RNA-binding proteins (RBP) shuttle between cellular compartments either constitutively or in response to stress and regulate localization, translation and turn over of mRNAs. In our laboratory, cytosolic proteome map of Phanerochaete chrysosporium was established and upon Pb exposure, the changes in cytosolic protein expressions were determined. The identified RBPs were a newly induced polyadenylate-binding protein (RRM superfamily) as well as two up-regulated proteins, namely splicing factor RNPS1 and ATP-dependent RNA helicase, all being very important candidates of post-transcriptional control in response to stress. This finding inspired us to conduct Real Time PCR studies in order to have a better understanding of the changes in the expression of corresponding genes at mRNA level in response to Pb exposure, thus the present study aims at examining the effect of lead exposure on the transcript levels of the genes coding for ATP-dependent RNA helicase, splicing factor RNPS1 and polyadenylate binding protein. As shown via expression analysis based on Real Time PCR, the mRNA level of splicing factor RNPS1 showed 2.68, 2.62 and 4.86 fold increases in a dose-dependent manner when the cells were grown for 40 h in the presence of 25, 50 and 100 &micro / M Pb, repectively. ATP-dependent RNA helicase mRNA level showed no significant increase in response to 25 &micro / M Pb exposure while increased 2 and 1.84 fold in response to 50 and 100 &micro / M Pb, respectively. Polyadenylate binding protein mRNA levels revealed no significant increase when exposed to 25, 50 and 100 &micro / M Pb. As to the mRNA dynamics as a function of duration of lead exposure, the mRNA level of this protein showed 2.54-fold increase upon 1 h exposure to 100 &micro / M Pb. Splicing factor RNPS1 mRNA level showed a significant increase of 19.22 fold at 2nd h of 50 &micro / M Pb exposure. Expression level of ATP-dependent RNA helicase was not affected by the time of exposure to Pb.

This thesis was aimed to analyze phenol oxidation by the bifunctional catalase/phenol oxidase of the thermophilic fungus Scytalidium thermophilum. Several reactive oxygen species (ROS) are continuously produced in fungi under oxidative stress. Depending on the nature of the ROS species, some are highly toxic and are rapidly detoxified by various cellular enzymatic mechanisms, including the production of catalase. S. thermophilum produces a novel bifunctional catalase-phenol oxidase (CATPO) which is capable of oxidizing phenolics in the absence of hydrogen peroxide. Phenol oxidases convert phenolic compounds to quinones, which are then polymerized mainly by free- radical mediated reactions. In this study, 14 phenolic compounds were selected according to their different chemical structures and functional properties and were analyzed as substrates of CATPO. Among 14 phenolic compounds, only in catechol, chlorogenic acid, catechin and caffeic acid distinct oxidation products were observed by HPLC. The oxidation products of catechol, caffeic acid, chlorogenic acid and catechin were characterized by LC-ESI-MS. Dimer, trimer, tetramer and oligomer formations were detected. While the maximum conversion efficiency, at 1 hour of reaction, was observed with catechin, minimum conversion efficiency was attained by caffeic acid, under the specified conditions. The oxidation products observed after oxidation of catechol, chlorogenic acid, catechin and caffeic acid by CATPO was compared with the same phenolic compounds oxidation products oxidized by laccase and tyrosinase. CATPO was incapable of oxidizing tyrosinase and laccase-specific substrates tyrosine and ABTS respectively. However, the oxidizing spectrum of substrates indicates that the nature of phenol oxidation by CATPO appears to resemble mainly those of laccase.

One of the main problems of current cancer chemotherapy is the lack of selectivity of anti-cancer drugs to tumor cells which leads to systemic toxicity and adverse side effects. In order to overcome these limitations, researches on controlled drug delivery systems have gained much attention. Nanoscale based drug delivery systems provide tumor targeting. Among many types of nanocarriers, superparamagnetic nanoparticles with their biocompatible polymer coatings can be targeted to an intented site by an external magnetic field. Thus, the drug can be carried to the targeted site safely. The aim of this study is to prepare poly(dl-lactic-co-glycolic acid) (PLGA) coated magnetic nanoparticles and load anti-cancer drug, doxorubicin to them. For this purpose, magnetite (Fe3O4) iron oxide nanoparticles were synthesized as a magnetic core material (MNP) and then coated with oleic acid. Oleic acid coated MNP (OA-MNP) was encapsulated into PLGA. Effects of different OA-MNP/PLGA ratios on magnetite entrapment efficiency were investigated. Doxorubicin loaded magnetic polymeric nanoparticles (DOX-PLGA-MNP) were prepared. After the characterization of prepared nanoparticles, their cytotoxic effects on MCF-7 cell line were studied. PLGA coated magnetic nanoparticles (PLGA-MNP) had a proper size and superparamagnetic character. The highest magnetite entrapment efficiency of PLGA-MNP was estimated as 63 % at 1:8 ratio. Cytotoxicity studies of PLGA-MNP did not indicate any notable cell death between the concentration ranges of 2 and 250 &mu / g ml-1. It was observed that DOX-PLGA-MNP showed significant cytotoxicity on MCF-7 cells compared to PLGA-MNP. The results showed that prepared nanoparticles have desired size and superparamagnetic characteristics without serious toxic effects on cells. These nanoparticles may be suitable for targeted drug delivery applications. The findings obtained from drug studies may contribute to further work.

