FBE- Moleküler Biyoloji-Genetik ve Biyoteknoloji Lisansüstü Programı - Yüksek Lisans

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  • Öge
    Investigation of antimicrobial resistance in wild-type and stress-resistant Saccharomyces cerevisiae strains
    (Institute of Science and Technology, 2018-06-07) Ismaeel, Riziq Nahidh ; Çakar, Zeynep Petek ; 521151116 ; Molecular Biology – Genetics and Biotechnology
    Fungi are organisms that live in various habitats like air, soil and water. Some species live as symbionts and some are parasites. Fungi kingdom consists of mushrooms, molds and yeasts. They all contain rigid cell walls. Yeast are spread worldwide, they are mostly in the phylum ascomycota, and only a few are within the phylum Basidiomycota. There is a high number of economically important types of ascomycete yeasts. These types are commonly used in bread, wine, and beer production; and are selected from Saccharomyces cerevisiae strains. Saccharomyces cerevisiae is a eukaryotic unicellular microorganism. It has a completely sequenced compact genome which makes it a suitable microorganism for easy manipulation of the genome and also ease of cultivation. In this study, ethanol-resistant, coniferyl aldehyde-resistant, caffeine-resistant, propolis-resistant and antimycin-resistant mutant S. cerevisiae strains which were previously obtained by inverse metabolic engineering strategy, were analyzed against different antimicrobials and possible relations between resistance to those antimicrobials were investigated. For this analysis, spot assay method was applied to reference strain (905) and mutant strains under respirative and fermentative conditions against rapamycin, erythromycin, kanamycin and antimycin-A antimicrobials. Antibiotics are generally known and referred to as a subset of anti-infective agents which are derived from bacterial or fungal sources that are used to treat bacterial infections. Antibiotics are generally used medically to treat or prevent bacterial infections. Most antibiotics inhibit the replication or completely kill bacteria. The name 'antimicrobial' is a general term for any compound that inhibits some vital functions or kills microorganisms including chemicals such as antibiotics, antifungal agents, antivirals, and antiseptics. Fungi and viruses may also pose some risks to humans and are targeted by antifungals and antivirals, respectively. Rapamycin is a macrocyclic antibiotic. It is produced by the soil bacterium Streptomyces hygroscopicus found on Easter Island soil. Rapamycin was discovered as a potent antifungal agent, but initially, it exhibited an undesirable and immunosuppressive effect, leading to its development as a clinically useful drug. Rapamycin is the inhibitor of Target of Rapamycin (TOR) pathway. This pathway has important roles such as nutrient sensing, chronological aging, apoptosis and energy metabolism. There is a recent discovery which states that the mTOR inhibitor rapamycin can extend the lifespan of mammals which has created great excitement, as it represents the first demonstration of the pharmacological extension of maximum life in a mammalian species. Since then, rapamycin effects on the lifespan of mammals have been confirmed by some additional studies. Kanamycin is mostly known and referred as kanamycin (A). It is an aminoglycoside bactericidal antibiotic which is found in oral, intravenous, and intramuscular forms. It is used to treat and cure a wide range of infections. Kanamycin is isolated from the bacterium Streptomyces kanamyceticus, and its mostly and commonly used form is kanamycin sulfate. Aminoglycosides function by binding to the bacterial 30S ribosomal subunit and causing misreading of t-RNA. By this way, the bacterium cannot synthesize the proteins vital for bacterial growth. Aminoglycosides are generally used against infections of aerobic, gram-negative bacteria such as Pseudomonas, Acinetobacter, and Enterobacter. Additionally, some mycobacteria are susceptible to aminoglycosides, including bacteria that cause tuberculosis. Infections caused by gram-positive bacteria can also be treated with aminoglycosides, but other antibiotics are more potent and less harmful to the host. Erythromycin is a bacteriostatic macrolide antibiotic produced by Streptomyces erythreus. By binding to the 50S ribosomal subunit, it inhibits protein synthesis. The binding to the 50S ribosomal subunit inhibits peptidyl transferase activity and interferes with the translocation of amino acids during the cycling and coupling of proteins. Erythromycin is released from the bacterial