The protein assemblies may be the key in making next generation materials mimicking Nature`s design. Peptides as smaller domains of proteins become crucial components in constructing new types of materials having the versatility that we observe in Nature. Inorganic material specific peptides have attracted increasing interest in the recent years as novel bio-surface functionalization agents. Such functionalization can recruit biological cues to the material's surface and promote the otherwise challenging coupling of distinct materials. Their modularity also offers a unique opportunity to link them to numerous other functional proteins. Herein, we provide the details for their design and recombinant incorporation into functional proteins and enzymes to achieve addressable self-organization. In the scope of this dissertation, we first constructed an AuBP1 peptide genetically fused to the C-terminus of maltose-binding protein (MBP) using two different linkers to produce MBP-AuBP1 hetero-functional constructs in order to accomplish the biological self-assembly on gold surfaces. Using various spectroscopic techniques, surface plasmon resonance (SPR), and localized surface plasmon resonance (LSPR), we verified the binding of MBP-AuBP1 fusion protein on gold substrates. The AuBP1 peptide tag were demonstrated to direct the organization of recombinant MBP protein onto various gold surfaces via efficient control of the organic–inorganic interface at the molecular level. Furthermore, using a combination of soft-lithography and self-assembly techniques, AuBP1 peptide tag controlled the formation of protein assemblies onto gold nanoparticle arrays with high molecular packing density and patterning efficiency following a series of simple, reproducible steps. This model system offers layer-by-layer assembly capability upon specific AuBP1. In our second design, we developed multifunctional monitoring molecule through genetic fusion of gold binding peptide (AuBP2) to a red fluorescence protein, DsRed-Monomer. AuBP2 was utilized as a material specific linker to construct novel bio-imaging, drug delivery, and targeting bio-nano hybrid systems. The AuBP2c peptide tag was enabled the self-immobilization of the DsRed protein onto a variety of nanoscale gold surfaces. Functional DsRed monomer generated by protein engineering methods using AuBP2 allowed variety of properties for biological sensing as well as analytical applications. The red emission of DsRed protein offered new opportunities at the nano- to micro-scale of a material surface such as multicolor labeling and fluorescence resonance energy transfer (FRET) applications. Our designed fusion protein with a fluorescent tag provided quick and accurate visualization of the self-immobilized proteins on the gold surface. Additionally, DsRed constructs have a selective and reversible binding affinity to copper. This unique binding property of DsRed protein to copper ions and gold nanoparticles results in tunable quenching of its fluorescence activity. Given the importance of gold nanoparticles and copper ions in several application areas, DsRed-AuBP2 was proposed as a bio-sensing modality through its bi-functional properties, fluorescence and self-assembly. We also tested the efficiency of the DsRed-AuBP2 protein as an integrated component in nanofiber system. The fibers were formed in the presence of the protein provided the integration of gold nanoparticles along the fiber length through Au-binding peptide tag. These nanofibers that feature gold nanoparticles with proteins did not affect the red fluorescence property of the protein, rather protein allowed the visualization of the protein construct along the fibers. Engineering proteins that can decorate gold nanoparticles well integrated into PEO polymer fibers offers several opportunities to create versatile structural templates for metallization in formation of conducting nanofibers. Our final set of design included a fusion enzyme that is designed to have gold binding activity. Formate dehydrogenase, FDH, was fused to an AuBP2 fusion tag to construct FDH-AuBP2. This fusion enzyme was utilized to develop a circuit-based electrode system that provided verification of the redox catalytic ability of the self-immobilized enzyme on gold electrode by subsequent addition of formate. We achieved this validation following the self-immobilization of FDH-AuBP2 onto a gold electrode surface and monitored the conversion of formate to CO2 electrochemically. The circuit-based system we constructed consisted of two gold electrodes submerged in a buffer solution. Prolonged catalytic activity of the enzyme was observed by subsequent addition of format into solution. The engineered multi-functional biomolecular constructs developed were demonstrated to self-organize, forming functional hybrid hierarchical entities. However, no all-encompassing solution exists to control the orientation of the enzymes on surfaces using inorganic binding peptide tags. Each peptide and enzyme system requires detailed evaluation following genetic conjugation to investigate the performance of the chimeric functions. Our results demonstrate the engineered inorganic binding peptides as a promising platform technology by their role in producing bi-hybrid nanostructured systems including fluorescent biosensors for metals, bioelectrodes for biobatteries, and nanofibers for conductive nanomaterial. Based upon an in-depth investigation of the limited research currently published in this growing field, we have developed a strategy for the design of new protein constructs as well as their over-expression as recombinant products. Moreover, we provide a valuable approach to generate bioactive nanostructures for the engineering protein complexes by co-expression, purification, and characterization, coupled with bionanotechnological applications. Our engineering approach to conjugate proteins with inorganic binding peptides to create nanodevices propels this technology forward, one step closer to becoming a reality. Our fabrication process is based upon a bio-enabled self-assembly technique and is, herein, proven capable for engineering multilayered protein and nanometallic assemblies that utilize modular goldbinding peptides, such as AuBP1 and AuBP2. Overall, the reported findings in this dissertation demonstrate a great potential to design novel nanostructured materials that incorporate the engineered proteins that are designed in the scope of this thesis. Our multi-functional engineered proteins display high binding affinity and specificity to various gold surfaces at the nano- to micro scale under ambient conditions. These novel platforms can be used for a variety of purposes such as preparing protein micro/nano-arrays, biosensors, biobatteries, and reagents for molecular imaging and targeting. Our established model constitutes biological routes for biofabrication of various protein arrays, plasmon-active nanometallic assemblies, and devices that feature controlled organization, packing density, and architecture.
Schizochytrium sp. mikroalginden biyokütle üretimi ve üretilen biyokütlenin içeriğinin tayini
Biotechnology has been working for the goods of livings in order to develop procceses and products for years. Human well-being takes the first place through these studies especially the production of essential metabolic molecules. Omega fatty acids are one of those significant molecules and getting more attraction at last decades. The process of obtaining these fatty acids from microalgae instead of cold marine fishes is a populer topic as well. There are many species metabolizing these omega fatty acids for example Crypthecodinium cohnii, Schizochytrium sp and Nannochloropsis sp. Schizochytrium sp. was known to belong fimgi family but then substituted to under Thraustochytrids family as heterekont algae with the help of modern genetic and molecular methods. This alga produce high amount of DHA, which is an essential fatty acid for our metabolism. The current source of this polyunsaturated fatty acid is cold marine fishes but this system has many deficiencies like not being sustainable and environmentally friendly. In this study, all the experiments were conducted to produce a high amount of Schizochytrium sp. microalgae biomass and so to obtain fatty acids within this one cell organism. The aim of the study was to develop a sustainable, environmentally friendly, scalable and non-toxic process in order to growth Schizochytrium sp. to obtain omega fatty acids. There are heterotrophic and phototrophic algae metabolising high amount of lipids, proteins and carbohydrates. Phototrophic algae production was populer at the beginings because the sun energy and salty water were free sources in the production processes. But then it was realised that the amount of biomass and lipid content was very low. Also this system dependent to sun and other environmental factors. This means that system has negative points as the fish based systems. Therefore, the heterotrophic methods are designed and many microalgae species are isolated which are able to grow heterotrophically and can produce high amounts of lipids, proteins and carbohydrates. In this study Schizochytrium sp was acquired from ATCC and then stock cultures were prepared, one for seed stock and the others for working stocks. Two-stage growth method was used, firstly the main stock was inoculated to a complex medium for 48 hours under 25o C and then those cells were inoculated to defined mediums. Lastly, these cells were incubated for 48 hours and then the resulting content was centrifuged to obtain the biomass produced. This biomass was dryed then the resulting biomass is weighted. Then this biomass was smashed and subjected to hexane extraction in order to obtain the oil. Additionally, nile red staining also processed to quantify the total oil. Results showed that a large-scale production method could be implemented with fermenter conditions to produce high amount of biomass and lipids.
Ailesel behçet sendromu ailelerinde hla-b5 geninin genetik ve epigenetik analizleri
Behçet's Syndrome (BS) is a chronic multisystemic inflammatory disorder, primary symptoms of which are oral and genital ulceration and uveitis. The disease is also chracterized by inflammation in tissues and organs throughout the body such as vessels, lungs, kidneys, joints, gastrointestinal and central nervous system. BS usually occurs in second and third decades of the life with a severe disease course. It exists most commonly in Mediteranean and Middle East populations. In Turkey, frequency of the disease changes between 20 and 420 of 100.000. BS risk factors include genetic predisposition which is mainly based on familial aggregation and increased HLA-B51 carrier rate as well as infectious agents, environmental causes and yet undefined immunological mechanisms. Even though HLA-B51 allele has shown to be the most associated marker for BS, the presence of healthy individuals who carry HLA-B51 allele and similarly BS patients without HLA-B51 allele have led us question other mechanisms for BS causation. Epigenetic mechanisms have gained exceeding importance for their effects on DNA and chromatin structures without altering DNA sequence. DNA methylation is one of the mostly studied epigenetic mechanisms. HLA-B gene has shown to comprise a CpG island (CpGi) which span 1346 nucletides where CG ratio is 66.6 %, therefore methylation of cytosine residues may lead to a change in gene expression levels. We previously investigated global methylation and HLA-B locus specific methylation in a group of 4 MZ BS twins and 4 DZ BS twins; which suggested an involvement of an epigenetic regulation in HLA-B exon 1-2 region. BS twins had a higher methylation levels for HLA-B gene (p=0.0024). In this thesis we wanted to further our preliminary findings in familial BS cases and more directly examine the influence of HLA-B51 carrier rate and its methylation upon the presence of the clinical phenotype. Therefore, involvement of genetic and epigenetic roles of HLA-B51 region in BS would be better analysed and the sample size would be increased by including familial BS cases with their affected and unaffected relatives. 100 BS patients were contacted from Cerrahpaşa Medical Faculty, Rheumatology Polyclinic between 2013 and 2015 and asked if they have an affected relative. Within those 1800 index patients, 150 patients had a family member with BS. Among those, 15 families accepted to enroll in the study. So the study consists of 15 index patients; 17 affected relatives and 26 unaffected relatives. Peripheral blood samples were collected and genomic DNA was isolated from leukocytes. Patients and relatives were genotyped for HLA-B51 alleles using sequence specific PCR method. HLA-B51 positivity ratio was found to be 12/15 for index patients, 13/17 for affected relatives , 22/26 for unaffected relatives. Among BS and healthy family members, HLA-B51 frequency did not show a statistically significant diffrence. For healthy controls, who are not related with families, HLA-B51 positivity was 8/25. However, HLA-B51 positivity for BS patients was statistically higher when compared to healthy controls (p=0.0005). After determining HLA-B51 positivity, methylation profiles on HLA-B gene were analyzed and compared between groups using Real-time PCR based OneStep qMethyl Kit. For exon-1 and exon-2 region of HLA-B gene, BS patients had statistically higher methylation levels compared with healthy individuals in the family (p=0.0065). Methylation levels were not significantly different among HLA-B51(+) and HLA-B51(-) individuals for index patients, relatives with BS and healthy relatives. Observed results were also correlated with the data from our previous studies. These findings suggested us a room for epigenetic modifications which might be independent from HLA-B51 positivity.
