This study was aimed at designing a cell carrier from an intelligent polymer to achieve loading of mechanical stress for the purpose of improving the tissue engineering capability in vitro. Ethyleneglycoldimethacrylate (EGDMA) crosslinked poly(Nisopropylacrylamide) (pNIPAM) films were prepared by radical polymerization with ultraviolet light (UV) in the presence of photoinitiator 2,2&#039 / -azoisobutyronitrile (AIBN) in isopropanol/water (1:1). Patterns were formed on the surface of the polymers by using silicon wafers with microridges (2 &amp / #956 / m) and grooves (10 &amp / #956 / m) that were prepared by photolithography technique as the template. The surfaces of the films were also modified by adsorption of ELP-RGD6 polypeptide. Bone marrow stem cells (BMSCs) isolated from 6 week old Sprague-Dawley rats were seeded onto the pNIPAM films with different surface topography and chemistry and cultured under static and dynamic conditions. Dynamic conditions were generated by cyclic temperature changes (15 min at 29&deg / C, 30 min at 37&deg / C) for 10 times a day during 5 days starting on the second day post-cell seeding. ELP-RGD6 on the films enhanced initial cell attachment but had no effect on proliferation in long term culturing. However, for the dynamic culturing, ELP was crucial for both retaining cells attached on the surface when the surface became hydrophilic and resulted in weakened cell attachment, and for better communication between cell and material which enhanced the ability of pNIPAM films to transfer mechanical stress on the cells. Dynamic conditions improved cell proliferation but decreased differentiation. Presence of the patterns also influenced the differentiation but did not affected proliferation.

The effects of increasing nitrogen and phosphorus loading on submerged macrophyte development was tested in a mesocosm experiment for three months. Experiment consisted of three NO3-N loadings with factorial of two PO4-P loadings in a fourfold replicated design. Twenty four enclosures placed at one meter depth were isolated from the lake but kept open to sediment and atmosphere. Each enclosure stocked with ten Myriophyllum spicatum shoots with underyearling fish to reduce zooplankton grazers. Biweekly sampling and weekly nutrient additions were performed for three months. Mean total nitrogen (TN) concentrations sustained in nitrogen treatments through out the experiment were 0.52, 1.99, 8.07 mg/l. Both phosphorus treatments converged to a mean concentration below the targeted level, ranging between 0.05-0.1 mg/l TP. In comparison to mesocosm studies in temperate lakes, higher assimilation rates for nutrients were observed in Lake Pedina. Due to extraordinarily high evapotranspiration and drought in 2007, the water level decreased 0.6 m in enclosures. Total macrophyte biomass remained indifferent to nutrient treatments with continuous growth and failed to validate any direct or indirect negative effect of increasing nutrient concentrations. Phytoplankton biomass differed significantly among factorial treatments but remained low, while periphyton biomass differed among nitrogen treatments. In comparison with other studies the phytoplankton biomass remained low and the periphyton biomass became high for reference TP concentrations, indicating a competitive advantage of periphyton over phytoplankton on nutrient utilization in the enclosures. Zooplankton:phytoplankton biomass ratio was low throughout the experiment and zooplankton community mainly consists of smaller species, reflecting high predation pressure.