cell membrane and is reversibly attached to the 50S subunit of the bacterial ribosome. This prevents bacterial protein synthesis. Depending on the concentration of the drug, erythromycin may exhibit bacteriostatic or bactericidal action in the infection site and the sensitivity of the related organism. Additionally, erythromycin may enhance the actions of other drugs by the inhibition of microsomal metabolism, leading to toxicity of these other medications. Erythromycin is available in the form of different esters, including erythromycin estolate, erythromycin lactobionate, erythromycin ethylsuccinate, and erythromycin gluceptate. Antimycin-A is an antifungal produced by Streptomyces species. It is the mitochondrial complex III inhibitor. Antimycin binds closely to a pocket in 1 of 5 of the major electron transport proteins. Antimycin binds at the stage where ubiquinol, referred to as (coenzyme Q,) which usually binds to produce electrons to the level of (O2) that is bound to a close iron-containing enzyme. This explains why the electron transporter is blocked at some stages, the bound oxygen is exchanged to superoxide, a very reactive form of oxygen. Antimycin interacts specifically with the complex system of protein structures related to the electron movement. Biochemists make use of this specific binding of antimycin to avert the electron flow and to investigate the chemical details of aerobic respiration. With these investigations, significant information has been revealed about how antimycin adds at the enzyme level, down to the molecular level and the specifics on how the side chains on the antimycin can impact and influence the electron movement at the protein stage. Antimycin is an active ingredient in Fintrol®, a registered product, used as an industrial agent (pesticide). As a result of the antifungal properties, it has been demanded for commercial applications in agriculture. Antimycin is highly toxic to fish species. However, it is used in many fisheries. It has also been used as a commercial toxic agent to get rid of the unwanted fish species in catfish farms. Cross resistance analysis revealed that all mutant strains showed similar behavior against erythromycin and antimycin-A stress. Additionally, under fermentative conditions, only rapamycin affects the growth of the yeast. The aim of this study was to determine the antimicrobial resistances of some stress-resistant S. cerevisiae mutant strains and to identify possible relationships between cross-resistances to different antimicrobials and a variety of stress types. The results could be useful for future research on antibiotics and antifungals resistance.
  • Öge
    Evolutionary engineering and physiological analysis of antimycin-a resistant Saccharomyces cerevisiae
    (Institute of Science and Technology, 2018-06-07) Topaloğlu, Alican ; Çakar, Zeynep Petek ; 521151101 ; Molecular Biology - Genetics and Biotechnology
    Saccharomyces cerevisiae is a well known eukaryotic model organism in terms of its clearly known genetic and metabolic basis and the ease of cultivation. Its genome was fully sequenced at 1996 and it was the first eukaryotic organisms which the genome was fully sequenced. Additionally, S. cerevisiae genome has high homology genes with the human genome. Furthermore, it is also a good model organism to study mitochondrial functions because of its special properties. Most of the discoveries about mitochondria were found on S. cerevisiae model organism. Most importantly, it is a facultative microorganism which can survive under mitochondrial dysfunction and it has several survival mechanisms against mitochondrial DNA mutations. Mitochondria is an organelle which has the main function of producing energy by oxidative phosphorylation. In addition to this function, it has also other responsibilities like thermogenesis, apoptosis, lipid and sterol synthesis and calcium storage. It is a double-membrane organelle and includes its own mitochondrial DNA (mtDNA) and ribosomes which are structuraly different than cytoplasmic ones. As its structure and functions, it is more likely to prokaryotic microorganism. Functional disorders of mitochondria might cause mitochondrial diseases which are observed in the most energy-requiring systems, mainly nervous and muscle system. Those disorders typically cause cardiovascular and neurodegenerative disorders, vision and hearing loss, diabetes, kidney and liver failures. Although many discoveries were made on the pathogenesis of mitochondrial disorders in the last two decades, there are still no effective treatments to cure those