Conventional cancer chemotherapies cannot differentiate between healthy and cancer cells, and lead to severe side effects and systemic toxicity. In the last decades, different kinds of controlled drug delivery systems have been developed to overcome these shortcomings of chemotherapeutics. Magnetic nanoparticles (MNP) are potentially important in cancer treatment since they can be targeted to tumor site by an externally applied magnetic field. In this study, it is aimed to synthesize folic acid conjugated / polyethylene glycol (PEG) coated magnetic nanoparticles with appropriate size, surface chemistry, magnetization and biocompatibility to be used in biomedical applications. First MNP were synthesized, then covered with oleic and PEG / and finally conjugated with folic acid. A detailed characterization of synthesized nanoparticles was done by TEM, XRD, FTIR, VSM and XTT analyses. MNP synthesized by the rapid addition of ammonium hydroxide exhibited more spherical nanoparticles with a narrower size distribution. Agglomeration tendency of naked nanoparticles was prevented by oleic acid addition during the synthesis. Both naked and surface treated MNP have been found to exhibit superparamagnetic behavior both at room temperature (23

The largest class of cell surface receptors in mammalian genomes is the superfamily of G protein-coupled receptors (GPCRs) which are activated by a wide range of extracellular responses such as hormones, pheromones, odorants, and neurotransmitters. Drugs which have therapeutic effects on a wide range of diseases are act on GPCRs. In contrast to traditional idea, it is recently getting accepted that G-protein coupled receptors can form homo- and hetero-dimers and this interaction could have important role on maturation, internalization, function or/and pharmacology. Bimolecular fluorescence complementation technique (BiFC) / is an innovative approach based on the reassembly of protein fragments which directly report interactions. In our study we implemented this technique for detecting and visualizing the GPCR interactions in yeast cells. The enhanced green fluorescent protein (EGFP) fractionated into two fragments at genetic level which does not possess fluorescent function. The target proteins which are going to be tested in terms of interaction are modified with the non-functional fragments, to produce the fusion proteins. The interaction between two target proteins, in this study Ste2p receptors which are alpha pheromone receptors from Saccharomyces cerevisiae, enable the fragments to come in a close proximity and reassemble. After reassembly, EGFP regains its fluorescent function which provides a direct read-out for the detection of interaction. Further studies are required to determine subcellular localization of the interaction. Moreover, by using the fusion protein partners constructed in this study, effects of agonist/antagonist binding and post-translational modifications such as glycosylation and phosphorylation can be examined. Apart from all, optimized conditions for BiFC technique will guide for revealing new protein-protein interactions.

Obesity is a metabolic disorder that results in elevated levels of free fatty acids and triglycerides in the blood circulation, which further leads to accumulation of lipids within various tissues. Like in other similar metabolic disorders, obesity is thought to be originated from structural and regulatory changes in macromolecular assemblies. This current study aims to investigate the effects of obesity on macromolecular alterations in order to characterize Berlin fat mouse inbred (BFMI) lines which arenew models for the obesity research that may contribute to understanding of spontaneous obesity without induction of a high fat diet. Attenuated Total Reflectance - Fourier Transform Infrared (ATR-FTIR) spectroscopy was used to characterize content and structure of macromolecules in male and female control (DBA/2J) and BFMI lines / namely BFMI856, BFMI860 and BFMI861, in two different adipose tissues / inguinal fat (IF) which is subcutaneous adipose tissue (SAT), gonadal fat (GF) which is visceral adipose tissue (VAT). Structural and compositional alterations in proteins, lipids, saturated and unsaturated lipid contents, nucleic acid, collagen and glycogen contents and variations in the lipid chain length were determined. BFMI860 and BFMI861 lines were found to be the most affected lines since they showed the indications of lipid peroxidation and insulin resistance more severely as they had lower glycogen content in all tissues and lower unsaturated lipid content especially in IF adipose tissues. Moreover, structural changes in lipids were observed especially in male GF adipose tissues of BFMI856 and BFMI861 lines. Protein content decreased significantly specifically in female IF adipose tissues but no change was observed in the structure. Furthermore, BFMI852 line was found to be affected different than other lines and had an effect on especially female IF. To conclude, obesity induced changes differ in BFMI lines according to the gender, adipose tissue type and distinctness in the strains.