Interaction between transcription factor p53 tumor suppressor and p60-katanin (KATNA1) promoter
Cytoskeleton is a network of filamentous structures which are spread throughout the cytoplasm. Cytoskeleton play roles in vital functions such as movement of the cells, cell shape, mitosis, cytokinesis, intracellular transport of molecules. Cytoskeleton consists of three distinct polymeric fibers. Microfilaments are required for maintenance of cell shape, cytokinesis, separation of the dividing cell into two and cell surface preparation for adherence. Other component of cytoskeleton, intermediate filaments provide mechanical strenght and mechanical linkage. The final member of cytoskeleton is microtubules that have crucial roles such as drawing the chromosomes apart in mitosis, shaping morphological structure, intracellular traffic to carry vesicules, cell motility, formation of axons and dentrites and therefore, the neuronal structure. Microtubules are structural polymer proteins and consist of α-tubulin and β-tubulin heterodimers. Microtubules are dynamic structures resulting from rapid polymerization and depolymerization of tubulin monomers by a process known as "dynamic instability". Reconfiguration of the microtubules by these mechanisms is responsible for microtubule growing and shrinking. When dynamic instability, speed and time for branching for special neuronal morphology are considered, it can be easily understood that cells need another mechanism to explain the movement of intracellular microtubules. "Cut and run" model proposes that microtubules are cut into small pieces by severing enzymes; katanin, spastin and fidgetin. These enzymes are members of AAA family of ATPases. Because microtubules lose their ability to move when they are long, microtubule severing is important in terms of movement capacity. Katanin and spastin have same working mechanism, but, they provide different severing approach. Spastin encoded by SPG4 gene. Katanin is a heterodimer of p60-katanin encoded by KATNA1 gene and p80-katanin encoded by KATNB1 gene. p60-katanin has the enzymatic activity and it has AAA ATPases region but p80-katanin enzyme do not have the enzymatic activiy. According to studies, katanin is associated with neurologic diseases. Katanin is ubiqutiously expressed in nervous system and its inhibition or overexpression impairs axon formation. Thus, it is clearly understood that katanin has vital role for microtubule reorganisation and axon growing. Hypophosphorylated form of tau protein normally provides stabiliziation of microtubules and protects microtubules from the severing enzymes. Hyperphosphorylation of tau results in dissociation of tau from microtubules and thus, microtubules become accesible for severin enzyme katanin. The enzymatic activity of katanin is proportional to level of enzyme so regulation of katanin protein level via transcription mechanism comes into prominence. p53 is important for many cellular mechanisms as a tumor supressor in processes such as cell cycle regulation, metabolism, apoptosis, DNA replication, immunity responses, proliferation and differentiation. In our previous study, we have shown that neuronal processes were retracted upon PKC activation following increases in both p60-katanin and p53 levels in neurons. This result led us to analyze changes in p60-katanin, which is organizing the neuronal cytoskeleton. We showed that cells which have enhance p60-katanin protein level also had increase in p53 transcription factor, which is related on neuronal differentiation. In this study, we aimed to identify regulatory DNA sequences of KATNA1 gene and possible regulation of p60-katanin by p53 trasnscription factor. For this purpose, we first decided to characterize regulatory regions of KATNA1 gene. Primarly, the putative transciptional regulatory regions of p60-katanin were identified by bioinformatic methods. p60-katanin regions (336 bp promoter, 448 bp 5'UTR, 784 bp promoter + UTR, 2682 bp intron + UTR and 3000 bp promoter + UTR + intron) were cloned for the identification of their regulatory activities on p60-katanin expression by Luciferase assay. Then, p60-katanin promoter, p60-katanin UTR, p60-katanin promoter + UTR and p60-katanin promoter + p60-katanin UTR + p60-katanin intron regions were amplified by PCR and after required restriction all regions prepared. We identified that 5'-UTR region enhanced transcriptional activity of the reporter gene, but not putative promoter region and the presence of the intron decreased the activity. Thus, expression of katanin-p60 seems to be regulated via 5'-UTR, and intron-1 might have repressor elements. When potential gene regulatory regions were analyzed by bioinformatics software, presence of a CpG island that comprising promoter and 5'-UTR regions (from -615 to -918 bp) was identified. Next, we identified p53 consensus sequence on KATNA1 gene bioinformatically. Therefore, it is thought that KATNA1 gene could possibly be regulated by p53 transcription factor. We started with analyzing the binding of p53 to the corresponding promoter region. To further confirm the specificity of the p53 binding to KATNA1 promoter was confirmed by Chromosome Immunoprecipitation (ChIP) and also by Electrophoretic Mobility Shift Assay (EMSA) using oligonucleotides including related transcription factor binding sites. On the other side, our observation indicated that, p53 acts as an activator or repressor KATNA1 gene promoter. The result for KATNA1 gene was confirmed in mRNA level by performing real time PCR.
Ailesel Akdeniz ateşi, gut ve erişkin still hastalarında MEFV mutasyon analizi
The "autoinflammatory" term is first defined and developed in 1999 by a group of researcher who were studying hereditary periodic fever syndromes. The main cause of the syndromes is mutations/variations in genes involved in innate immunity and their alteration of the signaling or cytokine activation pathways. Familial Mediterranean fever (FMF, OMIM ID: 249100 and 134610) is one of the most common and best characterized inflammatory diseases that is inherited autosomal recessively and characterized by recurrent attacks of fever, abdominal pain, rashes, and arthritis while amyloidosis is the most severe complication of the autoinflammatory disorder. Gout is common inflammatory arthritis caused by hyperuricemia and the deposition of uric acid crystals in tissues and fluids within the body which results with metabolic, cardiovascular and renal morbidity especially in men over age of 40. Adult-Onset Still's Disease (AOSD) is a rare inflammatory disorder of unknown etiology and characterized by daily spiking fever, arthritis and rash. MEFV, MEditerranean FeVer gene is the first autoinflammatory gene that is introduced as a candidate gene for FMF. To date, 305 sequence variations are identified on MEFV gene. Still new mutations/variations and their association with FMF are understudied. Mutations/variations are located in exons 1, 2, 3, 5, 9 and 10 of MEFV. The majority of the mutations/variations are missense changes and clustering in exons 2 and 10. There are five common mutations/variations; V726A, M694V, M694I, M680I in exons 10 and E148Q in exon 2. MEFV transcripts encode a 781 amino acids protein called Pyrin or Marenostrin (P/M). The main function of P/M protein is the regulation of caspase-1 activation, relatively interleukin-1β (IL-1β) production. The association between FMF and MEFV gene mutations/variations is well established in the previous studies, still the existence of patients without mutations/variations and P/M role were not clarified. The aim of this study was to examine the specificity of MEFV gene to FMF or to general inflammatory pathway by comparing with other autoinflammatory diseases. MEFV exon 2 and 10 variations were explored in a group of FMF (N=75), Gout (N=30) and AOSD (N=28) patients and compared the frequencies between disease groups and healthy controls (N=54). MEFV gene product P/M is indicated to have a role in inflammatory pathway. The aim of the protein studies was the quantification of P/M levels in FMF (N=22) patients compared to healthy controls (N=9). DNA and protein samples were obtained from whole blood. We first compared all mutations/variations between the patient and HC groups. MEFV gene mutations/variations were detected in 73 (97.3%) patients in FMF group, 21 (75%) in AOSD, 20 (66.6%) in Gout and 35 (64.8%) in HC group. MEFV mutations/variations were detected in 16.2% of alleles in FMF, 6.8% in Gout, 5.4% in AOSD patients and 7% in HC. The difference in the frequency of the MEFV mutations/variations between the patient groups and control group was statistically significant (p=<0.0001). The common haplotype A165A-D102D-G138G was observed frequently in all groups, but the frequency was higher in FMF group (61.8%) compared to Gout (30%), AOSD (22.6%) and HC (33.6%) (non-significant). One of the most common variations E148Q in exon 2 was observed at a frequency quite similarly between groups. The frequencies were 5.3%, 5.0%, 7.1%, 5.6% in FMF, AOSD, Gout patients and healthy controls, respectively (non-significant). In exon 2, only R202Q variation was found to be more frequent in FMF patients (42%) compared to other groups (19-25%) (FH: p<0.0001, FG: p=0.0266, FS: p=0.0032). Out of 16.2% of all observed mutations/variations in FMF, R202Q was observed in 2.2%, whereas E148Q in 0.3%. There was also significant difference in pathogenic mutations/variations between FMF and other groups. Out of 5.2% of all alleles, they were observed 3.5% in FMF, whereas 0,5-0,6% in other groups. Within these pathogenic mutations/variations, out of 19.7%, only E148Q (4.6%) and M694V (11.2%) were observed in all groups. The most prominent pathogenic mutation/variation was M694V. It was observed 8.9% in FMF, 1.3% in Gout, 0.4% in AOSD and 0.6% in healthy controls as being the most frequent mutations/variations (p=<0.0001). As a further aspect, increased M694V frequency in Gout and AOSD patients compared to HC requires further analysis. Second part of this study, we wanted to investigate the quantification of P/M levels in FMF (N=22) patients compared to healthy controls (N=9). Our preliminary results indicate approximately two fold decrease of P/M levels in FMF patients compared to HC group (p=0.3920), which is the opposite of the previous results. To investigate the relationship between mutations/variations and P/M levels, MEFV mutation/variation analysis were also performed in protein study group. The allele frequencies of M694V in FMF and HC were 34.1% and 5.6%, respectively (p=0.0247). E148Q, R202Q and R761H were also observed in both groups, however NS. The average P/M quantity of the patients carrying M694V mutation/variation was 0.23, whereas non-carriers had 0.33. One of the FMF patients was in her/his attack period when the sample was collected and the P/M level was highest even though he/she was not M694V mutation/variation carrier. We have observed much higher P/M level and total mutation/variation carrying allele in attack patient compared to attack free patients. The protein study requires optimization with enlarged sample sizes and replicated for more accurate statistical results.
İstiridye mantarı: Pleurotus ostreatus kullanılarak mikoremediasyon yöntemi ile topraktan ağır metal giderimi
According to fossil records, fungi diverged from other life around 1.5 billion years ago and probably fungi colonised on Earth during the Cambrien, which is long before human beings evolved on it. The term "mycology" is derived from Greek word "mykes", meaning mushroom which is a branch of life science that refers the study of fungi. In general, mushrooms are responsible for decomposing of organic molecules to provide continuance of life by recycling organic wastes and returning of nutrients back into the ecosystem. These features of fungi reveal a term called as "mycoremediation". Increasing human population and supporting unconscious consumerism by a politics of capitalist world economy caused the pollution of the environment with synthetic compounds which has become a major problem all around the world. These synthetic compounds are called as xenobiotics which do not occur naturally in the biosphere so are not easily degraded by the natural microflora and fauna. Biological approaches based on the environmental biotechnology are focusing on the development of "environmentalist technologies". Further, these clean technologies focus on the use of metabolic pathways of organisms for the remediation of waste. One such biological method is mycoremediation (fungal remediation). The mushrooms and other fungi act as enzymatic machinery for degradation of a wide variety of waste/pollutant. However mushrooms, basidiomycetous fungi, are becoming more popular nowadays for remediation purposes as a bioremediation tool. The white rot fungi in all the fungi species, are known to degrade polyaromatic hydrocarbons (PAHs), chlorinated aromatic hydrocarbons (CAHs), polycyclic aromatics, polychlorinated biphenyls, polychlorinated dibenzo(p)dioxins, the pesticides and some azo dyes. White-rot fungi are also used in bioremediation of polluted soils and accumulation of heavy metals and involved in mineralization, biodeterioration, biodegradation, transformation and co-metabolism. Phanerochaete chrysosporium, Pleurotus ostreatus, Pleurotus pulmonarius, Pleurotus tuber-regium, Lentinus squarrosulus etc. are white-rot fungi, so far used in bioremediation researches. Heavy metals are natural constituents of the earth crust. Their non- biodegradable, non-thermodegradable features contribute to environmental pollution mainly and show toxic effects on organisms. The most common heavy metals found at contaminated sites, in order of abundance are Pb, Cr, As, Zn, Cd, Cu and Hg. Those metals are important since they are capable of decreasing crop production due to the risk of bioaccumulation and biomagnification in the food chain. There is also the risk of superficial and groundwater contamination. Cadmium, mercury and lead can describe as three big poisinous metals which are not known essential role for living organisms. Cadmiums chemical similarity to Zn which is an essential micronutrient for plants and animals, probably responsible of its toxicity. Mercury, Hg, is the only liquid metal at standard temperature, and major source of Hg contamination is caused by coal combustion. Mostly Pb contamination in nature arises during the improper disposal of Pb storage batteries. The aim of this study is to remediate contaminated soils from three most toxic heavy metals using Pleurotus ostreatus mycelium. Soil and mycelium heavy metal analysis were performed by Bureau Veritas Mineral Laboratories, in Canada. Soil Pb and Cd analyses were performed using ICP-MS analysis. In this study, bioaccumulation percentage of was studied with three different concentration of Pb, Cd and Hg for two different incubation period. The results showed that bioaccumulation percentage increased by incubation period for lead, cadmium and mercury. While SET I mycelium analyses showed that increasing concentration of lead caused a decrease for its bioaccumulation, in SET II bioaccumulated concentration of Pb did not showed a significant difference for all concentrations. According to bioaccumulation percentage calculations for Pb, it showed a decrease in order of 2.5 ppm, 5 ppm and 10 ppm. Cadmium showed the lowest bioaccumulation percentage in Content 2, comparison to Content 1 and Content 3, both SET I and SET II. Heavy metal analyses of mycelium samples showed that the most bioaccumulated heavy metal by Pleurotus ostreatus was mercury. The mycelium samples belonged to 10 ppm mercury contained soil samples showed 54 % Hg bioaccumulation and 85 %, in SET I and SET II, respectively. The heavy metals which were used in this experiment in order of bioaccumulation percentages were Hg > Cd> Pb.