Acrylamide is an industrially produced chemical with known neurotoxic, reproductive toxin and carcinogenic effects. The carcinogenicity associated with acrylamide is mostly attributed to its metabolism by liver CYP2E1. However, studies investigating the effects of acrylamide on CYP2E1 enzyme are limited. In this study, it was aimed to investigate in vivo interaction of carcinogenic acrylamide on microsomal cytochrome P450 enzyme activities, and protein levels, and on cytosolic NQO1 and GST enzyme activities of rabbit liver, kidney and lung of acrylamide-treated rabbits. The in vivo protective effect of resveratrol, a phenolic compound, was also investigated on acrylamide toxicity. New Zealand male rabbits were treated with acrylamide and resveratrol, separately in different doses and conditions. Their combined effects were also investigated. CYP2E1-dependent p-Nitrophenol hydroxylase, NDMA N-demethylase and aniline 4-hydroxylase activities were found to be significantly increased in acrylamide-treated rabbit liver (1.80-3.0 fold) and kidney (1.6-fold). Rabbit liver and kidney CYP2E1 protein levels (determined by western blot analyisis) also increased approximately 2-fold due to acrylamide treatment. In rabbit liver, resveratrol was found significantly effective in decreasing both acrylamide-induced CYP2E1-dependent enzyme activities (approximately 1.5-1.80 fold) and CYP2E1 protein levels (approximately 1.5-1.70 fold). Additionally, resveratrol significantly decreased acrylamide-induced CYP2E1 protein level (2-2.5 fold) in rabbit kidney. However, no significant change was observed in rabbit lung CYP2E1-dependent enzyme activities and CYP2E1 protein levels due to acrylamide, resveratrol or their combined treatments. Furthermore, it was found that acrylamide treatment significantly increased CYP3A6-dependent erythromycin N-demethylase enzyme activity (1.85-fold) and CYP3A6 protein levels in rabbit liver (1.69-fold). No change was observed in CYP2B4-dependent benzphetamine N-demethylase enzyme activities of rabbit liver, kidney and lung by in vivo acrylamide, resveratrol or their combined treatments. Moreover, total GST and GST-Mu activities of rabbit kidney (1.5-fold, respectively) and total GST activity of rabbit lung (1.6-fold) were increased significantly only in resveratrol treated group. NQO1 enzyme activity of rabbit kidney was significantly increased by acrylamide treatment (1.6-fold). The results of the present study have demonstrated for the first time that acrylamide induces rabbit liver and kidney CYP2E1-dependent enzyme activities and CYP2E1 protein levels. The induction of CYP2E1 enzyme activity and protein level by acrylamide treatment can stimulate formation of other toxic compounds and procarcinogens metabolized by CYP2E1 which in turn further potentiates the risk of hepatotoxicity, mutagenicity and carcinogenicity. In the present study, it was also demonstrated for the first time that acrylamide treatment also increases CYP3A6 enzyme activity in rabbit liver which may lead to alterations in drug metabolism. The results of this study have also suggested that resveratrol may have protective effects on acrylamide induced toxicity / however, further in vivo studies are required to clarify the effect of resveratrol on both acrylamide-induced toxicity and anti-oxidant enzymes.

Resveratrol is one of the promising naturally occurring polyphenolic compound found in red wine having antioxidant and anti-carcinogenic properties. However, in vivo studies investigating the effects of resveratrol on antioxidant enzymes are limited. In the present study, we investigated, for the first time, the influence of resveratrol on liver and kidney antioxidant enzymes and oxidative stress markers in acrylamide treated and control rabbits. New Zealand male rabbits were treated with acrylamide and resveratrol, separately in two different doses and conditions. Their combined effects were also investigated. While, acrylamide treatment significantly decreased the glutathione peroxidase (GPx) activity in liver (1.24-fold), it was significantly increased (1.20 &ndash / 1.40-fold) by combined effect of resveratrol and acrylamide in liver and kidney. Furthermore, alone resveratrol administration increased (~1.37 &ndash / fold) GPx activity in kidney. Although, glutathione reductase (GR) was found to be significantly increased (~1.30-fold) in two different dose of resveratrol treated rabbit liver, it was not changed in acrylamide and their combined treatments. Despite, glutathione (GSH) content was decreased around 1.6 fold as a result of acrylamide treatment in rabbit liver and kidney cytosols, GSH level was returned to normal levels by resveratrol tretment in rabbit liver and kidney. Furthermore, acrylamide treatment significantly increased the SDH activity in blood serum (1.68-fold) and in liver (1.27-fold) with respect to control. On the other hand, resveratrol treatment brought this activity nearly normal level in acrylamide treated rabbits.. Besides, sorbitol deydrogenase (SDH) was found to be decreased (3.13-fold) significantly in rabbit liver cytosol as a result of single dose of 100 mg/kg b.w. resveratrol treatment. Moreover, catalase activity and MDA level were not affected from either resveratrol or acrylamide and with their combination effect in investigated rabbit organs. An important liver damage marker enzyme other than ALT and AST, SDH was characterized in terms of substrate, cofactor and enzyme concentration in rabbits which have been not investigated before and found to be 200 mM, 141 &micro / M and 0.5 &micro / L, respectively in rabbit liver. Furthermore, the Km value was first calculated in liver of New Zealand rabbits as 55,5 mM. In addition to these, in vitro effects of resveratrol on GST activity was also studied throughout this study. Resveratrol was shown to be a noncompetitive inhibitor for liver cytosolic GST against substrate CDNB with Ki of 175 &micro / M. On the other hand, resveratrol was shown to be a competitive inhibitor for liver cytosolic GST against substrate GSH with Ki of 55 &micro / M. The results of the present study have demonstrated for the first time that resveratrol induced some of the antioxidant enzyme activities and as well nonenzymatic antioxidants in rabbit liver and kidney. The results of GPx, GR, SDH activities and GSH level have also suggested that resveratrol may have protective effects on acrylamide induced hepatoxicity and renal toxicity. Therefore, it may be a therapeutic approach for the oxidative stress-related diseases such as cancer. However, further in vivo studies are required to clarify the effect of resveratrol on both acrylamide-induced toxicity and bioavailability in the body.

