Master Degree / Yüksek Lisans Tezleri
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Master Thesis The Effects of Newly Synthesized Compounds on the Hiv-1 Reverse Transcriptase and Blood Cells(Izmir Institute of Technology, 2002) Yılmazer, Özgür; Güneş, HaticeAcquired Immunodeficiency Syndrome (AIDS) is a result of replication of Human Immunodeficiency Virus (HIV-1) in an infected host. Reverse transcriptase (RT) enzyme of HIV-1 is a multifunctional enzyme in the life cycle of the virus. Even though many compounds have been developed against different aspects of HIV-1, RT enzyme is a prime target for the development of drugs against HIV-1 because eucaryotic cells do not have RT activity. In order to develop new therapeutic agents against HIV-1, nineteen newly synthesized compounds were analyzed for their effects on inhibition of RT activity as well as their effects on viability, proliferation, and activity of peripheral blood mononuclear cells (PBMC). Finally, mutagenic effects of the compounds were investigated. Results indicated that AVM 002 and AVM 014 were the most promising compounds with 51% and 43% inhibitory effects, respectively on RT enzyme at 100.M concentration. The compounds AVM 001, AVM 010, AVM 011, AVM 015 and AVM 019 also showed inhibitory effects between 24% and 40% at 100.M concentration.AVM 002 did not cause any toxic effect on cell viability, proliferation and activation until 500.M concentration. Similar to AVM 002, AVM 014 did not show any toxic effect on the cell viability until 500.M and 1000.M at the and of 24 hour incubation; however, AVM 014 at 500.M and 1000.M resulted in 2-fold decrease in the cell viability after 48 and 72 hour incubation, compared to the control. Unlike AVM 002, AVM 014 gave rise to 3,4-fold decrease in cell activity at the end of second and third day incubation. Moreover, among all of the tested compounds, AVM 010 was the most toxic at 500.M and 1000.M compared to the control. Furthermore, all the compounds did not show any mutagenic effect on Salmonella typhimurium TA 100 and TA 102 strains.In summary, the results indicate that AVM 002 and AVM 014 is the best candidate to be improved in order to reach higher RT enzyme inhibitory effect and decreased cytotoxicity profile by slight modifications in compound structure.Master Thesis Biochemical and Molecular Characterization of Extracellular Enzyme Producing Staphylococci Isolated From Different Origins(Izmir Institute of Technology, 2006) Appak, Sıla; Güneş, HaticeStaphylococci are pathogenic bacteria known to cause diseases among diferrent organisms including human. The two species Staphylococcus aureus and Staphylococcus epidermidis are well defined in human diseases although their exact mechanism of pathogenesis is still not fully understood. These pathogenic bacteria could be isolated from soil, water, air, as well as from the living organisms and they are both pathogenic and saprophytic.Extracellular enzymes of the organisms are used for the industrial purposes. The isolation and characterization of these enzymes are crucial steps in biotechnology. The extracellular enzymes derived from the bacteria serve for many purposes in the industry. In this project 128 Staphylococcus sp. were used. Of these 128 bacteria, 12 were isolated from patients, 40 were isolated from the foodhandler.s hygiene detections, 27 were isolated from pygeons and 49 of them were reference strains. They were searched for the presence of some of the industrially important extracellular enzymes: protease, lipase, cellulase, xylanase, amylase, laccase, urease, DNase and pectinase with biochemical tests. They were also searched for the presence of the lipase, protease and thermonuclease amplifications by PCR. The bacteria apart from the refence strains were also tried to be identified by 16S-ITS-rRNA RFLP analysis. The results would indicate the extracellular enzyme production among these pathogenic bacteria and would also be used as a guide in further studies to correlate between Staphylococcal pathogenity and enzyme production.Master Thesis Test of Biomaterials in Biological Systems(Izmir Institute of Technology, 2001) Sudağıdan, Mert; Güneş, HaticeCeramic, metallic, polymeric and composite materials are generally used as biomaterials in order to improve human health. In addition to desired mechanical properties of biomaterials, biocompatibility is important in the treatment or replacement of body parts. Prior to the