Ailesel multipl skleroz'da bağlantı analizi ve genom çapı ilişkilendirme çalışması
Multiple Sclerosis (MS) is an immune-mediated, neuroinflammatory and neurodegenerative disorder affecting the central nervous system (CNS), and characterised by multifocal lesions in white and grey matter with demyelination, axonal transection, neuronal degeneration, gliosis, and perivenular inflammatory cell infiltrates. MS is a complex disease, which develops in genetically susceptible individuals under specific environmental influences. Early observations from classical genetic studies have shown that MS has a genetic background with a broad range of heritability estimates (25%-76%) reported by different studies. Early linkage analyses revealed a strong association of HLA-DRB1 locus of the class II human leukocyte antigen (HLA) region with MS. Subsequent linkage and candidate-gene based analyses have identified different HLA allele associations and a non-HLA association, interleukin 7 receptor alpha (IL7RA) gene. Further chip-based genome wide association studies (GWAS) have identified a total of 110 non-HLA associations, most of which are related to immune pathways, supporting the immune basis of MS. However, current knowledge on MS genetics can explain only about 27% of the predicted MS heritability, leaving much to be explored. We have previously conducted a proteome study in our MS cohort that have been collected in Istanbul University, Cerrahpaşa Faculty of Medical, Neurology Department since 2007. The proteome study identified pathological pathways in MS including renin-angiotensin, aldestorene-regulated sodium reabsorption, complement-coagulation and notch signalling patways with potential biomarkers. In the current study, to our knowledge for the first time, we wanted to correlate genomic data from familial MS pedigrees and unrelated patient/control groups with the proteome data. To this end, first we conducted a linkage analysis in MS patients and their affected and unaffected relatives. 10 multiplex MS families with 35 individuals were included in the analysis and SNP genotyping on the Illumina CytoSNP 300K array was performed for genomes of each individual. NPL scores were calculated for each of 3118 informative SNP markers spaced at an average of 1 cM intervals using SimWalk multipoint NPL analysis. Fine mapping of regions showing NPL scores higher than 1.7 was performed for each SNP markers spaced at every 0.2 cM, revealing that the most promising loci for linkage were mapped to 13q13.3 and 21q22.2, with NPL scores of 1.82 and 1.85, respectively. From the resulted loci, Interferon (Alpha, Beta, and Omega) Receptor 1 (IFNAR1) 18417, Interferon (Alpha, Beta, and Omega) Receptor 2 IFNAR2 11876 polymorphisms, and Mab-21- Like 1 (MAB21L1) CAG repeat number were selected as candidate genes for further analyses. Selected regions were amplified by polymerase chain reaction (PCR) and genotyped in 27 unrelated patients with MS and 10 healthy controls of Turkish origin. Statistical analyses were performed to calculate genotype and allele frequencies, revealing a significant association of IFNAR2 11876 GG genotype with increased risk of MS (P = 0.027, OR 3.64 [95% CI 1.09 – 12.1]). We further conducted a GWAS comprising of 11 unrelated MS cases that had been included in the proteomic analyses and in the linkage study, and 60 healthy controls of Turkish origin, revealing 14 SNPs with significant association (P < 10-4), and 106 SNPs showing suggestive association with MS (P < 10-3). Subsequently, chromosomal regions from the linkage analysis and SNPs from the GWAS were analysed in order to observe a correlation with the previous proteomic findings. One gene with significant (INS-IGF2, P = 4.39E-07), and eight genes with suggestive associations (PRKCE, MAPK9, RBPJL, ADAMTSL1, NR6A1, NOTCH2, IL1R1, NTN1) from the GWAS were found to involve in pathways those shown to be affected in MS subtypes, and there were three genes common between the GWAS and linkage results (CLDN14, RUNX1, LINC00598). When individual proteome data of each patient involved in the genetic analyses was observed, a total of 20 proteins having altered expression level in one or more patients were also found to have significant or suggestive association in the GWAS. Among them, CNTN5 had the only significantly associated SNP markers (P = 4.71E-05 and P = 7.79E-05). Using a multi-disciplinary approach that combined genetic, proteomic, and bioinformatic analyses, we identified several candidate genes, whose possible roles will be explored in our further studies.
Bacillus subtilis GntR ailesine ait LutR transkripsiyon faktörünün doğrudan kontrolü altındaki genlerin CHIP ve EMSA yöntemleriyle belirlenmesi
LutR transkripsiyon faktörü GntR familyasının bir üyesi olup B. subtilis'te lutR geni tarafından kodlanan global düzenleyici bir proteindir. lutR geni ilk keşfedildiğinde "yvfI" olarak adlandırılmış fakat laktat kullanımında görevli olduğu belirlendiğinde "lutR" (Lactate utilisation gene) adıyla tekrar adlandırılmıştır. B. subtilis LutR proteini, E. coli FadR regulatör proteini ile homologdur. LutR protein dizisi, GntR süperfamilyasının FadR alt familyasındaki farklı türlere ait ortologlarıyla da önemli homolojiye sahiptir. Ayrıca FadR benzeri proteinler birçok metabolik yolun regülasyonunda rol alırlar. GntR familyası DNA'ya bağlanmak için Heliks-Turn-Heliks (HTH) motifi kullanan regulator proteinlerden oluşan bir HTH-tip protein süperfamilyasıdır. Farklı çalışmalardaki CDD analizlerinde, GntR familyasında oldukça yaygın olan "FadR C-terminal ligand bağlanma bölgesi"nin, LutR C-terminalinde de yer aldığı tespit edilmiştir (PFAM 07729). Buna ek olarak LutR N-terminalinde bulunan 44 amino asitlik bölge ile GntR wHTH domain arasında önemli bir benzerlik olduğu belirlenmiştir (PF00392). GntR familyasına ait üyeler N-terminalde HTH motifi içeren bir DNA bağlanma domaini (DBD) ve C-terminalinde bir efektör bağlanma veya oligomerizasyon (EBD) domaininden oluşurlar. Bu proteinler DNA'ya bağlanmak için kullandıkları heliks-turn-heliks (HTH) motifi ile karakterize edilirler. DNA-bağlanma domaininin dışındaki bölgeler çok farklılık gösterirken, küçük bir β-tabaka ile beraber 3-heliksli bir çekirdekten oluşan DNA-bağlanma bölgesi bütün GntR familyasında oldukça iyi korunmuştur. Bacillus genusu üyeleri arasında antibiyotik üreten ana suş B. subtilis'tir. Yaklaşık 30 kadar antibiyotik üreten B. subtilis'e ait antibiyotikler oldukça farklı antimikrobiyal aktivite göstermektedir. Bunlardan bazıları gram pozitif, bazıları gram negatif mikroorganizmalara etki ederken, bazıları daha geniş spektrumda etkilidir. B. subtilis genomunda yaklaşık 350 kb'lık bir bölge antibiyotikleri kodlayan genleri içermekte olup, toplam genomun %4-5'ini oluşturmaktadır. Yapısal olarak oldukça değişken olan B. subtilis antibiyotiklerinin en önemli özelliklerinden biri fonksiyonel açıdan sadece antimikrobiyal ajan olarak görev yapmayıp, ayrıca hücre morfolojisi ve fizyolojisinin düzenlenmesinde de etkili olmalarıdır. Basilisin [L-alanine-(2.3-epoxycyclohexanone-4)-L-alanine] non-ribozomal yolla sentezlenen dipeptit yapıda bir antibiyotiktir. B. subtilis'te basilisin biyosentezinden polisistronik bir operon olan bacABCDEF (eski adı ywfBCDEFG) ve monosistronik bir gen olan ywfH sorumludur. Basilisin, N-terminalde L-alanin ve C-terminalde non-proteinojenik L-antikapsin rezidülerinden oluşur. Antibiyotik etkisinin görülmesi için L-antikapsin rezidüsünün serbest kalması gerekir. Bu, ancak basilisin bir peptidaz tarafından proteoliz edildiğinde gerçekleşir. Basilisin bir peptidaza maruz kalmadan önce bir peptit permeaz tarafından sitosöle transfer edilir ve L-antikapsin burada bir peptidaz tarafından serbest bırakılır. Dipeptit antibiyotik basilisin biyosentezi; B. subtilis'de global düzenleyiciler olarak görev yapan ComA, SpoOA, AbrB, ScoC, ve CodY proteinlerinin kontrolü altındadır. Bu kompleks regülasyon ağına, GntR tip transkrisiyon faktörü LutR'ında katıldığı belirlenmiştir. Akabinde L-laktat kullanımından sorumlu lutABC (eski adı yvfV-yvfW-yvbY) operonunun da LutR'ın kontrolü altında olduğu bulunmuştur. Son olarak, grubumuz tarafından yürütülen çalışmalar neticesinde; LutR ın, L-laktak kullanımı ve basilisin biyosentezinin yanı sıra, karbonhidrat kullanımı ve transportu, nitrojen metabolizması, fosfat alımı, yağ asidi ve fosfolipit biyosentezi, protein sentezi ve translokasyonu, hücre duvarı metabolizması, enerji üretimi, mobil genetik element transferi, fajla ilgili genlerin indüksiyonu, sporilasyon, sporilasyonun gecikmesi ve kanibalizm ve biyofilm oluşumu gibi pek çok farklı fizyolojik ve metobolik süreçte görev yapan birbirinden bağımsız birden fazla geni/operonu kontrol eden pleiotropik bir global regülatör olduğu bulunmuştur. N-terminalinde helix-turn-helix motifi içeren diğer bir protein SinR'dır. SinR proteini B. subtilis'te geç üreme sürecinde kompetans ve motilite gelişimini aktive ederken sporilasyon ve ekzoproteaz üretimini inhibe ederek çift yönlü düzenleyici rol sergilemektedir. SinR, Spo0A gibi pleitropik bir düzenleyici anahtar gibi hareket eder ve adaptif cevapların gelişmesini sağlayarak gereksiz olan potansiyel diğer cevapları engeller. lutABC operonu, hem LutR hem de SinR proteininin kontrolü altındadır. LutR ve SinR proteinleri birlikte hareket ederek lutABC operonunu baskılamaktadırlar. Biyofilm olarak bilinen uzun hücre zincirlerini bir arada tutan ekstrasellüler matriksten sorumlu epsA-O ve yqxM-sipW-tasA operonlarına ait 18 genin de SinR tarafından baskılandığı bilinmektedir. Bu tez çalışmasının temel amacı LutR transkripsiyon faktörünün, B. subtilis genomunda, doğrudan bağlanarak düzenlediği genlerin belirlenmesidir. Bu amaçla E. coli pQE60::lutR suşunda heterolok olarak üretilen ve saflaştırılan LutR proteini ile B. subtilis PY79 suşuna ait kromozomal DNA kullanılarak, amacımıza göre modifiye ettiğimiz in vitro kromatin immünopresipitasyon (ChIP) yöntemi uygulanmıştır. Sonrasında, LutR proteininin, uygulanan modifiye-ChIP yöntemi ile yakalanan aday genlere doğrudan bağlandığını doğrulamak için EMSA yöntemi kullanılmıştır. In vitro olarak doğrulanan söz konusu etkileşimlerin, gen ifadesi üzerinde pozitif veya negatif yöndeki etkisini belirlemek amacıyla da, ayrıca RT-qPCR yönteminden yararlanılmıştır. ChIP yöntemi kullanılarak yapılan bu çalışmada LutR proteininin doğrudan kontrol ettiği genlerden yuxO (comB, comAB) geni belirlenmiştir. Bunlara ilaveten, Dr. Öykü İrigül-Sönmez'in doktora tez (2012) çalışması kapsamında lutR geninin aşırı ifade edildiği, PY79 (amyE::Pspac::lutR lutR::Tn10::spc) suşunda gerçekleştirilen mikroarray analizleri sonucunda, lrpB, yodT, rpsP, yutK, nasA, yvsG, ytsD ve yvnB genlerinin, LutR'ın direk kontrolü altında olabileceği belirlenmiştir. Bu bulguların kesinlik kazanması için bu tez kapsamında uygulanan EMSA analiz sonuçları, söz konusu lrpB, yodT, rpsP, yutK, nasA, yvsG, ytsD ve yvnB genlerinin, direk LutR proteini tarafından kontrol edildiğini göstermiştir. Bunlara ek olarak, grubumuz tarafından sürdürülen daha önceki farklı araştırmaların sonuçları, SinR global regülatör proteinininin, test ettiğimiz LutR'ın direk kontrolü altında bulunan tüm genlere bağlanma kapasitesinin bulunduğunu göstermiştir. Bu bilgi doğrultusunda tez kapsamında yapılan EMSA analizlerinde LutR ve SinR proteinleri birlikte kullanılarak, LutR ve SinR'ın birlikte düzenledikleri olası yeni genlerin aydınlatılması amaçlanmıştır. Bu kapsamda yapılan EMSA analizleri sonucunda; LutR ve SinR proteinlerinin yuxO, lrpB, yodT, rpsP, yutK, nasA, yvsG, ytsD ve yvnB gen transkripsiyon seviyelerini birlikte düzenledikleri belirlenmiştir. Son olarak, deneysel analizlere paralel bir şekilde, karşılaştırmalı in silico analizler ve karakterizasyon çalışmalarıyla LutR proteinine ait biyoinformatik verilerin elde edilmesi amaçlanmıştır. Bu amaçla iki analiz yolu takip edilmiştir. İlk analiz yolunda, çeşitli biyoinformatik araçlar yardımıyla farklı türlere ait LutR proteinlerinin fizikokimyasal, yapısal ve filogenetik özellikleri incelenmiş ve LutR'ın türler arasındaki benzerlik ve farklılıkları ortaya çıkarılmıştır. İkinci analiz yolunda ise sadece B. subtilis 168 suşuna ait LutR proteininin, kristallografik analizleri tamamlanmış ve yapısı iyi bilinen GntR-HTH-tip transkripsiyon faktörleri FadR ve YvoA ile PyMOL programı yardımıyla yapısal olarak karşılaştırılması yapılmıştır. Ayrıca bu tez çalışmasında kullanılan ChIP yöntemi için oluşturulan prosedür, prokaryotik sistemlere yönelik olması ve antikor kullanılmaksızın uygulanması açısından metodolojik bir yenilik sunmaktadır.