diseases. Most of the genetic and functional information about mitochondria was obtained by the studies with S. cerevisiae as the model organism. Today, most of the mitochondrial diseases caused by genetic mutations are researched on S. cerevisiae models by using recombinant DNA technologies and rational metabolic engineering techniques. However, such experimental design has some limitations, such as requiring detailed genetic background knowledge, rejection of recombination by the organism and obtaining unexpected phenotypic results. Because of these limitations, inverse metabolic engineering has been introduced as an alternative approach. In light of this information, in the present study, it was aimed to obtain antimycin-A resistant S. cerevisiae mutants by evolutionary engineering, an inverse metabolic engineering strategy, and to complete physiological analyses that are the first steps to enlighten the molecular basis of this resistance. Antimycin-A is a mitochondrial complex III inhibitor which binds to bc1 complex and inhibits electron transfer. It is the analog of quinone and binds Qi site of the bc1 region (Quinone binding site) Binding of antimycin-A to this site blocks further electron transfer from Qo site and electron transfer to complex IV is stalls. As a result, cells cannot produce enough energy via mitochondrial oxidation and are obliged to find alternative metabolicpathways to survive. Additionally, antimycin-A cause increased Reactive Oxygen Species (ROS) generation by disrupting redox reactions on the mitochondria. Other than antimycin-A, there are various mitochondrial inhibitors such as atpenin, oligomycin, stigmatellin or cyanide which are widely used in research. While deciding the mitochondrial inhibitor among others, many factors were considered. Thus, considering the high affinity to bind mitochondrial complex III, the power of inhibition at low concentrations and safety of use, it was decided to use antimycin-A as the mitochondrial inhbitior in this study. In this study, antimycin-A resistant mutants were selected successfully from both reference strain and EMS-mutagenized reference strain population. As the selection method, gradually increasing stress level application protocol was used in succesive batch cultures under respirative conditions to make cells dependent on mitochondrial functioning. After 52 passages, antimycin-A concentration was increased to 6.6 nM and mutant individuals were randomly picked from the final populations. Twelve mutants were chosen from the reference strain selection and 15 mutants were chosen from the EMS-mutagenized reference strain population. After those mutants were screened at different antimycin-A concentrations, two mutants with the highest resistance levels were chosen. It was observed that mutant individuals obtained from the reference strain selection were resistant to 15 nM antimycin-A, while mutants obtained from the EMS-mutagenized reference strain population were resistant to 50 nM antimycin-A. This study, based on the literature information, is the first one where antimycin-A resistant S. cerevisiae mutants were obtained by evolutionary engineering strategy. To determine whether the gained antimycin-A resistance was permanent or not, genetic stability test was applied. Except for one mutant individual obtained from the reference strain selection, all resistant mutants tested were genetically stable. Additionally, antimycin-A resistant mutant population obtained from the reference strain selection might indicate possible mutagenic effects of antimycin-A. Within the frame of physiological characterization experiments, cross-resistance analyses results revealed that, antimycin-A resistant mutants were also cross-resistant to caffeine, propolis, coniferyl aldehyde and ethanol. Similarly, mutants resistant to these chemicals except for the ethanol-resistant mutant were also found to be cross-resistant to antimycin-A in a previous study. This implies some similar molecular stress resistance mechanisms between these mutants. Resistance to ethanol is also an expected result, because the selection procedure was applied by using ethanol as the sole carbon source. In this study, antimycin-A resistant S. cerevisiae mutants were obtained by evolutionary engineering strategy and the physiological characterization experiments were performed. For further studies, genomic and transcriptomic analyses could be performed to identify alternative pathways which by-pass the effects of the nonfunctional mitochondrial complex, and possible pharmaceutical targets could be identified.