Cancer is a group of diseases in which normal cells are converted to cells capable of autonomous growth and invasion. In the chemotherapeutic control of cancer, drugs are usually given systemically so they reach toxic levels in healthy cells as well as cancer cells. This causes serious side effects. Another important problem with chemotherapy is resistance developed to cytotoxic drugs (multi drug resistance). Doxorubicin (Dox) occupies a central position in the treatment of breast cancer. However doxorubicin induced cardiac toxicity is associated with a high incidence of morbidity and mortality. Resistance of malignant tumors to Dox is another important cause of treatment failure in patients with cancer. One approach to overcome Dox-related toxicity is to use polymeric drug carriers, which direct the Dox away from heart tissue, and allow usage of lower dosages. In this present study two different anti-cancer drug delivery methods were evaluated. Dox was encapsulated in PLGA microparticles by single and double microemulsion solvent evaporation techniques. The highest entrapment of doxorubicin within PLGA microspheres obtained by optimization of process parameters. A sustained release of doxorubicin was obtained for 20 days. Several protein transduction domains are known to have the ability to pass through biological membranes. One such peptide is HIV-1 TAT. In this study TAT was evaluated for its ability to carry Dox into Dox resistant MCF-7 tumor cells. Dox peptide conjugate was more potent than free drug. The concentration of drug in resistant cancer cells was increased indicating a partial reversal of drug resistance.

Scytalidium thermophilum produces a novel phenol oxidase, which has turned out to be a bifunctional catalase-phenol oxidase (CATPO) during the course of this work, by other researchers of our group. Therefore, in the beginning of the studies, substrate specificity and inhibitor assays were conducted on the crude enzyme, followed by production, purification, cloning, expression, and mutagenesis and crystallography studies for further functional and structural analysis of CATPO. Accordingly, substrate specificity and inhibitory tests applied for crude enzyme characterisation presented the similarity of the phenol oxidase nature of CATPO essentially to catechol oxidase. Production studies were performed to investigate the effects of different factors including induction time, growth temperature, exogenous iron and hydrogen peroxide addition. In view of that, CATPO is constitutively produced in a growth associated manner. However, some phenolic compounds enhance its production. In this study, 15 phenolic compounds were tested for their ability to affect CATPO production. Of the phenolic compounds tested, catechol, resorcinol and vanillic acid caused repression of CATPO production. On the other hand, caffeic acid, myricetin and resveratrol enhanced CATPO production. As a biocatalyst, the efficiency of CATPO was examined and found to be a good candidate for getting pharmaceutically important drug intermediates. Its dual mechanism was analysed through side-directed mutagenesis. Two conserved residues (His101 and Val142) were mutated to discriminate catalase and phenol oxidase activities. Spectroscopic and mutagenesis studies exhibited the presence of heme d centre. Lastly, its structure was analysed by X-ray crystallography and found to have a tetrameric structure.

The kidney is one of the most affected organs of body from diabetes. Diabetic kidney disease is a complication of diabetes seen in 30-40% of diabetic person. The aim of this work is to contribute the useful information in the therapy of diabetes. It is very important to know the role of antioxidants at the molecular level during diabetes. The protecting role of antioxidants against lipid peroxidation, the effect of cellular antioxidant enzyme systems, understanding the changes of membrane fluidity, lipid order and protein structure which are resulted from antioxidant treatment, determining the effective therapeutic dose with the help of biochemical methods are very important in order to understand the effect of antioxidants at molecular level. In this thesis work, the Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) was used in order to study the diabetic kidney disease at the molecular level, which is encountered as a complication of diabetes. Furthermore, the protecting and possible therapeutic role of selenium in the course of diabetic kidney disease was investigated. To conclude, the kidney plasma membranes were severely deteriorated due to diabetes with respect to its lipid, protein and carbohydrate structure and content, which were corrected after selenium treatment. The diabetes causes diminishment of whole membrane fluidity, which was normalized with the selenium administration. This is the first study demonstrating the effect of diabetes on kidney plasma membrane and the effect of selenium on stz-induced diabetic kidney plasma membranes using spectroscopic tools. The study revealed serious therapeutic and preventing capacities of selenium on diabetic kidney plasma membranes which needs confirmation of future researches. Furthermore, the dosage of selenium given to diabetics should be investigated in detail and proved with biochemical and clinical data.