Sistemik lupus eritematozus ve skleroderma hastalarında anti-insan lökosit antijen antikorlarının incelenmesi
Human leukocyte antigen (HLA) is the human form of Major Histocompatibility Complex (MHC). Generally, HLA is a system of gene locus that encode for proteins on the surface of cells that are responsible for the regulation of the immune system. It is located on chromosome 6, encoded cell surface antigen presenting proteins. Anti-HLA antibodies are normally not occurring produced against self or non-self HLA antigens to form a immunologic response in blood transfusion, pregnancy and organ or tissue transplantation. Systemic lupus erythematosus (SLE) is an autoimmunity disorder which inflammation along with tissue damage affecting multi organs such as skin, kidney, joint are the main cause of it. SLE is a multisystem autoimmune disorder that has variable clinic manifestation. Prior physiologic mechanism of SLE is polyclonal B cell activation and production of autoantibodies against nucleus, cytoplasmic and cell surface antigens. Although the main cause of SLE pathogenesis remains unclear, there have been several clinical and serological factors that define predisposition and risk for SLE. Systemic Sclerosis (SSc) that is characterized with vascular damage caused by immune system dysfunctions and collagen accumulation in several tissues and organs can cause smooth muscle atrophy result of neurological function loss and then motor function loss. Generally anti-HLA antibodies can cause acute and chronic rejection in transplantation. Lately, there have some studies that associated between autoimmunity and anti-HLA antibodies. According to new theory, it has been indicated that there may be a similarity between alloimmune responses occur during transplantation and autoimmunity for self-antigen. Microchimerism is a well-known phenomenon effecting after solid transplantation, pregnancy and blood transfusion. The analogy between autoimmune diseases and alloimmune response in transplantation may explain role of microchimerism related with fetal or maternal origin. Therefore, in our project, SLE and SSc were selected as a model autoimmune disease to investigate the relationship between autoimmunity and anti-HLA antibodies. First of all, three different group including SLE, SSc patients and healthy controls were formed. The clinical and demographic data were collected and analyzed. Then, anti-HLA antibodies screening were done to all groups with flourometric methods. After analysis of anti-HLA antibodies screening, statistical analysis were done. Also, anti-HLA antibody identification analysis has been applied to anti-HLA antibody positive SLE, SSc patients and healthy controls. PRA ratios and CREG groups were determined statistically. According to our findings, the frequencies of both class I and II anti-HLA antibodies were increased in both SLE and SSc patients when compared to controls. Also, anti-HLA antibodies frequencies were similar in both SLE and SSc patients. We also found that typical parameters for SLE patients such as photosensitivity, thrombocytopenia, anti-RNP positivity, anti-Ro positivity, anti-nuclear antibody (ANA) had slightly higher frequency than SSc patients. On the other hand, SSc parameters frequency such as digital ulcers, anti-centromere antibody, pulmonary hypertension, and pulmonary fibrosis were higher in SSc patients. Also, in comparison of degree of positivity of anti-HLA antibodies we found that patients with high positivity for anti-HLA antibodies in both SLE and SSc group was found higher than low positivity individuals in same groups. In conclusion, anti-HLA class I and II antibodies were found as a significantly increased in patients of SLE and SSc that were chosen as model autoimmune diseases than healthy controls in which microchimerism may play a role. In order to focus the role of microchimerism in this relationship, more targeted studies should be done.
Evrimsel mühendislik yöntemiyle elde edilmiş tuza dirençli bir Saccharomyces cerevisiae mutantının moleküler ve fizyolojik karakterizasyonu
Yeast is an excellent model system for molecular biology, cell morphogenesis, chromosome stability , and even aging. Saccharomyces cerevisiae CEN.PK 113-7D is widely used for metabolic engineering and systems biology research in industry and academia. A consortium of German yeast researchers developed the isogenic family of CEN.PK strains by crossing different laboratory strains of S.cerevisiae in the 1990s. The haploid MATa strain CEN.PK113-7D, has been extremely popular for studies in systems biology and is a preferred laboratory strain for industrial biotechnology research. Maintenance of alkali cation homeostasis is a key process in most types of cells, including yeasts. Sodium chloride is very important for human biology. Na+ and K+ are essential for human nutrition and also Li+ is an essential nutrient in rodents and goats. The budding yeast S. cerevisiae is able to grow in the presence of a broad range of external concentrations of K+ (10 mM–2.5 M) and Na+ (1.5 M). The aim of this study was to investigate the effects of NaCl stress at physiological and transcriptomic level. S.cerevisiae was first mutagenized by ethyl methanesulfonate (EMS) and then exposed to salt stress (NaCl) by Sezgin T. (2010). An individual was chosen from the population and compared to wild type to investigate how it gained resistance to salt stress. Specific growth curves of wild type and "T8" in YMM were calculated by growing these strains in 5 mM LiCl, 0.5 M NaCl, 0.7 M NaCl and 0.9 M NaCl. In all of these conditions, T8 grew better than the wild type. Spot test results of T8 and wild type (CENPK-7D) showed that resistance to sodium and lithium are obvious, however potassium resistance was not observed. NaCl resistant mutant "T8" was resistant to sodium and lithium, while the wild type as well as T8 grew in the presence of potassium. It couldbe inferred that "T8" had resistance to cation toxicity, but not to osmotic stress. Flame Atomic Absorption Spectrometry (FAAS) results showed that high extracellular Na+ and Li+ concentrations increase intracellular Na+ and decrease intracellular K+ , in both wild type and T8. Apperantly, cells maintain the total monovalent cation content low after K+, Na+ and Li+ stress. NaCl resistant mutant "T8" did not exhibit significantly increased lithium accumulation, compared to the wild type strain. The lithium resistance of the wild type was not caused by increased lithium efflux. "T8" accumulated slightly more Li+ than the wild type in double stress conditions. FAAS results showed that T8 has higher K+/Na+ ratio at every stress condition compared to the wild type. Difference of K+/Na+ ratio between T8 and the wildtype is obvious at 5 mM LiCl. K+/Na+ ratio of T8 was 4.21 and of wildtype was 1.97 at 5 mM LiCl stress. ENA6 and NHA1 genes' expression were lower at LiCl stress, compared to NaCl stress. The reason why K+/Na+ ratio is higher at LiCl stress can be the lower expression of these efflux genes so that T8 may not have active efflux at LiCl stress as at NaCl stress. Glycogen and trehalose are two important glucose stores of the yeast S. cerevisiae, and production of them change in response to a number of environmental stress conditions. The disaccharide trehalose accumulates during salt adaptation. Assessment of glycogen and trehalose content showed that amount of trehalose and glycogen in T8 are prominently high compared to the wild type without stress conditions. Lithium triggers the amount of glycogen and trehalose more than sodium. It could be explained by higher toxicity of lithium. Trehalose accumulation is higher in T8 than glycogen without and with sodium and lithium stress. In our NaCl-resistant evolved S. cerevisiae strain "T8", production of glycerol is higher and production of ethanol is reduced compared to the wildtype. The metabolism of the evolved strain is redirected toward glycerol and away from ethanol production. The cross-resistance of T8 was assessed by growth on LiCl, ZnCl2, H2O2, CoCl2, KCl, ethanol, CuCl2, MnCl2, CrCl3, AlCl3YPD and YMM plates.T8 has significantly high cross resistance to lithium.T8 has slight resistance to Co+2, Mn+2 Ni+2, Zn+2 and is sensitive to aluminum. The S. cerevisiae genome contains three genes encoding alkali metal cation/H antiporters (Nha1p, Nhx1p, Kha1p) that differ in cell localization, substrate specificity and physiological function. Sequencing of NHX1, NHA1, and KHA1 were checked to be compared with mutant strain "T8". Sequencing results showed that EMS mutagenesis and NaCl stress did not affect the sequences of NHX1, NHA1, and KHA1 genes. The transcriptional response to high NaCl concentration stress in the yeast S. cerevisiae was analyzed by using DNA microarray to understand yeast strains showing tolerance to high salt stress. In microarray results, several hundred genes were identified associated with NaCl tolerance involving a broad range of functional categories including energy; cell rescue, defense and virulence; metabolism; cellular transport, transport facilities and transport routes; cellular communication/signal transduction mechanism. FLO11 gene encoding GPI-anchored cell surface glycoprotein(flocculin) which is required for flocculation and biofilms was the most highly expressed gene in T8. In "T8" microarray results, HOG pathway is not induced. "T8" had slower glucose uptake and did not accumulate high levels of succinate and acetate. In microarray results,T8 has induced ENA6 expression, however NHA1 is not induced. In Real-time PCR results, both ENA6 and NHA1 genes are induced. This may result from the differences in sensitivities of microarray and real-time PCR, the latter being more sensitive. Microarray results indicated that "T8" has many genes overexpressed compared to the wild type even in the absence of NaCl stress. These upregulated and downregulated genes adapted this mutant to high sodium and lithium stresses. Nha1 was cloned to be overproduced and it is checked by complementation assays. Overexpression of Nha1p showed that this protein complements better at pH 3.5. Specific single site mutations [G372S, D266H, D267A, D266H/D267A, D266N/D267N, N176, D177N ] on Nha1 were applied to understand these influence of this mutations on pH. In this study it was shown that under S. cerevisiae strains with enhanced glycerol and reduced ethanol yields could be obtained by evolutionary engineering for NaCl-stress resistance.This study is the first one that uses EMS and increasing NaCl stress levels for the selection of S. cerevisiae strains by evolutionary engineering. Additionally, the results of this thesis work suggest that FLO11 may have an important role in high NaCl-resistance in S.cerevisiae.