  • Öge
    Recombinant expression, purification and characterization of TNFR1
    (Fen Bilimleri Enstitüsü, 2020) Öz, Yağmur ; Dinler Doğanay, Gizem ; 637756 ; Moleküler Biyoloji-Genetik ve Biyoteknoloji
    Receptor/ligand interactions are the mechanisms that form the basis of many signaling pathways. Establishing the correct interaction is crucial for ensuring regulation at the cell level and for the cell's proper functioning. Cytokines are low-molecular-weight glycoproteins that are secreted by different cells in the body, mainly cells of the immune system and which coordinate and propagate immune responses within the body. They have specific effects on the interactions and communications between cells. Cytokines can be divided into pro-inflammatory (such as tumor necrosis factor-alpha (TNFα), interleukin-6) or anti-inflammatory (interleukin-10). The cytokine TNF-α is expressed by a great variety of cells and forms homo-trimers in circulation that have wide biological effects. It is known that TNF-α has important effects, especially in autoimmune diseases. TNF-α acts by binding to specific receptors on the cell surface, stimulating secondary signal pathways. As a result of the stimulation, the signal cascade continues in three main ways: two of them are effective in the inflammation related to nuclear factor (NF) and the third is effective in the process of apoptosis. Today, the use of TNF-α receptors as inhibitors in the treatment of diseases that develop due to the increase of TNF-α is one of the approaches used in the clinical field. TNF-α has two forms in the body, the first of which is the membrane-anchored form (26 kDa), while the second is the secreted form (17 kDa). Firstly, TNF-α is synthesized as a trimeric molecule in which each subunit is corded to the cell surface through a membrane anchor. These membrane tethers are cut by the TNF-α converting enzyme (TACE / ADAM17) which is major sheddase. TNF-α achieves all its different cellular effects by its binding to either the TNF-α receptor 1 and 2 (TNFR1 and TNFR2). TNFR1 and TNFR2 are transmembrane I type proteins located on the cell surface. TNFR1 can interact with both soluble and membrane-bound forms of TNF-α, while TNFR2 can only be stimulated by membrane-bound TNF-α. TNFR1 consists of three main domains: extracellular, transmembrane, and intracellular domain. Signaling proceeds by the recognition of TNF-α trimers by endogenic TNF receptors (TNFR) 1 and 2, which form trimers themselves before complex formation with TNF-α. The intracellular downstream signal pathway begins as a result of conformational changes occurring in the intracellular domain by the interaction of TNF-α in the trimer structure with the extracellular domain of TNFR1. It has been proven that anti-inflammatory antibodies and antigen-binding fragments (Fab) against TNF-a successfully suppress TNF-a induced inflammation in inflammatory autoimmune diseases. In the field of biotherapeutic medicine, there are approaches regarding the use of TNFR1's extracellular domain as a therapeutic tool. In light of these approaches, we aimed at bacterial production, purification, and characterization of the extracellular domain of TNFR1 (hereafter TNFR1). In our study, the plasmid containing the TNFR1 gene was transferred into the host cell of which is Escherichia coli (E. coli) BL21DE3. Protein expression was achieved by IPTG induction. Inclusion bodies formed in the cell as a result of high protein expression were isolated from cell lysates and solubilized. The target protein was purified from the protein mixture by immobilized metal affinity chromatography (IMAC). Purification efficiency was confirmed by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) and immunoblotting techniques. Blue native polyacrylamide gel electrophoresis (BN-PAGE) was used to understand the native structure of the pure protein and it was found that the protein was in dimer structure. In order to secondary structure estimation of the purified protein, circular dichroism (CD) which is defined as the unequal absorption of left-handed and right-handed circularly polarized light was used. According to measurements in the far-UV range, beta structures were found to be dominant in the protein structure. In the measure of protein purity, capillary electrophoresis (CE-SDS) and high-performance liquid size exclusion chromatography (HPLC-SEC) methods, which are more sensitive techniques for protein characterization, were used. The purified protein was determined to be> 95% purity by CE-SDS. It was confirmed that the protein in the HPLC-SEC was dimeric according to the molecular weight and size of the pure protein. A Pull-down assay was utilized for the determination of the functionality of the recombinant protein. Binding experiments were performed with both the receptor in the cell lysate and the purified ones. As a result of the pull-down assay, the receptor was found to bind to TNF-α. The sample of the binding assay with purified receptor was also analyzed by the HPLC-SEC method and complex formation was observed. Based on the result of the experimental findings, it was found that the methods developed for the engendering and purification of the recombinant TNFR1 protein can be used to obtain the pristine product that maintains its bioactivity.
