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.

Here, we provide results of mid/late Holocene fresh water Ostracoda analyses from coastal modern lake basins in the Thrace region of Istanbul and Sarikum Lake, on the Black Sea coast near Sinop. While neither diatoms nor Cladocera are abundant in the sediments, Podocopian (fresh water) ostracods preserved well, but with discontinuous occurences during the mid/late Holocene. Un-noded forms of Cyprideis torosa, along with other Podocopian ostracods, dominated the sediments of all three lakes. Studying these three lagoonal basins along the Black Sea and Thracian coasts of Turkey allows reconstruction of long term, regional environmental histories, using the following methods. Loss-on-ignition (LOI) analyses at 1 cm intervals of short and long cores provide stratigraphic cross-correlation and calculations of organic matter, carbonate and mineral weight. At 5 cm intervals, spectrally-inferred chlorophyll-a contents by visible reflectance spectroscopy (Michelutti et al., 2010), provide estimates of algal production. Trace element analysis (Mg/Ca and Sr/Ca) using ICP-AES (coupled plasma atomic emission spectroscopy) is applied to fully calcified adult specimens of un-noded forms of Cyprideis torosa shells (which dominate the uppermost 145 cm of Terkos Lake). 210Pb and 137Cs dating of short cores, and AMS 14C dating of long cores, are used to infer sediment accumulation rates and to place specific ages on inferred environmental changes. Benthic foraminifers, gastropods, bivalves, single valves of fossil Glochidia, and Charophyte seeds are the other biological indicators observed within the sediment archive. Based on these data: 1. Terkos Lake sediments contain records of multiple, sub-millennial scale marine incursion events, over the last 2.8 ka, inferred to be the result of severe storms or tsunami on the Black Sea, including the tsunami in AD 1598 and AD 557-543 / 2. short core sediments from Sarikum Lake reveal sharp decreases in organic matter, carbonate, and increases in algal production and sand amount that suggest a storm or more recent earthquake / such as the Great Erzincan Earthquake (26 December, 1939) or the Bartin earthquake (3 September, 1968) while four more such events appear in the undated sediments of the Sarikum Lake long core / and 3. a large earthquake in AD 447 that affected the entire Sea of Marmara (Leroy et al., 2002) does not appear in the B&uuml / y&uuml / k&ccedil / ekmece Lake sediment record, but there is evidence for a significant hiatus in these deposits before the development of the dam (AD 1989) and after the youngest AMS date (2400 cal yrs BP). This suggests that B&uuml / y&uuml / k&ccedil / ekmece Lagoon was an environment of net erosion prior to its artificial impoundment, either from gradual processes or from scouring by one or more tsunami.

Whooping cough (pertussis) is a highly contagious respiratory infection caused by Bordetella pertussis. It becomes widespread among adolescent and adults as well as infants. Although availability of effective pertussis vaccines seems to decrease the incidence of the disease, B. pertussis circulation in population has not been eliminated. It is thought that the antigenic drifts in major protective antigens and continued circulation of B. pertussis strains will result in gradual loss of the efficacy of the current pertussis vaccines. Therefore, development of more effective acellular pertussis vaccines with conserved protective proteins is a convenient strategy to provide a better protection against whooping cough. In this study, immune responses against putative peptidyl-prolyl cis-trans isomerase (PPIase) which was shown to be immunogenic in B. pertussis for the first time by our immunoproteome group and FimX whose expression was found higher in our local Saadet strain were determined in mice. The genes encoding FimX and putative PPIase were amplified by PCR, cloned into pGEM&reg / -T Easy vector and sequenced. The genes were then introduced into pET-28a (+) vector and they were expressed in Escherichia coli BL21(DE3) cells. The recombinant proteins were purified by His-tag affinity chromatography and dialyzed. After Western blot analyses, 20 &micro / g and 80 &micro / g recombinant FimX and 80 &micro / g recombinant putative PPIase were used to immunize BALB/c mice (16-18 g) at day 0 and 21. The mice were challenged intranasally with 2.5 x 109 live B. pertussis Saadet cells. Before second immunization and challenge, the sera were collected to carry out ELISA for measurement of serum-specific IgG levels. According to ELISA results, IgG levels in the mice immunized with 20 &micro / g and 80 &micro / g recombinant FimX were found significantly higher than in control groups at both first and second vaccinations (p&lt / 0.01). On the other hand, immunization with 160 &micro / g recombinant putative PPIase provided a significant increase in IgG level (p&lt / 0.05) only at second vaccination. The lungs of the mice were removed at day 2, 5, 8 after challenge and bacterial colonization was determined. No significant decrease in bacterial colonization was observed in the lungs of the mice immunized with 20 &micro / g and 80 &micro / g recombinant FimX and 80 &micro / g recombinant putative PPIase with respect to control groups. After respiratory challenge and second immunization (at day 30) with 20 &micro / g and 80 &micro / g recombinant FimX, the spleens of the mice were removed and a spleen cell culture was obtained. Supernatants were collected after induction of the cells with the recombinant protein and cytokine ELISA was carried out to measure IFN-&gamma / level. No significant difference was observed between control and vaccinated mice in terms of IFN-&gamma / production.