Multipl skleroz klinik alt tiplerinde moleküler yolakların ve biyobelirteçlerin araştırılması
Multiple sclerosis (MS) is a neuro-inflammatory and neuro-degenerative disease of the central nervous system (CNS), in which myelin sheaths and axons are damaged. Inflammatory demyelination and other pathological changes disrupt the ability of the nervous system to communicate resulting the wide variety of signs and symptoms. Heterogeneous clinical presentation and course ıf the disease, without an effective diagnostic laboratory test, make it difficult to draw firm conclusions about the disease's states, drug response and in general prognosis which may lead to more severe forms in some cases. The purpose of this study is to find out molecular biomarkers and pathways associated with the etiopathogenesis of the disease and its major subclinical forms. To this aim, proteomic, bioinformatics and genomic approaches were used to reveal differentially expressed proteins and their related pathways in cerebrospinal fluids of patients with clinically isolated syndrome (CIS), relapsing remitting MS (RRMS) and progressive MS (PPMS). Cerebrospinal fluid (CSF) samples of 65 CIS, 72 RRMS, 42 PPMS and 42 controls including healthy subjects and other neurological disease samples were analyzed. For all samples, 2D-PAGE analyses were performed. At least 2 times differently expressed protein spots were identified by PDQuest® software and removed from the gel for MALDI-TOF-MS protein identification analysis. The results of proteomic studies were further confirmed by both ELISA and western blot methods for selected candidate proteins. KEGG pathways analysis was performed with genes corresponding the proteins to identify disease relevant specific molecular pathways. Further linkage and genome-wide association study (GWAS) were performed by using single nucleotide polymorphism (SNP) genotyping on the Illumina CytoSNP 300K array in 36 individuals from 10 informative MS families having more than one affected member. Nonparametric linkage (NPL) scores were calculated for each of 3118 SNP markers spaced at an average of 1 cM intervals using SimWalk multipoint NPL analysis. Subsequent fine mapping of regions showing higher NPL scores was performed. GWAS analysis was performed by using 18 MS and 60 healthy subjects data by comparing the 298114 different genome wide SNPs. Comparison of disease groups with controls identified a total of 151 proteins that are differentially expressed in clinically different MS subtypes. KEGG analysis using PANOGA tool revealed disease related pathways including aldosterone-regulated sodium reabsorption (p=8.02x10-5) which is important in the immune cell migration, renin-angiotensin (p=6.88x10-5) system that induces Th17 dependent immunity, notch signaling (p=1.83x10-10) pathway indicating the activated remyelination and vitamin digestion and absorption pathways (p=1.73x10-5). Linkage study including 10 MS families showed suggestive evidence of linkage (NPL scores above 1.7) to chromosomal regions of 13q12.2-14.11 and 21q22.12-22.3. Fine mapping of these regions revealed that the most promising loci for linkage were mapped to 13q13.3 and 21q22.2, with NPL scores of 1.82 and 1.85, respectively. Detailed search of these regions revealed inflammation and neurodegeneration associated genes some of which previously implicated in MS and also promising novel genes yet to be identified like N4BP2L2 and TRPC4 were found within the suggestive regions. On the other hand, GWAS analysis revealed disease associated SNPs with significant associations on disease pathogenesis associated genes like INS-IGF2 (p=4.39x10-7 MAF = Case:Control = 0.36/0.03), MGMT (p=9.66x10-6 MAF = Case:Control = 0.32/0.04). Individual comparison of genomic and proteomic data revealed 9 different genes correlated with 14 different proteins, in 4 different molecular pathways that are associated with proteomic data. Those genes and proteins are the most prominent molecular parameters to understand disease mechanisms and needed to be further verified with various studies. To conclude, proteomic investigation of CSF samples belonging to different MS phenotypes indicated that all MS clinical forms share common biological pathways such as renin-angiotensin system (RAS) and complement and coagulation cascade (CCC) pathways. There are also clinical subtypes specific and pathophysiology related pathways, which may have further therapeutic implications. Furthermore, our study implicated importance of salt metabolism in MS pathogenesis for the first time. On the other hand, genomic results also showed disease pathogenesis associated genetic variations and chromosomal regions. Our suggestive results provide a framework for deep sequencing to identify new susceptibility genes and novel variants associated with risk of MS.
bsLDH enziminde Q102R ve Q203L çift mutantının substrat özgüllüğü ve aktivatör gereksinimine etkileri
Enzymes are biological catalysts that play role in chemical transformations, thus they are important for several branches of industries. The L-lactate dehydrogenase (LDH) from Bacillus stearothermophillus(bs) is substantial for the synthesis of pharmaceuticals and agrochemicals as it provides the production of chiral building blocks. It enables the reaction that generates hydroxy acids from their corresponding oxoacids. Moreover, because Bacillus stearothermophillus is a thermophilic bacterium, the enzyme is highly stable to heat. However, the enzyme has a limited substrate specificity. On the other hand, the enzyme is activated by an expensive activator, fructose 1,6 bisphosphate (FBP). FBP also shows undesirable co-factor complications for industrial processes. Besides, bsLDH activity is inhibited by excess substrate (pyruvate), which is once more a deleterious feature owing to its industrial use. All these characteristics make bsLDH an applicable target for protein engineering. Previous studies have provided significant developments over these unwanted properties by using rational and randomized mutagenic methods. For instance, by Wilks et al., a Q102R substitution (found in the naturally occurring malate dehydrogenases) on the bsLDH sequence has been shown to convert the substrate specificity from lactate to malate by 3 orders of magnitude. In another research, Allen and Holbrook succeeded in producing a mutant (6A) which is almost fully activated in the absence of FBP. This 6A mutant had three amino acid replacements: R118C, Q203L and N307S, resulting in a 70-fold activation. Normally FBP activates the enzyme by enabling structural rearrangements which turns the protein into tetrameric form from dimeric form. Because the Q203L is located at the dimer-dimer interface, this substitution is thought to have realized the substrate switch by altering the dimer-tetramer equilibrium. By our laboratory, similar protein engineering applications have yielded significant effects on bsLDH as well. For example, by D38R replacement, using PCR based overlap extension mutagenesis, the substrate inhibition was decreased threefold by Binay and Karaguler. In another study, recombinant colonies, formed by random mutagenesis were screened and a more efficient malate dehydrogenase, compared to Q102R variant, was produced. Our aim in this study is forming a double mutant form of bsLDH enzyme and investigate its effect both on the activator need and on substrate specificity. In order to achieve this, Q102R and Q203L mutants were constituted by site-directed mutagenesis in bsLDH sequence which was found in pQE2 vector. To be used in the PCR reaction, appropriate oligonucleotides were designed to generate Q102R and Q203L mutations. Following the PCR with these oligos, mutant dsDNAs were selected by Dpn I digestion. After that, mutated DNA was transformed into E.coli strain BL21 E. coli genotype fhuA2 [lon] ompT gal [dcm] ΔhsdS competent cells. Plasmid DNA was purified following the transformation and samples were sequenced to ensure to have achieved the correct mutations. According to the data obtained, Q102R and Q203L mutations are formed in bsLDH sequence. Mutated DNA was amplified in large-scale E.coli culture and the culture was precipitated in order to obtain protein from cells. Desired protein with 6xHis-tag was sorted from the protein pool by Ni-NTA resin system which selectively binds His-tagged proteins. Thereafter, by Sodium Dodecyl Sulphate Gel Electrophoresis (SDS-PAGE), the protein was visualized at the region of its molecular mass (35kDa) and have ensured to obtain the enzyme. By ultrafiltration, smaller proteins and other contaminants were removed and a purer enzyme was attained. With different concentrations of pyruvate as substrate, wild type and mutant enzyme kinetic measurements were realized in the absence and presence of FBP. Wild type data were found quite consistant with the previous studies. But we could not get detectable results in mutants with pyruvate. Actually, this phenomenon agrees with the Q102R mutant feature, which gives rise to a malate dehydrogenase upon mutation. Further experiments will be made with malate and oxaloacetate substrates to more precisely elucidate the double mutant effect.