  • Öge
    Physiological investigation of Rhodobacter sphaeroides
    (Institute of Science and Technology, 2013) Özmeral, Özge ; Çakar, Zeynep Peter ; 352302 ; Molecular Biology - Genetics & Biotechnology Programme
    Rhodobacter sphaeroides is an ?-3 purple non-sulfur eubacterium with an extensive metabolic organization. Under anaerobic conditions it is able to grow by performing photosynthesis, respiration and fermentation. Photosynthesis occurs only under anaerobic conditions since photosynthetic apparatus of R. sphaeroides is harmed in the presence of oxygen. Photosynthesis may be both photoheterotrophic and photoautotrophic. While R. sphaeroides performs photoheterotrophic photosynthesis using organic compounds as both a carbon and a reducing source, R. sphaeroides performs photoautrophic photosynthesis using carbon dioxide as the sole carbon source and hydrogen as the source of reducing power. In addition, R. sphaeroides can grow both chemoheterotrophically and chemoautotrophically. Regarding biotechnology applications, R. sphaeroides is used both in industry and in medicine. Industrial application of R. sphaeroides comprises hydrogen gas and polyhydroxybutyrate (PHB) production and bioremediation of heavy metals. R. sphaeroides can also be used in the production of vitamin B12, coenzyme Q10, 5-aminolevulinic acid (ALA) and porphyrin. Like other microorganisms, R. sphaeroides has different defence mechanisms to give response against different stress types that occurs frequently in its microenvironment. Defence mechanisms of R. sphaeroides vary according to the types of stress. For instance, while under osmotic stress condition, R. sphaeroides gives response by increasing cardiopilin levels which is an anionic phospholipid playing an important role in energy conversion in cells of R. sphaeroides, as for, under oxidative stress condition it gives response increasing its carotenoid levels.
  • Öge
    Disease gene identification using linkage and exome analysis
    (Institute of Science And Technology, 2020-06-15) Yavuz, Derya ; Turanlı, Eda Tahir ; 521171119 ; Molecular Biology-Genetics & Biotechnology Programme ; Moleküler Biyoloji-Genetik ve Biyoteknoloji
    Rare disorders are described as conditions with prevalence less than 1 in 2000 people. Approximately 300 million people are affected with rare disease worldwide and in Turkey this number is estimated as 5 million. More than 6000 rare diseases are identified to date and 80% of these are monogenic disorders. Disease onset is usually during childhood with chronic, progressive and debilitating nature. Patients life expectancy is lower than unaffected population. Extensive clinical examination is required for definite diagnosis. For these reasons it is crucial to reveal underlying mechanisms of rare disorders. Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, chronic autoinflammatory disorder that affects children between age of 2-17. It is characterized by aseptic,symmetric and recurrent bone lesionsthat tend to cluster around metaphysis of long bones such as femur, tibia, vertebral column and pelvic floor. Although there is no definitive data on prevalence of CRMO, it is estimated to be 1-2 cases per million. Though having an unclarified etiology and pathogenesis, families with multiple affected members suggest genetic background on disease development. Identification of disease-causing genes - LPIN2 and IL1RA in Majeed syndrome and deficiency of interleukin-1 receptor antagonist (DIRA) which are defined as chronic nonbacterial osteomyelitis (CNO) further strengthen the involvement of genetic features. Takayasu arteritis (TA) also known as pulseless disease is a rare, systemic large vessel vasculitis that predominantly affects women younger than 40 years old. Its major clinical symptoms are occlusion and stenosis of aorta and its main branches accompanied with loss of radial pulse whereas involvement of pulmonary, coronary, renal arteries and aneurysmal formations are also encountered. Severity of the clinical progression depends both on the vessels afflicted and ethnicity of the patient. Highest prevalence of TA is reported in East Asia, especially Japan with up to 40 cases per million. In Turkey, it is postulated to be the second mostly encountered vasculitis with a prevalence up to 12.8/million accompanied with 1.11/million incidence. In spite of undefined etiology, several factors including infections are suggested to be involved in TA pathogenesis. Monozygotic twins diagnosed with TA and familial aggregation observed in some cases indicate