The induction of hepatic cytochrome P4501A1 and its monooxygenase activity 7-ethoxyresorufin O- deethylase, (EROD) in fish by PAHs, PCBs and dioxins has been suggested as an early warning system &ldquo / most sensitive biochemical response&rdquo / for assessing environmental contamination conditions. In this study, the degree of induction of cytochrome P4501A1 protein as determined immunochemically and CYP1A1 associated EROD activity in fish were utilized as biomarkers of exposure to PAHs, PCBs and related organic pollutants along the izmir Bay on the Aegean Sea Coast after the Great Canal Project. Three different fish species were used throughout this study, namely leaping mullet (Liza saliens), annular seabream (Diplodus annularis) and common sole (Solea vulgaris) which were representatives of pelagic, benthopelagic and benthic fish, respectively. Fish were sampled in November 2002 and October 2003 from different sites of the Bay. The mullet caught from Harbor, &Uuml / &ccedil / kuyular port site, and Pasaport region displayed highly elevated EROD activities which were 2258&plusmn / 840 (n=15), 2011&plusmn / 490 (n=4), 1813&plusmn / 287 (n=11) pmole /min/mg protein, respectively and were 104, 80 and 79 fold higher than that of fish obtained from the reference point (25&plusmn / 9 pmole/min/mg protein / n=4). Mullet caught along the pollutant gradient at three other sites (Hekim Island, inciralti, and Zeytinalani) exhibited less but highly significant induced EROD activity. EROD activities of common sole sampled from Fo&ccedil / a open sites (107&plusmn / 20 pmol/min/mg protein, n=5) and site16A (80&plusmn / 12 pmol/min/mg protein, n=9) were found to be very low and the latter was accepted as reference site. The highest EROD activity were seen in fish captured from inciralti which was about 6.3 times higher than those obtained from reference site. Common sole caught from the mouth of Gediz River and Hekim Island exhibited also highly elevated EROD activities. Annular seabream was tested to monitor CYP1A inducing chemicals for the first time in this study. The highest EROD activity (1376&plusmn / 279 pmol/min/mg protein, n=8) were detected in fish samples collected from Harbor region. An inverse relationship was found between distance to the harbor region and EROD activities of annular seabream captured from other sampling sites. In this study for the first time, major cytochrome P450 dependent mixed function oxidase activities such as benzphetamine N- demethylase, ethylmorphine N-demethylase and aniline 4- hydroxylase, were characterized in annular seabream. Changes in the P450 1A1 protein level were determined by immunochemical analysis to monitor the pollutant based induction in all fish species and good correlation was obtained between EROD activity and CYP1A protein content. Fish from polluted sites had both highly induced EROD activity and cytochrome P450 1A content. Chemical analysis of total PAH concentration in sediment and liver tissues of some fish sample were also carried out. Although, izmir Great Canal Project has been active since 2000 to treat and protect the izmir Bay from the contamination of domestic and industrial wastes this study clearly demonstrated that the level of PAH, PCB and dioxin type persistent organic contaminants are still very high especially in the Inner and Middle Bay. This has implications for human fish consumption from contaminated areas, as well as for the health status of aquatic organisms.