İnsan nöral kök hücrelerinde NFI transkripsiyon faktörlerinn, NFIB hedef genlerinin ve NFIB ekspresyonunu düzenleyen miRNA'ların karakterizasyonu
The transcription factors of the nuclear factor one (NFI) family play an important role in regulation of gene expression during nervous system development. NFI family has four members (NFIA, NFIB, NFIC and NFIX) that are highly homologous in their N-terminal DNA binding domains, but vary in their C terminal transcription activation/repression domains. NFIs can induce or repress transcription of different genes in different cell types in a context dependent manner. Each NFI member has multiple alternative splicing isoforms; however, functions of these isoforms of NFIs have not been well described. NFI proteins recognize consensus sequence TTGGC(N5)GCCAA and bind double strand DNA as homo- or heterodimers. In addition, the post-translational modifications (glycosylation or phosphorylation) of NFI proteins can potentially affect or modulate their function. NFIs are expressed in an overlapping but distinct expression pattern in the developing embryo suggesting a role in regulation of development. All NFIs except for NFIC are expressed in the embryonic central nervous system while expression is restricted to stem cell niches in the adult brain. Deletion of NFIA, NFIB and NFIX leads to distinct phenotypes involving different brain regions such as neocortex, hippocampus, and hindbrain, altering different developmental processes like axonal outgrowth and guidance, differentiation, neurogenesis or gliogenesis. Previously, we have found that while all three neural NFI proteins are expressed in the precerebellar systems of the hindbrain, only the absence of NFIB lead to a delay in the development of precerebellar nuclei. To understand how NFIB affected neurogenesis in the precerebellar system, potential NFIB target genes were identified in precerebellar neuroprogenitors by mRNA expression profiling. Here, we set out to study regulation of downstream targets of NFIB as well as upstream regulators of NFIB in the human neural stem cell culture system derived from H9 human embryonic stem cell line. To this end, we first characterized the expression of NFI family members and three target genes (CDO, COL2A1 and FGF19) selected according to their expression patterns and known functions. Expression of NFIB, NFIC, NFIX, CDO and COL2A1 was significantly reduced while NFIA and FGF19 were up-regulated during neuronal differentiation. NFIB-4, NFIB-3 and/or NFIB-1 isoform mRNA expression was detected in this cell line. We found that NFIB-3 and NFIB-1 protein expression also decreased in differentiated hNSCs. To identify novel upstream regulators of NFIB, we first determined miRNA binding sites on the NFIB 3'UTR and looked for those miRNAs that are upregulated in hNSCs during neural differentiation. We analyzed expression of miR-153, miR-30a, miR-124a, miR-92b, miR-130a and miR-363 and found miR-153, miR-124a and miR-30a expression increased while miR-363 decreased in differentiating hNSCs. Upregulated miRNAs were selected for further study. 3'UTR fragments carrying binding sites of these miRNAs were fused to a reporter gene and screened for inhibition by miRNA mimics. Preliminary data indicate that miR-153 and miR-124a can directly repress translation of NFIB by interacting with 3'UTR_D (6418-6425) and 3'UTR_B (1842-1848) regions on the NFIB. Site directed mutagenesis of miR-153 and miR-124a binding sites appear to render these sites resistant to miRNA mimics, underlining the specificity of this repression.
MODY'nin moleküler patogenezinde miRNA analizi
Maturity onset diabetes of the young (MODY) is a monogenic form of Diabetes mellitus (DM) characterized with abnormal beta cell function, autosomal dominant inheritance, hyperglycemia, lack of auto-immunity in non-obese young patients. The prevalence of MODY changes with the population, but MODY3 associated with HNF1A gene mutations is the most common form of MODY. The main purpose of the study was to discover miRNAs that might be involved in the molecular pathogenesis of MODY by discovering the specific miRNAs that are regulated by HNF1A transcription factor.
Farklı prıon proteini varyantlarının yanlış katlanma mekanizmalarının moleküler dinamik simülasyonları ile araştırılması
Transmissible Spongiform Encephalopathies(TSE) are fatal neurodegenerative diseases. Examples are: bovine spongiform encephalopathy (BSE) in cow, scrapie in sheep and Creutzfeldt Jakob disease (CJD) in humans. A misfolded version of a protein named prion causes these diseases. Properly folded protein(PrPC) is rich of α-helix. The misfolded protein (PrPSc) contains less α-helix and is mostly comprised of β-sheets. Encounter of PrPC and PrPSc catalyzes the misfolding and disease propagation. The disease is transferred between individuals via transfer of these proteins. PrPSc can create oligomers or fibrils. The N-terminus of this protein is disordered and it is believed that it binds Cu2+. The ordered part at the C-terminus is comprised of 3 α-helices and 2 β-strands in the correctly folded state. In the literature, the part of the protein which causes the disease is controversial. Also, the misfolded shape and misfoling pathways are unknown. The predominantly accepted idea is that helices 2 and 3 (H2 and H3) at the C-terminus create a β-sheet by misfolding. Existance of more than one misfolded shape and pathway is also possible. However, the 3-dimensional PrPSc shape is still unknown and there is a need for researches on this subject. This study will contribute to enlighting of these misfolding pathways by using molecular dynamic simulations. One of the most common form of TSE is scrapie. For scrapie development, the 136th, 154th and 171st residues are important. This study focuses on 3 variants. First of these variants is weakly resistant wild type (ARQ: A136, R154, Q171), the second one is the most susceptible mutant (VRQ: V136, R154, Q171) and the other one is the most resistant mutant (ARR: A136, R154, R171). Simulations have been made with generalized Born continuum solvation method and Amber ff10 force field at 310 and 330 Kelvin. Simulation durations are 400-700ns. Fluctuated regions (except termini) of the proteins in all 3 simulations at 310K are: 1) H1 and the loop between H1 and β-strand 1(residues 135-149); 2) the loop between β-strand 2 and H2 (residues 168-177); 3) C-terminus of H2 and the loop between H2 and H3 (residues 186-204). Movement of the region between 168-177 is similiar in all 3 variants. In the other two regions, the magnitude and direction of the movements are found to be different in all 3 variants. ARR which is the most resistant variant has the lowest mobility. VRQ, the most susceptible variant, is the most mobile variant in the simulations. Therefore, it is seen that there is a relation between susceptibility and mobility. Especially, in VRQ, H1 is relocated with respect to H3. Position of H1 in the other two variants (ARR, ARQ) is not too far from crystal structures. Hence it is seen that valine as the 136th residue has an effect regarding the reposition of H1. The other two variants have alanine instead of valine as the 136th residue. In order to accelerate the conformational changes, simulations have been performed at 330 K and H1 has changed its position in all these simulations, including ARR and ARQ. In addition, VRQ has deformed its β-sheet in the 330 K simulations. These results are compatible with the 'banana peeling model' which suggests that relocation of H1 is necessary for the conversion of H2 and H3 to β-sheet.
Olası koşaperon MZB1 proteininin Grp94 şaperonu üzerindeki etkisi
Grp94 is Hsp90 paralog, residing in endoplasmic reticulum (ER), implicated in efficient expression of various proteins secreted and presented on cell membrane. As an essential eukaryotic chaperone, a set of studies have already shown that Grp94 involves in maturation of Toll-like receptors, integrin family members and antibody secretion. Since the condition of ER significantly differs from that of the cytosol, Grp94 must have been uniquely evolved from its cytosolic counterparts in order to deal with distinct client profile of ER. It is known that vast majority of Grp94 clients possess at least one disulphide bond whose formation is crucial to attain 3-D structure and stability for many ER proteins. Besides, ER is much more oxidizing than cytosol due to its enzymatic components catalysing disulphide formation. Despite of having same domain organisation with that of other Hsp90 family members, in which N terminal domain preceding M domain and finally C-terminal domain functioning in dimerization, Grp94 has significant conformational differences correspond to its cytosolic counterparts. Most importantly, unlike other Hsp90, in the presence of AMP-PNP or ADP, Grp94 adopts same twisted V conformation which seems to make N-terminal dimerization unfavourable. It must be noted that binding of ATP makes Hsp90 family members undergo some conformational changes in which N- terminal domains of each protomer dimerize resulting in ATP hydrolysis. Furthermore, unlike its cytosolic counterparts, Grp94 does not show different affinity profile for ATP and ADP. These data has been leading an intriguing question regarding to the extent in which Grp94 differs from its cytosolic homologs in undergoing conformational changes during its ATPase cycle. It is long standing enigma as to whether Grp94 is regulated by a co-chaperone which may mediate client loading or remodel ATPase cycle to function in maturation of a set of ER proteins. Even though some studies have shown that a set of protein interact Grp94 along with clues concerning physiological relevance, our current understanding of the precise nature of interaction in these presumptive proteins with Grp94 and their regulatory effects on Grp94 conformational changes are still infant. pERp1/MZB1 is relatively newly discovered protein residing in ER. Its function has not been fully understood, which presumably act as unique oxidoreductase or chaperone. In previous studies, it has been demonstrated that pERp1 interacts with ER chaperones, Grp94 and BiP. In the context of this thesis, wild type Grp94 and a flanked at both terminal domains by partial ubiquitin version of Grp94 are compared by subjecting to ATPase assay and circular dichroism spectroscopy. The main aim is to check whether ubiquitin tagged Grp94 has no significant difference from wild type in ATPase kinetics and secondary structure. It must be noted that this version was important to do further investigation on Grp94 by single molecule study. Moreover, titration of pERp1 has been performed to calculate EC50 which is an indicator of binding affinity. Apart from steady state kinetics, citrate synthase aggregation assay and analytical size exclusion chromatography are applied to the respective proteins.
Kuraklık stresi altında Brachypodium distachyon yapraklarının proteom analizi
Among the environmental stress factors, drought is the one that has the most severe effect on agricultural production. Hence, improving the yield under drought stress is one of the most important challenges in agriculture. Drought or water deficit has a direct impact on the cellular metabolism of plant. This impact significantly reduces the growth index and crop yield. A number of studies highlighted the existence of a complex network in the cell upon drought stress. Changes in protein expression play the major role in this complex network. The main strategy to improve crop yield is to integrate all the properties that are required to sustain yield under drought stress and accumulate the most powerful genes and proteins in elite genotypes without any harmful effect on the potential crop yield. This approach will provide the improvement of new plant cultivars that are stable and exhibit high yield potential under drought conditions. In plants, there are number of studies on transcriptome level to elucidate the mechanisms underlying stress response. However, alterations in mRNA levels do not always correlate well with protein levels in the cell due to the post-transcriptional and post-translational modifications. Therefore, transcriptome studies are insufficient for understanding the complex network of stress response, causing a lack of knowledge for the enlightenment of the mechanisms related to stress response. Brachypodium distachyon, model plant for monocot species, exhibits a close relationship to agriculturally and economically important crops. It is also known that Brachypodium distachyon displays high variation in the response against drought stress. This variation will potentially lead to identify new stress-responsive genes and thus proteins. Ongoing transcriptomic analyses in Brachypodium distachyon are available. However, requirement of proteomic analyses is an emerging subject for this plant species. In this study, changes in protein repertoire of Brachypodium distachyon leaves after exposing individuals to time-dependent drought stress (4, 8 and 12 day application of drought) were described. Based on two-dimensional difference gel electrophoresis (2D-DIGE), constructed with 3 replicates, and combined mass spectrometry (MS) experiments, a proteomic approach was carried out for this purpose. The comparison of the resulting proteomic data highlighted differentially expressed proteins (DEPs) upon drought stress. 37 differentially expressed proteins were detected via statistical evaluation of relative spot densities and among these one, thirteen DEPs were identified by MS and classified according to their functions. The role and relation of DEPs in drought response of Brachypodium distachyon were discussed. The biological functions of DEPs included roles in photosynthesis, protein folding, antioxidant mechanism and metabolic processes. It has been highlighted that there is a significant degree of overlapping between metabolic alterations induced by drought stress. The identified proteins in this study could serve as potential biomarkers for the selection of drought-tolerant Brachypodium distachyon plants as well as its relatives like wheat, barley and rye. Furthermore, identified proteins will contribute on the studies on development of drought-resistant crop species such as wheat, oat and barley, which are close relatives of Brachypodium distachyon.
ATP hidrolizinin HSP70 şaperon proteini tarafından katalizlenmesi
Molecular chaperone proteins assist the newly synthesized proteins to fold into the native structure. Many chaperone proteins are also heat shock proteins, which assists refolding of the proteins denatured under stress conditions, with Hsp70 being one of them. In this study, the ATP hydrolysis mechanism of Hsp70 has been investigated using the QM/MM ONIOM method with M06-2X for the QM part and the AMBER force field for the MM part. Two main models were created depending on the protonation states of D201. Particular attention has been paid to the role of K70 because this residue is crucial for ATP hydrolysis. In addition, the possibility that K70 is involved in acid catalysis has been investigated. Other candidates for acid catalysis were D194 and a water molecule between Mg2+ and K+ and E171 is used as a base candidate. In the crystallographic structure, a Mg2+ ion is coordinated to the beta-phosphate group. It is suggested that shift of Mg2+ to a position between the beta- and gamma-phosphate groups. This mechanism is also tested. Our results showed that proton transfer to ATP occurs with nucleophilic water directly attacking the ATP and acid-base catalysis seems unlikely. Suggested mechanism about the shift of Mg2+ is also unlikely. In the models with a deprotonated D201, Pi tends to donate a proton to a nearby water or E171, which suggests that E171 can accept a proton after product formation. And when a proton is deleted on Pi, Mg2+ shift between the two phosphate groups is energetically favorable.