introduction of new biomaterials to the market, detailed biological tests are carried out to prevent any undesired side effects in the body. Both in vitro and in vivo tests are applied initially which is followed by the evaluation with clinical trials of the biological safety and performance.The aim of this study was to examine some biomaterials in biological systems.For this purpose, the effects of ceramic, metallic, polymeric and ceramic composite materials with different chemical and surface properties on the viability of peripheral blood mononuclear cells (PBMC) by trypan blue exclusion method, on the proliferation of PBMC by incorporation of bromodeoxyuridine to DNA in the proliferating cells and the activation of PBMC by MTT test were investigated. Furthermore, the effects of biomaterials on the secretion of proinflammatory cytokines (IL-1. and IL-6) from PBMC were examined by using ELISA kits. The alteration of conductivity and pH in different solutions were determined to elucidate dissolution properties of ceramic pellets. In addition, AMES test (Salmonella typhimurium reverse mutation test) for the determination of mutagenic potentials and the agar diffusion method for examination of anti-bacterial effects of biomaterials were applied. Adhesion of pathogenic bacteria to the surface of biomaterials was investigated by staining bacteria and examining under the optical microscope.Except for HA 800 C pellets, all samples showed positive results for biocompatibility compared to the controls without biomaterials. The dissolution of HA 800 °C pellets in the culture medium changed the ionic environment that led to a decrease in the viability, proliferation and activation of PBMC. However, BSA-coated HA 800 °C pellets increased the cell viability with respect to uncoated HA 800 °C pellets. Polished metallic samples and other metallic and polymeric samples showed high percent cell viabilities during 48 hours. After 72 hours, most probably because of released ions and particles to the environment, a decline in the viabilities of PBMC was determined. A negative correlation between increasing extract concentration and the cell viability was observed for all ceramic samples especially after 48 and 72 hours treatments.Cytokine secretion analysis after treatment of PBMC with biomaterials indicated that HA 800 °C, HA-Alumina 1250 °C and HA-Zirconia 1250 °C pellets led to a decrease in IL-1. secretion and BSAcoated HA 800 °C pellets in the presence of LPS. Stainless steel, titanium alloy and cirulene pellets caused low levels of IL-1. secretion. In addition, BSA-coated HA 800 °C pellets increased IL-6 secretion compared to uncoated pellets. In metallic samples, low IL-6 levels were obtained with and without LPS stimulation.Moreover, the proliferation and activation of PBMC in the presence of biomaterials were evaluated. HA 800 °C, HA-Alumina 1250 °C and HA-Zirconia 1250 °C samples had inhibitory effects on the proliferation of PBMC in the presence of Con A. In the activation of PBMC, HA 800 °C and HA 900 °C samples showed the lowest values at all incubation periods. Moreover, other ceramic samples showed lower cell activation than the control cultures after 48 and 72 hours treatments. In addition, the cell activation was observed at 24 hours after treatment with the ceramic extracts at low concentrations.Furthermore, investigation of dissolution properties of ceramic samples indicated that only HA 800 °C pellets led to significant increases in the conductivity of cell culture medium and deionized water. Moreover, a slight increase in the pH levels of solutions was obtained in the presence of HA 800 °C samples, but not in the other samples.Finally, none of the extracts of biomaterials in PBS had mutagenic effect on Salmonella typhimurium TA100 strain when they were compared to the mutagenic material (sodium azide) and the negative controls. In addition, all tested ceramic powders, ceramic pellets, metallic and polymeric materials had no anti-bacterial effects on both gram-negative strains (E. coli, P. aeruginosa, K. pneumonia, Proteus spp.) and gram-positive strains (S. aureus and S. pyogenes). In bacterial adhesion studies, it was found that surface roughness and other surface properties play important roles for attachment, adhesion and formation of biofilm by bacteria on the surface of material.As a result, although the ceramic samples sintered at low temperatures resulted in a decrease in the viability of PBMC, all