the role of genetic factors in disease phenotype. Various genome-wide association studies (GWAS) in cohorts of different size and ethnicity replicated significant association of HLA-B*52 and IL12B to TA. Susceptibility loci FCGR2A/FCGR3A, MLX, HLA-B/MICA and HLA-DQB1/HLA-DRB1 also show significant association with TA. In this study we present three Turkish consanguineous families with multiple members diagnosed with TA and one consanguineous family with two brothers diagnosed with CRMO. Clinical diagnosis and follow up of TA families were done at Cerrahpaşa Medical Faculty whereas CRMO patients were diagnosed and followed in Pamukkale University Medical Faculty. Individuals' were informed and their written consent for participating in the study was obtained accordingly to the ethics committee approval (MBG.22/2014). The aim of the study is to identify disease causing genes by using gene mapping techniques as linkage and exome analyses. Linkage analysis and homozygosity mapping were used to identify non-recombinant homozygous genomic regions that were shared between affected individuals but not with healthy members. By exome analysis, possible candidate genes were investigated. Linkage analysis and homozygosity mapping of CRMO1 family revealed 54 regions that are larger than 200 kb with LOD score > 1.50 that were shared among the affected siblings. Exome analysis on these regions did not reveal any homozygous, non-synonymous and rare variants. Therefore, exome analysis was conducted without basis on linkage regions revealing 10 variants out of which only CARNS:c.1145C>T had a closer association with CRMO phenotype when compared to the rest. However, the region it lied was homozygous in all family members eliminating the variant from being a candidate. Two sisters diagnosed with TA are present in all three families under investigation. TA1 and TA2 families indicate autosomal recessive inheritance model. Affected sisters of TA3 family are half siblings (sibs) who share the same mother but different fathers. CRMO1 family two brothers were diagnosed with CRMO. Genomic DNA samples obtained from peripheral blood were used for genotyping via genome-wide single nucleotide polymorphism (SNP) array where ~710,00 fixed markers were genotyped using Illumina Infinium OmniExpress microarray. By performing multipoint parametric linkage analyses under recessive inheritance model and homozygosity mapping using the SNP data, non-recombinant regions that were shared among the affected individuals but not with the healthy members were detected. Exome sequencing data were used to fine map these regions to variant resolution and investigate the presence of a potential candidate variants involved in the TA or CRMO phenotype. Both linkage and exome analyses were conducted under the recessive inheritance model. Variants in the functional regions (exonic and splicing) with minor allele frequency (MAF) < 0.01 were further examined with bioinformatics tools. Expression sites and levels, pathways, interactions, evolutionary conservation of the genes harboring candidate variants were studied to unravel any potential involvement in the immune system or inflammatory response that could lead to the TA or CRMO phenotype. Linkage analysis of TA1 family revealed 59 regions larger than 200 kilobases (kb) with maximal LOD score 3 1.50. Homozygosity mapping confirmed 41 of these to be shared between affected individuals. Co-joint multipoint parametric linkage analysis of TA1 and TA2 families indicated 123 linkage regions larger than 200 kb with LOD score 3 1.50. Nine of these were shared between all affected members of both families. Exome analysis of these sites revealed 9 variants in total of which 5 were in TA1-04 which are RELN: c.103301976_103301984dup, ANXA8L1: c.449C>T, EHBP1L1: c.1321C>T, MYH14: c.565C>T and SHANK1: c.3947G>A. The remaining 4 variants in TA2-01 are AP4B1: c.767C>T, MST1L: c.811dupG, AGAP: c.G8A and ZNF829: c.1173A>T. Variant filtration and prioritization of the exomes of TA3 family members, revealed no common variant that are found in affected sibs. There was no gene that got hits that were present in both TA patients. Although our results were not indicative of a gene involved in the TA development, this is the first study integrating linkage analysis, homozygosity mapping and exome analysis where three consanguineous families with multiple affected members were included to identify possible disease-causing variants. Variants detected in TA patients should be further studied to unravel their possible roles in TA pathogenesis.