Metagenomik yöntem kullanılarak selüloz parçalayan yeni enzimlerin keşfedilmesi
Fossil fuel is a finite energy source and use of fossil fuels releases harmful chemicals to environment. To fulfill energy requirement, alternative energy sources that can replace fossil fuel must be developed. A high potential environmental friendly alternative is bio-fuel produced from cellulosic biomass. Despite of high number of biofuel researches, production of sustainable and economical biofuel remains a challenge due to recalcitrance of cellulosic materials. The major obstacle for industrial-scale production of biofuel is the inefficient degradation of plant material due to absence of enzymes that catalyze efficient hydrolysis at optimum conditions. Fully degradation of cellulose requires synergetic action of a complex of enzymes for optimized saccharification. Cellulases, which catalyze cellulolysis, are classified into three groups according to their structure and mechanism of action: endoglucanases, exoglucanases, and β-glucosidases. Unfortunately, commercial biocatalysis is insufficient due to the lack of enzymes with optimal performance for specific applications. A powerful approach, which draws attention lately, for discovery of new enzymes with optimum performance is screening of natural diversity. Microorganisms, which adapted to a wide range of environmental conditions, serve as a good source. Metagenomic approach, which comprises the direct extraction of genomic DNA from environmental samples, can prevent loss of diversity resulting from cultivation, and allows recovery of genomes of all microorganisms present in a given niche. In this study, the aim was identification of novel cellulolytic genes using metagenomic approach. Environmental samples used in the study comprised water samples from Dalaman Spring, Dalaman Lake, Emet Hot Spring, and Kuzuluk Hot Springs, straw samples that were incubated in water for enrichment of cellulolytic microorganisms, and grasshoppers obtained from TUBITAK MAM campus and commercial farm. The metagenomic DNA extraction and DNA clean-up (if required) were performed using commercial kits. The isolated DNAs were used for construction of metagenomic library with next-generation sequencing technology. Bioinformatic tools were used for assembly of reads, phylogenetic studies, and gene annotations. Total of 63 candidate cellulase genes were selected from annotated for further analysis. Candidate genes, which were amplified from metagenomic DNA, were cloned into E. coli BL21 (DE3) for production of recombinant protein. 22 of 63 candidate proteins was expressed successfully and screened for biochemical activity against cellulose. Two different substrate, CMC and pNPG, were used for screening of endoglucanases, exoglucanases, and β-glucosidases. Activity assay revealed one active enzyme against CMC, while no active protein that hydrolyzes pNPG was found.
Tersine metabolik mühendislik yöntemi ile aluminyuma dirençli Saccharomyces cerevisiae eldesi
Although Al3+ is non-essential and toxic to life, it is commonly encountered in water, foods, medicines and beverages. Al3+ has been reported to interact with organic molecules in vitro, however, molecular mechanisms of aluminium toxicity/tolerance are not well-known. Aluminium is also related with human neurodegenerative diseases such as Alzheimer and Parkinson. The yeast Saccharomyces cerevisiae is a model organism which has been used widely in molecular biology and biotechnology for a long time. In addition to research, it has also been used for many industrial applications. It can make fermentation which is very important in baking and wine industry. S.cerevisiae can be in haploid and diploid form and its genome has a high degree of homology with those of higher eukaryotes. This makes it important in understanding molecular mechanisms of human-related diseases. In this study, S. cerevisiae was used as a eukaryotic model organism to investigate aluminium tolerance mechanisms. An evolutionary engineering strategy based on batch selection at increasing Al3+ concentrations was employed to obtain aluminium-resistant S. cerevisiae mutants: initially, S. cerevisiae CEN.PK 113-7D wild type strain was chemically mutagenized to increase the genetic diversity of the initial population for selection. 905 (Wild Type) and 906 (EMS mutagenized wild type) were screened under different aluminium levels such as; 0.05, 0.1, 0.4, 0.6, 1, 2, 5, 10, 25, 50 mM to determine the initial aluminium stress level to be used during increasing stress level selections. A batch selection strategy was then applied to obtain aluminium-resistant S. cerevisiae mutants by gradually increasing aluminium-stress levels from 0.5 to 21.5 mM AlCl3 through 43 passages. The 43th last population was spread on plates and 12 individual mutants were randomly chosen. They were tested for their aluminium resistance by using spot assay procedure. According to spot assay results, 4 highly resistant mutants (named as Alu9, Alu10, Alu11, Alu12) were chosen for further analysis. In order to quantify the aluminium resistances of the mutants in detail Most Probable Number method was applied to those 4 mutant individuals. They were then tested for the genetic stability of the Al-resistance.Results showed that the mutants are genetically stable. Cross-resistance tests were also applied using a variety of other metal and non-metal stress types to test if the aluminium-resistant mutants developed cross-resistance or sensitivities to other stress types. For this purpose, the following stresses were tested: 8%(v/v) Ethanol, 0.2 mM NiCl2, 0.7 mM H2O2 , 0.25 mM CuSO4, 1 ml/L phenyl ethanol, 1.3 M MgCl2, 2.5 mM CrCl3, 10 mM MnCl2, 30 mM FeSO4, 80 mM H3BO3, 300 mg/ml Propolis, 0.5 M CaCl2, 2 mM GaNO3, 75 µM AgNO3, 0.20 NiCl2, 10 mM NaCl, 3 mM ZnCl2, 1 mM CoCl2, 2 mM LiCl, pH 4, 25 mM FeCl2. However, no significant cross-resistance or sensitivities to other stress types were detected, except for a slight level of LiCl,GaNO3 and ethanol tolerance, and a slight sensitivity to AgNO3. According to the experimental results, the best individual mutant (Alu10) was chosen for further detailed analyses at physiological and molecular levels.
Helicobacter-aktive B(HAKT-B) hücrelerinin CD4+ T hücreleri ile fonksiyonel etkileşimleri
Helicobacter pylori is a spiral-shaped, gram-negative bacterium that infects the gastric mucosa of more than half of the world's population. The infection initially occurs in childhood, becomes persistent and the chronic infection leads to gastric inflammation. A major virulence factor of H. pylori is the cytotoxin-associated gene A (CagA) protein and this CagA protein interacts with several intracellular components of signal transduction and activates some crucial signaling pathways. Helicobacter has developed a variety of mechanisms to persist in the gastric mucosa. Gastric epithelial cells (GECs) are primary target for H. pylori infection, therefore they are the first point of contact for H. pylori and activate an innate immune response through TLRs. Rather than being a strong TLR4 ligand, H. pylori LPS is thought to activate TLR2 on gastric epithelial cells. Animal and cell culture experiments suggested that ligands in Helicobacter species can bind to TLR2 and activate NF-κB in epithelial cells. In mouse studies, Helicobacter felis (H. felis) is mainly used because it is more immunogenic than H.pylori in mice. B cells and their cytokines have important roles in Helicobacter infections by balancing between the infection and T cell driven gastric immunopathology. The ability of B cells to interact with pathogenic T cells and to produce anti-inflammatory cytokines such as IL-10 is crucial to dampen harmful immune responses. It has been found that B-cells exposed to Helicobacter sonicate produced large amounts of the regulatory cytokine IL-10. Using mouse models of Helicobacter-induced gastric premalignant pathology, it is shown that IL-10 secretion by purified B cells absolutely required MyD88 signaling and TLR-2. The TLR-2- dependent Helicobacter activation of B cells differentiates them into IL-10 and TGF-β producing regulatory B cells. Both IL-10 and TGF-β have crucial effects on T cell differentiation. When co-cultured with Helicobacter-activated B cells, naive CD4+ T-cells are shown to produce IL-10 and differentiate into T regulatory 1 (Tr1)-like cells. In addition to that, it is suggested that Breg cells contribute to regulatory T-cell induction by producing TGF-β. At the same time, studies have shown convincingly that TGF-β is required for Th17 differentiation in vitro and in vivo. In previous studies in our laboratory, Helicobacter-activated total B cells were separated into two subgroups: IL-10+ B cells and IL-10- B cells. The experimental results showed that Helicobacter-activated IL-10+ B cells are the source of the IL-10 production while Helicobacter-activated IL-10-B cells are mostly TGF-β positive. However, it was not clear if the Helicobacter-acitvated-IL-10+ B cells or the Helicobacter-activated-IL-10- B were specifically causing the Tr1 differentiation. Taking account that Bregs are known for producing IL-10 and TGF-β which are key cytokines in T cell differentiation, the interaction between Helicobacter-activated B cell subgroups and CD4+ T cell differentiation was investigated. By magnetic isolation techniques, CD19+B cells and CD4+T cells were isolated from the spleens of C57BL/6 mice with high purities (with an average of 90% and 93%, respectively). Following the B cell isolation, cells were treated with Helicobacter felis sonicate (10 µg/ml) for 24 hours. For the last 5 hours of incubation, to induce an optimal IL-10 production and secretion, PMA (50 ng/ml) and ionomycin (500 ng/ml) were added. After the in vitro stimulation of B cells, IL-10 producing B cells were labeled and the IL-10+B and IL-10-B cells were separated. To observe the interaction between the Helicobacter-activated-B cell subgroups and CD4+ T cells, isolated CD4+ T cells were put on co-culture in 1:1 ratio with the Helicobacter-activated-IL-10+ B cells and Helicobacter-activated-IL-10- B cells, for 24 hours. The cell surface markers and intracellular cytokine productions were examined by flow cytometry. While the supernatants of the co-culture groups were subjected to ELISA tests, the cell pellets were used for gene expression analyses. The intracellular staining of IL-10 of T cells co-cultured with Helicobacter-activated B cell subgroups showed that about 15% of T cells co-cultured with HACT-IL-10+ B cells produced IL-10 while almost 20% of the T cell population was IL-10 positive when T cells were co-cultured with HACT-IL-10- B cells. In addition, according to IL-10 ELISA results, CD4+T cells co-cultured with IL-10+B cells and IL-10-B cells secreted twice IL-10 when compared to only T cells. For IL-10+B cell and T cell co-culture, a part of secreted IL-10 came from B cells while most of the IL-10 secreted from IL-10-B cell and T cell co-culture originated is suggested to be from T cells. CD25 has been used as a marker to identify activated T cells as well as some regulatory T cell subsets in mice; while CD62L has been known to rapidly shed from lymphocytes upon cellular activation. Both CD25 and CD62L levels in co-culture groups showed significant differences compared to T only groups. Furthermore, the differences in T cell CD25 and CD62L levels together might indicate that T cells co-cultured with Helicobacter-activated IL-10-B cells are more activated/differentiated compared to the T cells interacting with IL-10+B cells. In literature, it has been shown that B cells which were activated by Helicobacter induce IL-10–producing CD4+CD25+ Tr1–like cells in vitro. In addition, it has been identified that the co-expression of CD49b and LAG-3 distinguishes Tr1 cells from Th1, Th2 and Th17 cells. Flow cytometry results of CD4-CD49b-LAG3 stainings showed that almost 20% of T cells co-cultured with IL-10+B cells express CD4-CD49b-LAG3 surface markers while only around 10% of CD4+ T cells co-cultured with IL-10-B cells express CD49b and LAG3 together. CD4+ T cells obtain distinct functional properties in response to signals sent by commensal and pathogenic microbe-activated cells of the innate immune system. Th17 cells secrete interleukin-17 (IL-17), IL-17F, and IL-22 and have significant roles in protecting the host from bacterial and fungal infections, particularly at mucosal surfaces. Data of four independent experiments showed that the approximately 15% of CD4+ T cells co-cultured with IL10-B cells turned into IL17 producing CD4+ T cells while surprisingly about 8% of T cells also produced IL-17 when co-cultured with IL10+B cells. IL-17 ELISA results were parallel with the IL-17 intracellular cytokine staining analyses. Ror gamma T and IL-17 relative gene expression levels in co-culture groups showed similar results with flow cytometry and ELISA results. Main research focus of this study was to investigate the interaction between HACT-B cell subgroups and CD4+ T cells and understand the effects of these B cell subgroups on T cell differentiation ex vivo. The results revealed that both Helicobacter-activated IL-10+B and IL-10-B cells induce IL-10 production from CD4+T cells and it might suggest that T cells differentiate into Tr1-like cells in both co-culture conditions. Although the CD25 and CD62L expression on T cell surfaces show significantly higher activation/differentiation in Helicobacter-activated IL-10-B and T cell co-cultures, regulatory type indicator CD4-CD49b-LAG3 surface marker co-expressions were higher on T cells in the Helicobacter-activated IL-10+B and T cell co-cultures. Furthermore, intracellular IL-17A levels and relative gene expression experiments of IL-17A and RorgammaT showed significantly higher results in IL-10- B and T cell co-culture groups, as expected. On the other hand, interestingly IL-17 production was also observed in T cells co-cultured with IL-10- B cells. The reason behind the IL-17 and RorgammaT expressions in IL-10+ B–T cell co-culture groups was suggested to be the IL-6 and TGF- β produced by IL-10 negative B cell population in the IL-10 positive co-culture group. This study has contributed to the literature through providing a first step to show the ex vivo interactions of Helicobacter-activated B cells subgroups, IL10+ HACT B cells and IL-10- HACT B cells, with CD4+ T cells.