tested biomaterials showed positive results for in vitro biocompatibility evaluation.Master Thesis Investigation of Chromosomal and Plasmid Dna Profiles of Lactococcus Lactics Ssp. Lactis(Izmir Institute of Technology, 2005) Okuklu, Burcu; Güneş, HaticeLactic acid bacteria used in the manufacturing of cheese and other fermented milk products are known as starter cultures. Starters play an important role in the sensory properties of fermented milk products by lactic acid production which influences desirable quality characteristics. In dairy industry, diversity of starter strains of Lactococcus lactis is very limited. There is a considerable demand for novel starters with technologically desirable properties. Gene products responsible for lactose fermentation are encoded on plasmid DNA. Therefore, the plasmid stability of lactic acid bacteria is an important industrial property for starter culture. In the previous study, Lc. lactis ssp. lactis strains were isolated from an artisanal "comlek peyniri'" from Cappodocia and these strains were selected as good acid producers. The aim of this study was to investigate intraspecific diversity of 54 Lc. lactis ssp. lactis by choromosomal and plasmid profiling. In addition, the plasmid stability of 35 artisanal starters was determined by the examination -galactosidase activity. First of all, partial DNA sequencing of the 16S rRNA genes was carried out in order to confirm that the previously identified isolates were Lc. lactis ssp. lactis. The partial DNA sequencing and blast search results indicated that representative isolates showed 90 % homology with Lactococcus lactis. Based on chromosome and plasmid profiling results, 35 Lactococcus lactis artisanal starters exhibiting good acidifying activities were classified into ten different genotypes and nine plasmid groups. None of the reference strains of Lactococcus lactis ssp. lactis and Lc. lactis ssp. cremoris and Lc. lactis ssp. lactis biovar. diacetylactis were included into these genotypes and plasmid groups. The rest of the 17 isolates which were defined as low acid producers by technological methods were clustered into thirteen unique genotypes and seven plasmid groups. Only four of the isolates were found to contain the most stable plasmids by instability studies and these kept their lactase activities during prolonged subculturing. As a result, chromosome and plasmid DNA profiling allowed us to classify artisanal starter strains in certain groups. The strains with stable and instable plasmids will serve us to characterize and improve technological features of these artisanal starter strains in future studies.Master Thesis Isolation and Characterization of Bacillus Thuringiensis Strains From Different Grain Habitats(Izmir Institute of Technology, 2004) Apaydın, Özgür; Güneş, HaticeBacillus thuringiensis is a Gram positive, facultative anaerob bacteria that produces proteins toxic against different insect species. This feature makes it the most widely used biological control agent in agriculture. Since B. thuringiensis strains have great genetic diversity, the toxic behaviours of these strains differ from region to region. Native B. thuringiensis strains are isolated from different habitats and characterized to determine their toxic potential all over the world. The aim of this study was to isolate B. thuringiensis strains from different grain habitats in Central Anatolia and Aegean Regions, and to investigate their phenotypic and genotypic characterizations. Total 96 samples containing soil, grain, stored product dust, straw and various residues were collected from wheat farms, grain silos, haylofts and caves in Ereli/Konya, Takale/Karaman, Nikfer/Denizli, and Bozbük/Söke under aseptic conditions. Seven hundred bacteria were isolated from these samples by sodium acetate selection and heat treatment. For phenotypic characterization, 500 of these isolates were grown for 48 h and crystal protein production was observed by phase contrast microscobe during spore formation. One hundred and sixty three of the bacterial colonies were identified as B. thuringiensis. The isolates were divided into 5 different groups based on the shape of the crystals that they produced. Spherical type crystal morphology was mostly observed type among the others. For genotypic characterization, the cry gene content of the isolates were screened by polymerase chain reaction (PCR) analysis. In addition, chromosomal DNA analysis of 