The aim of the present study was to elucidate the possible relationship between the levels of various brain fatty acids and learning indices in aged and young mice classified as &ldquo / good&rdquo / or &ldquo / poor&rdquo / learners basing on their performance in a spatial learning task, the Morris Water Maze. The levels of several fatty acids including palmitic, stearic, oleic, linoleic, arachidonic acid (AA) and docosahexaenoic acid (DHA) were measured using gas chromatography separately in samples from four different brain areas: hippocampus, cortex, striatum and hypothalamus. The level of oleic acid in the cerebral cortex was significantly higher in young-good learners as compared to young-poor learners and higher in young-poor learners than in old-poor learners, with no significant difference in the concentration of this acid between old-good and old-poor learners. The most consistent correlation between animals&rsquo / learning capacity and brain fatty acid&rsquo / level was found for the arachidonic acid in the hippocampal region: AA level was significantly lower in young-good learners as compared to both young-poor learners&rdquo / and old-good learners&rdquo / with young-good learners showing significantly better performance than the two other groups. Interestingly, except hypothalamus, no significant between-group differences were recorded for the remaining fatty acids including DHA, in none of the four brain regions examined.

Amplification of target DNA in vitro via polymerase chain reaction (PCR) is a widely used scientific technique in molecular biology. This method relies on repeated heating and cooling cycles of the DNA and enzyme mixture, resulting with the enzymatic replication of the DNA. A heat stable Taq DNA polymerase and a thermal cycler that enables repeated heating/cooling cycles are the two key components of the PCR. In this study we have produced a high activity Taq DNA polymerase and used this enzyme to validate and optimize two newly developed thermal cyclers- a conventional and a capillary thermal cycler. Taq DNA polymerase gene was amplified from Thermus aquaticus DNA, was cloned and overexpressed using Gateway&reg / recombination cloning technology. Highly active Taq DNA polymerase enzyme was purified from E.coli and its activity was tested by PCR, using different sources of DNA. Our results showed that the enzyme activity of the produced Taq DNA polymerase was not significantly different from the commercial available Taq DNA polymerase. To further characterize the purified enzyme, endonuclease and nicking activities were also tested to be absent. The fidelity of the purified Taq DNA polymerase was also tested and found to be the same as the commercially available Taq polymerases. In this study, in addition to the production of a Taq polymerase, optimization studies for two new thermal cyclers, a conventional and a capillary, was also carried out. The conventional thermal cycler was found to be as efficient as the commercially available thermal cyclers in the 95% confidence interval. The capillary thermal cycler was tested as a proof of concept and our results showed that it works less efficiently due to the insufficient insulation and capillary tubes being longer than the capillary tube holder.

The eastern Mediterranean has been known as the most oligotrophic water body among the worlds oceans and as a result of limited nutrient inputs from terrestrial sources primary productivity and plankton succession are restricted by lack of nutrients especially by phosphorus and nitrogen. Within this domain, Mersin bay forms a so called hot spot (highly sensitive) area where a sharp contrast exists between the coastal area supplied by land-based nutrient sources and the nutrient limited open sea. Excess nutrient enrichment lead for eutrophication in the inner Mersin bay while altering the quality and quantity of flora from shore to offshore. Microorganisms are highly sensitive and profoundly affected by environmental disturbances and are widely used to assess the impact of environmental changes on ecosystem functioning. With this study, it is aimed to investigate responses of the smaller fractions of phytoplankton composed of heterotrophic bacteria, Synechococcus and flagellates to rapidly changing ambient biological, chemical and physical properties of shelf waters over an extended period between 2008 and 2010. Epiflourescence microscopy and image analysis setup were used to enumerate and measure size of cells for biomass estimates.