Helicobacter felis'in makrofaj polarizasyonu üzerine etkisi
Helicobacter pylori (H. pylori) has been identified and classified as type I carcinogen for gastric malignancies such as chronic gastritis, peptic ulcer and gastric adenocarcinoma. Even though more than half of the world's population is infected with the H. pylori, only a minority develops gastric complications and/or remain asymptomatic. The bacteria are rarely eliminated, colonization usually persists throughout life, and infection involves both innate and adaptive immune responses. Helicobacter felis (H. felis) is a gram-negative, spiral- shaped bacterium which was first isolated from the stomach of a domestic cat. It is more immunogenic on mice and zoonotic species of H. pylori. Hence, it is widely used in murine Helicobacter studies because it causes similar pathogenic effect on mice as H. pylori on humans. However, H. felis lacks Vacuolating cytotoxin A (VacA) and Cytotoxin-associated gene A (CagA) virulence factors. Despite lacking important H. pylori virulence factors, it causes more severe gastric inflammation than H. pylori does on mice. Neutrophils, dendritic cells and macrophages mediate innate immune response against H. pylori. Macrophages are plastic and heterogenic group of cells, which can polarize to different types under different stimuli. Polarization status of macrophages changes according to stimuli and the local microenvironment, allowing them to shape the local inflammatory status to adapt to outside stimuli. However, different stimuli do not exist alone in tissues and macrophages may not form clear-cut activated subsets or expand clonally. The various macrophage functions are associated with the stimuli variety, receptor recognition on the macrophage upon stimuli and the presence of cytokines. There are two distinct states of polarized activation for macrophages: the classically activated -M1 type- macrophages and the alternatively activated -M2 type- macrophage subsets. Cell markers alone do not fully define the many subpopulations of macrophages. Therefore, macrophages should be defined based on their specific functional activities. M1 type macrophages are the pro-inflammatory effector cells in innate immune response. Granulocyte macrophage stimulating factor (GM-CSF), lipopolysaccharide (LPS) and IFN- polarize macrophages towards the M1 phenotype which induces the macrophage to produce large amounts of pro-inflammatory cytokines, such as TNF-, IL-1, IL-6, IL-12/ IL-23. The antimicrobial functions of M1 macrophages are linked to up-regulation of inducible nitric oxide synthase (iNOS) that generates nitric oxide from L-arginine and substantial production of NO. M1 type macrophages are professional antigen presenting cells and phagocytes, they express high levels of MHC I and class II antigens, CD40, CD80 (B7.1), CD86 (B7.2) co-stimulatory molecules and secrete complement factors to facilitate complement-mediated phagocytosis. Even though cell markers alone are not enough to fully define the subpopulations of macrophages, it has been shown that M1 type macrophages express CD11c surface marker along with the specific macrophage identification markers such as CD11b and F4/80. M2 type macrophages function in immunosuppression and tissue repair. Exposure to IL-4 or treatment with M-CSF produces an "anti-inflammatory" meaning, alternatively activated M2 macrophages. M2 type macrophages are generally characterized by production of high levels of IL-10 and IL-1RA and low expression of IL-12/ IL-23, combined with high levels of scavenger, mannose (CD206). They repurpose arginine metabolism to ornithine and polyamine by arginase, which promotes growth. Meaning that, they do not produce NO. However, M2 macrophages can be further divided into subsets. M2a, M2b, and M2c based on their cytokine expression profiles. The M2a subtype is induced by IL-4 or IL-13. The M2b is elicited by IL-1R ligands or exposure to immune complexes plus LPS. The M2c subtype is characterized by production of IL-10, TGF- and glucocorticoid hormones. The fourth type of macrophage M2d (or TAMs) is characterized by an IL-10high IL-12low M2 profile. M2d's have phenotypic and functional attributes distinct from M2a-c. Their functions are immunosuppression, low tumoricidal activity and promotion of tissue remodeling and angiogenesis. There are several studies regarding effects of H. pylori on macrophages. In early studies, it has been shown that H. pylori induces the expression of inducible NO synthetase (iNOS) from macrophages along with pro- inflammatory cytokines such as IL-6, IL-8, TNF-α, IL-1-β. However, in recent studies, it has been shown that, in human gastric biopsy specimens from H. pylori positive individuals, CD163+ (alternatively activated; M2) macrophages were detected. Also, upon H. pylori infection, human monocytes secreted IL-1, IL-6, IL-10, and IL-12p40 (partially secreted as IL-23), but not IL-12p70, meaning that, M2 macrophages were up-regulated and secreted IL-10 but produced less of the pro-inflammatory cytokines than M1 macrophages. Furthermore, cytokines secreted from innate immune cells, antigen presentation from macrophages and dendritic cells and changes in the microenvironment, also activates the adaptive immune response against H. pylori. Pro-inflammatory Th1 and Th17 CD4+T cells mediate the pre-dominant adaptive response against H. pylori, the latter being induced by IL-23, IL-1, and IL-6. In spite of evidence for CD4+ T-cell-mediated protection against H. pylori infection in mouse models, Th1 and Th17 responses in humans are also present in chronic H. pylori infection as is IL-17. In mice, the induction of regulatory T (Treg) cells with the simultaneous suppression of Th17 cells may contribute to bacterial persistence. Also, recent findings revealed that murine splenic B cells produce and secrete IL-10 upon Helicobacter-infection in vitro as well as in vivo. IL-10 producing regulatory B cells restrain excessive Th1-type pro-inflammatory immune response and gastric immunopathology of C57BL/6 mice via suppression of CD4+ effector T cells. The interaction between regulatory B cells and T cells were also denoted as required for the function of regulatory B cells (Bregs). Bregs were shown to be able to convert CD4+ T cells into IL-10-producing T regulatory 1 (Tr-1) cell through direct interaction. Tr-1 cells and Bregs work in harmony in order to restore the immune balance in Helicobacter-infection by ameliorating excessive gastric immunopathology while preventing bacterial clearance in the gastric mucosa. Furthermore, in humoral response, H. pylori- specific serum IgM antibodies were present in H. pylori infected humans. Also, Serum IgA and IgG antibodies were directed toward many different H. pylori antigens. Above information shows that there is some information about the effects of H. pylori on macrophages but not definite characterization of H. pylori or H. felis on murine or human studies. Therefore, in this study, we investigated the effect of H. felis on polarization of two types of macrophages: bone marrow- derived macrophages and peritoneal macrophages to show the polarization status and their differences according to their surface receptor expressions and cytokine profiles. For that purpose, firstly bone marrow cells were isolated from leg bones of C57BL/6 mice and were differentiated to bone marrow- derived macrophages in the presence of M-CSF derived from L929 cell line. Peritoneal macrophages were collected from peritoneal cavity of thioglycollate induced C56BL/6 mice. After determining the percentage of differentiation and purity of isolation, cells were treated with LPS, H. felis sonicate, or left untreated as an internal control for 24 hours. Small portion of cell pellets were used for surface marker stainings of activation markers (CD40, CD80 and CD86) and of M1 and M2- type specific surface markers (CD11c and CD206, respectively). Also, their supernatants were collected for ELISA to measure IL-12/IL-23 (p40), TNF-, IL-1, IL-10, and NO secretion levels via griess reagent protocol. In addition to that, their pellets were collected for relative gene expression analysis of IL-12/IL-23 (p40), TNF-, IL-1, IL-10, IL-6 and iNOS using real-time PCR assay. The results of this study indicated that both LPS and H. felis sonicate- treated cells express high levels of activation markers of CD40 and CD80. CD86, which also is an activation marker, was the determining marker in identifying the activation status. CD86 marker expression suggesting the activation status, did not affect the polarization status of both bone marrow- derived and peritoneal macrophages. LPS treated bone marrow- derived macrophages were categorized as M1 type macrophages with the high CD11c and low CD206 expression, and H. felis sonicate- treated bone marrow -derived macrophages were categorized as M2 type macrophages with the high CD206 and low CD11c expression. However, high levels of CD11c surface marker expression was detected in LPS and H. felis sonicate- treated, thioglycollate induced peritoneal macrophages. Despite the high levels of CD11c expression, there was higher CD206 expression among H. felis sonicate- treated peritoneal macrophages when compared to LPS treated group. Therefore, as a result of surface marker expressions, there seems to be no correlation between the activation status and polarization status of peritoneal macrophages. After that, cytokine profiles of activated macrophages were also examined. Firstly, activation status of bone marrow- derived and peritoneal macrophages were assessed via CD86 marker expression. After that, cytokine profiles of activated macrophages were identified. As a result, LPS- treated BM- derived and peritoneal macrophages were polarized to M1 phenotype with high IL-12/IL-23 (p40), TNF-, IL-1, IL-6 cytokine expression and secretion, NO production and iNOS expression. Furthermore, H. felis sonicate- treated bone marrow -derived and peritoneal macrophages categorized as M2b phenotype with the high anti-inflammatory IL-10 production along with TNF-, IL-1, and IL-6 production. Also, IL-10 secretion observed from LPS- treated bone marrow- derived and peritoneal macrophages was thought to be the protective effect of IL-10 against LPS toxicity. However, production of pro-inflammatory IL-12/IL23 (p40) cytokine lead us to the conclusion that H. felis does not drive macrophages to polarize into only one phenotype of M2b, but there is probably M1 type macrophages mixed in the population. In conclusion, this study has contributed to the literature through providing definitive characterization of H. felis infected bone marrow- derived and peritoneal macrophages polarization, describing the surface marker and cytokine profiles for the first time.