34 isolates by Pulsed Field Gel Electrophoresis (PFGE) as well as plasmid DNA profiling for all isolates were also carried out. One hundred and three isolates were positive for 5 different cry genes (cry1, cry2, cry3, cry4, cry9) examined by PCR. Among all cry genes examined, cry1 and cry9 genes were mostly found in the isolates. Morover, plasmid profiling of the isolates indicated that a 15 kb DNA band was present in all the isolates; however, some of them had more than one DNA band at different sizes. Finally, chromosomal DNA profiling by PFGE showed different DNA patterns for isolates containing the same cry gene which suggest a high level of diversity among the B. thuringiensis strains isolated. Further studies related with extensive genetic characterization and toxic activity of each B. thuringiensis strain will give more comprehensive results on biodiversity of B. thuringiensis strains in Anatolia.Master Thesis Expression Profiles of Differentially Expressed Genes of Rat Mammary Adenocarcinoma in Various Tumor Cell Lines and Effects of Some Antioxidants(Izmir Institute of Technology, 2005) Certel, Seçil; Güneş, Hatice; Güneş, HaticeCancer is the most frequent reason of death in humans after heart disease. Most of the cancers are caused by mutations in tumor suppressor genes that play distinct roles in tumor formations. If a tumor suppressor gene is mutated, it loses its function to control cell division which may lead to overgrowth of cells that leads to tumor formation. It is important to identify the genes involved in tumor formation and metastasis to develop new cancer prevention and treatment methods. In a previous study, differentially expressed genes were identified between poorly metastatic and highly metastatic cell lines of rat mammary adenocarcinoma R3230AC. Eight cDNA clones from poorly metastatic CAb.D5 cell line and six cDNA clones from highly metastatic LN4.D6 cell line were identified (Gunes and Carlsen, 2003). The aim of this study was to investigate expression profiles of these differentially expressed genes in a set of different adenocarcinoma cell lines to find if they have any relation with metastasis. Cell culture and stocks of sixteen different cell lines were prepared and RNA from these cells were isolated. After synthesizing cDNA, RT-PCR analysis was carried out using primers specific for each cDNA clone. It was found that the gene clones FF-10 and SG-1 were expressed in non-metastatic cells but not in metastatic cells suggesting that they may have a tumor suppressive potential. Antioxidants are chemicals that prevents oxidation and free radical damages on cells. Because there is some evidence that antioxidants may prevent tumor formation, effects of antioxidants such as green tea catechins, beta carotene, lycopene as well as zeolite were examined on cancer cell growth and expression profiles of the differentially expressed genes. The cells were treated with different amounts of antioxidants and the cell growth was determined by MTT assay. The effects of antioxidants in gene expression were identified by RT-PCR analysis. At 100µM and higher concentrations, epigallocatechingallate (EGCG), epigallocatechin (EGC) and beta carotene significantly inhibited cell growth. Lycopene affected the cell growth at 3µM concentration. FH-2 expression decreased by 1.8-fold with lycopene treatment. In addition, EGCG increased the SG-1 expression by 1.14-fold. No effects of antioxidants as well as zeolite on the expression of other differentially expressed genes were observed. For further studies, investigation of expression profiles of differentially expressed genes in primary and secondary tumors of human will give more definitive results for the metastatic relevance of these genes.Master Thesis Isolation and Characterization of Bacillus Thuringiensis From Olive Tree-Related Habitats(Izmir Institute of Technology, 2005) Çınar, Çelenk; Güneş, HaticeAbstract:Bacillus thuringiensis (commonlyreferred to as Bt) is a Gram-positive, sporeforming soil bacterium that produces insecticidal crystal proteins during sporulation.These crystals are referred as Bt toxins or -endotoxins. The most important characteristics of the toxins are their insecticidalactivity against many insects. Since their insecticidal potential has been discovered,it has been produced commercially and used as microbial pesticides all over the world.Therefore, the aim of this study was to isolate Btfrom olive tree-related