Plants can not escape from biotic and abiotic stress factors such as, extreme temperatures, high light intensity, drought, UV radiation, heavy metals, and pathogen attack. Plants have versatile defens systems against such stress conditions. In this study, the role of glutathione S-transferases (GSTs) in cold stress conditions were examined. Glutathione S-transferases are the enzymes that detoxify natural and exogenous toxic compounds by conjugation with glutathione. Glutathione, an endogenous tripeptide, is important as reducing agent, nucleophilic scavenger, and alleviate the chemical toxicity in the plants by the reaction of GSTs. Glutathione conjugates can be transported to the vacuoles or apoplast and are generally much less toxic than the parent compounds. In plants there are four distinct families of the soluble GSTs, namely Phi (F), Type I / Zeta (Z), Type II / Tau (U), Type III / Theta (T), Type IV. By contrast with the mammalian families of GST, relatively little is known about the plant GST families. Up to date, there is not any study on GST isolation and characterization from Turkish red pine, in this respect, this study well play a frontier role the future research dealing with this topic. In this study, some properties of Turkish red pine GST activity towards CDNB (1-chloro-2,4 dinitrobenzene) were examined. The average specific activity of Turkish red pine GST towards CDNB was found as 200&plusmn / 50 (Mean&plusmn / SE, n= 18) nmole/min/mg cytosolic protein. GSTs in cytosol prepared from Turkish red pine needles retained its activity without loss for four weeks at -80&amp / #61616 / C. The rate of conjugation reactions were linear up to 0.8mg of Turkish red pine cytosolic protein and 0.4 mg cytosolic protein was routinely used. The Turkish red pine GST showed its maximum activity at pH 8.0 in 25 mM phosphate buffer and 42 &amp / #730 / C. The measurements were carried out at room temperature (RT) of 25 &amp / #61616 / C. Turkish red pine GST seemed to be saturated at 1 mM CDNB and 1 mM GSH concentrations. The Vmax and Km values of Turkish red pine GST for CDNB was 416nmole/min/mg protein and 0,8 mM, respectively, and for GSH 106.4 nmole/min/mg protein and 0.10 mM, respectively. Turkish red pine cytosol was applied on DEAE-Sepharose fast flow column but almost no purification was achieved with respect GST activity. In order to examine the effects of cold stress on Turkish red pine GST activity, the GST activity was determined in 240 seedlings at &ndash / 3&amp / #61616 / , 0&amp / #61616 / and 13 &amp / #61616 / C environmental temperatures. It was observed that GST activity was the highest at -3&amp / #730 / C and the lowest at 13&amp / #730 / C in both cold resistant and sensitive families with the exception of Yaylaalan and &Ccedil / ameli.

In the first part of this study, the effect of diabetes mellitus on the long bones (femur and tibia) of the streptozocin induced diabetic rats and the effect of selenium (Se) treatment on these bones are investigated at molecular level by Fourier transform infrared (FTIR) spectroscopy, light and electron microscopy. In the second part of this study, the effect of selenium and vitamin E deficiency or selenium toxicity on rat bones have been studied by FTIR spectroscopy. The results of the first part of the present study revealed that the changes observed in the mineral and matrix phases of diabetic bones, briefly, the increase in the mineral crystal size, the decrease in the acid phosphate and carbonate content, the increase in the ratio of pyridinoline [Pyr] cross-links to dihydroxylysinonorleucine [DHLNL] cross-links present in collagen I of the bone tissue as well as the increase in the lipid to protein ratio of the matrix are quite similar to those seen in osteoporotic patients and animal models and confirms the evidence of diabetic osteoporosis. Histologic studies carried out with light and electron microscopy supported these findings. FTIR spectroscopic analysis revealed that sodium selenite treatment had some restoring effects on the deviated properties of the microstructure of diabetic bones. The results of the second part of this study revealed that the deficiency of selenium led to increase in the crystal size of the bone minerals, decreases in acid phosphate and labile carbonate content and increase in the Pyr to DHLNL ratio as in the case of diabetic bones. These results can be indicative of the importance of selenium in glucose metabolism.The results of Se excess group are similar to those of Se deficient group except that toxic amount of selenium led to increase in the relative amount of acid phosphate. This can affect the pH of the mineral environment and lead to deformation of the bone tissue. It can be concluded from FTIR spectroscopic and light microscopic findings that both antioxidant deficient and excess diets cause almost similar defects in the mineral matrix phases of rat long bones. Overall results may reflect the importance of antioxidants for human life and if they are used in proper amounts they can be preventive for the complications of diabetes seen in bones as well as other organs. However, further investigations are necessary for the therapeutic usage of selenium, since the treatment of control group rat femurs with sodium selenite led to some structural defects.

In this work Agrobacterium (KYRT1::pTJK136) mediated gene transfer to lentil (Lens culinaris Medik.) embriyo apex and regeneration through micro-grafting in lentil was studied. In micro-grafting two different types root stock stem height and root stock preparations were optimized. According to the results half stem length was found to be more successful then the full. Also lentil root stock was more successful then the chickpea root stock. The types of root stock preparations studied were designated as Z and M. The Z type root stock was superior then the M type, when the micro-grafting, hardening and green-house stages were concerned. In study of Agrobacterium mediated gene transfer to lentil embryo apex the effect of the following parameters on the transformation efficiency were addressed / type and intensity of injury, type of pre-incubation media for injured explants, effect of evacuation, effect of L-cysteine during co-cultivation and Agrobacterium incubation duration. According to the results crushing type of injury was superior over the poking and sonication type of injuries. Following the injury Hogland`s solution was used as pre-incubation media prior to infection. The effect of evacuation parameter was found to be insignificant whereas the effect of L-cysteine during co-cultivation was negative on the transformation efficiency. According to the Agrobacterium incubation duration studies, 240 minutes, followed by 120 minutes of Agrobacterium incubation were the most efficient in terms of transformation efficiency. However, since there was no significant difference among the two 120 minutes chosen to be the optimum bacterial incubation duration.