habitats, and to determine the phenotypic andisolate Btfrom olive tree-related habitats, and to determine the phenotypic and genotypic characteristics of the isolates. To accomplish this purpose, 240 samples were collected in the Aegean Region. The phase-contrast microscopy results showed the presence of crystals in 54 environmental samples, corresponding to 100 Bt isolates. The crystal morphologies were spherical, bipyramidal, cuboidal, irregular pointed, and irregular shaped. The greatest proportion of samples yielding this organism was from the soil. The remaining were from olive leaf residue, green olive leaves, animal faeces,dust samples, and olive pomace. The isolates were characterized on the basis of biochemical characters, cry andcyt gene content, plasmid profiling, 16S-ITS rDNA RFLP.Biochemical tests included protease (caseinase and gelatinase), lecithinase, amylase, nuclease, urease, esculinase, arginine dihydrolyse activity; fermentation of sucrose, salicin, mannose, cellobiose, and maltose, production of acetyl-methyl carbinol, methyl red reaction. Polymerase chain reaction (PCR) has been applied for the identification of cry1, cry2, cry4, cry9, cry11, cry13, cyt1, and cyt2 genes. 68% of the isolates amplified cry1 gene; 57% amplified cry4; 20% amplified cry11; 26% amplified cry9; 20% amplified cry2 genes, andnone of the isolates harbored cry13 gene. Cyt1gene was found in 40% of the isolates while cyt2 gene was present in 80% of the isolates. The most abundant genotype of cry genes was cry1 and cry4. Most of the isolates(58%) possessed more than one cry gene. In addition, different combinations of cry andcytgenes were obtained. Plasmid profiling showed the presence of plasmids in all isolatesand the number of plasmids was usually morethan one. Also, the discrimation effect of 16S-ITS rDNA RFLP was tested to differentiate certain isolates and reference strains, which showed similar biochemical characteristics.Master Thesis Isolation of Bacillus Thuringiensis and Investigation of Crystal Protein Genes(Izmir Institute of Technology, 2002) Çetinkaya, Fatoş Tuba; Güneş, HaticeBacillus thuringiensis is a ubiquitous, gram-positive and spore-forming bacterium. During sporulation, it produces intracellular crystal proteins (cry proteins), which are toxic to insects. Because of its insecticidal activity, it has been used for nearly fifty years to control certain insect species among the orders Lepidoptera, Coloeptera, and Diptera. However, it is still necessary to search for more toxins to control other insect orders and to provide alternatives for coping with the problem of insect resistance. The genetic diversity of B. thuringiensis strains shows differences according to the regions where they were isolated. Thus, each habitat may contain novel B. thuringiensis strains, which have some toxic effects on target spectra of insects. The aim of this study was to isolate B. thuringiensis strains from different environments and to identify the crystalline protein gene content of the isolates. Sixty five samples including soil, stored product dust, insect cadavers, and dry leaf residues were collected from Akhisar/Manisa, İzmir, and Ereğli/Konya. Three approaches were applied for the isolation of B. thuringiensis: sodium acetate selection, heat treatment, and endospore staining. Polymerase Chain Reaction (PCR) method was used for the characterization of cry gene content of B. thuringiensis strains. The universal primers specific to cry 1, cry2, cry 3, and cry 9 genes were used to detect the type of cry gene carried by each environmental isolate of B. thuringiensis strains. In addition, 16S rRNA based PCR-restriction fragment length polymorphism (RFLP) was carried out to confirm B. thuringiensis strains. Finally, SDS-PAGE analysis was optimized to detect protein profiles of crystal proteins obtained from B. thuringiensis isolates. It was found that, 136 of 359 isolates showed B. thuringiensis-like colony morphology and subterminal endospore position. One hundred isolates were screened by PCR and 18 of them were found to contain cry genes (5 cry 1, 3 cry3, and 10 cry 9). However, the cry 2 gene was not detected from any isolates. 16S rRNA based PCR-RFLPfor 18 isolates gave the same restriction pattern as positive controls, indicating that all 18 isolates were B. thuringiensis. SDS-PAGE studies for Cry 9 proteins of the isolates exhibited different protein profile from positive control of B thuringiensis strain.