ABSTRACT PURIFICATION AND CHARACTERIZATION OF CYTOPLASMIC AND PROTEASOME ASSOCIATED CHYMOTRYPSIN-LIKE PROTEASES FROM THERMOPLASMA VOLCANIUM &Ouml / zdemir, F.inci Ph.D., Department of Biology Supervisor: Prof. Dr. Semra Kocabiyik September, 147 pages In this study, two novel cytoplasmic serine proteases were isolated and characterized from thermophilic archaea Thermoplasma volcanium. The first protease was purified by ion exchange and affinity chromatographies and identified as a chymotrypsin-like serine protease mainly based on its substrate profile and inhibition pattern. The presence of protease activity was analyzed by gelatin zymography which was detected as a single band (35 kDa). Optimum temperature was found to be 60oC for azocasein hydrolysis and 50oC for N-Suc-Phe-pNA hydrolysis. Optimum activity was observed in the pH range of 6.0-8.0 with a maximum value at pH 7.0. The Km and Vmax values for the purified protease were calculated to be 2.2 mM and 40 &micro / moles of p-nitroanilide released min-1.ml-1, respectively, for N-Suc-Phe-PNA as substrate. Ca2+ and Mg2+ at 4 mM concentrations were the most effective divalent cations in activating the enzyme. In the second stage of this study, 20S proteasome of Tp. volcanium with substantial chymotrypsin-like activity was purified and characterized. This enzyme complex was purified with 19.1 U/mg specific activities from cell free extract by a four-step procedure. SDS-PAGE analysis revealed two strong bands with relative molecular masses of 26 kDa (&amp / #945 / -subunit) and 21.9 kDa (&amp / #946 / -subunit). Tp. volcanium 20S proteasome predominantly catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residue Phe (chymotrypsin-like activity) in short chromogenic peptides. Low-level hydrolyzing activity was also detected carboxyl to basic residue Arg (trypsin-like activity). Chymotrypsin-like activity of Tp. volcanium 20S proteasome was significantly inhibited by chymotrypsin specific serine protease inhibitor chymostatin. When N-CBZ-Arg was used which is a substrate for trypsin, 20S proteasome was strongly inhibited by TLCK. The optimum temperature for Ala-Ala-Phe-pNA hydrolysis by the Tp. volcanium 20S proteasome was 55oC and the optimum pH was 7.5. The chymotryptic activity was significantly enhanced by divalent cations such as Ca+2 and Mg2+ at high concentrations, i.e. 125-250 mM. Keywords:Serine protease, 20S proteasome, archaea, thermophilic protease, Thermoplasma volcanium, chymotrypsin-like serine protease.

Some yeast strains secrete extracellular polypeptide toxins known to have potential growth inhibitory activity on other sensitive yeast genera but are immune to their own toxins. These yeast strains are termed as killer yeasts and their toxins are designated as killer proteins or killer toxins. Killer phenotypes are classified into 11 typical types (K1-K11). The toxic actions of yeast killer proteins on sensitive cells show differences and one of the most important toxic actions involves the selective functional damage by hydrolyzing major cell wall components. Because mammalian cells lack a cell wall, novel highly selective antifungals tend to be harmless to people by targeting important cell wall components specific to fungi. We have previously characterized the K9 type yeast killer protein isolated from Hansenula mrakii. This protein is stable at pH and temperature values appropriate for its medical usage. Antifungal activity of this protein was tested against 23 human pathogenic yeast and 9 dermathophyte strains. Pathogenic yeast strains found to be susceptible and both the MIC and MFC values ranged from 0.25 to 8 &micro / g/ml except C. parapsilosis and C guilliermondii isolates. 9 dermatophyte strains were not susceptible to this protein and MICs were &gt / 64 &micro / g/ml. According to the cell killing analysis toxin activity starts within the first 4 hours and complete cell death was observed for the 4, 8 and 16 times the MIC concentrations at 24 hour. The results obtained from this study might make the potential use of this protein possible as a selective antimycotic agent.

ABSTRACT Three different genotypes of sugar beet (diploid, triploid and tetraploid) / 4 varieties from diploid and triploid genotypes Soraya (KWS8123) and Leila (diploid), Visa (H68121) and Kassandra (triploid) and 2 lines from tetraploid genotype &Ccedil / BM315 and EA2075 (tetraploid) were used for investigating the effects of ploidy level on plant regeneration. Within three sugar beet genotypes, with respect to the treatments, triploids or tetraploids were found to respond to treatments significantly different when compared with diploids. The responses of polyploids were superior over the responses of diploids. Moreover, varieties from same genotype responded differently to treatments. Two types of calli were obtained / one white and friable with regenerative capacity and the other green and compact with no regenerative capacity. Concentration of sucrose on callus development was observed to be important. High concentration of sucrose (30 g/L) was found to cause discoloration and irresponsiveness of formed calli at callus enlargement and subsequent shoot regeneration stages. Therefore, low concentration (10 g/L) is advised to be used at these stages / although this caused less callus induction. Although initially used for the prevention of tissue discoloration, L-ascorbic acid inclusion into the medium was found to be positively affecting the regeneration capacity. When used at 20 mg/100 mL concentration, the only two spontaneous shoots from the tetraploid EA2075 line were obtained. Subsequently, these shoots were successfully rooted and whole plants were obtained. The effect of silver nitrate, in combination with L-ascorbic acid, on the prevention of sugar beet tissue discoloration was investigated. Unfortunately, the symptoms of discoloration did not diminish. Moreover, callus formation was reduced and the subsequent shoot recovery could not be achieved. Since a total of 3456 explants were used during this study, and only 2 whole plants were regenerated, the efficiency of plant recovery was calculated as a rather low value of 0.058 %.

The purpose of this study was to investigate the effect of gender and reasoning ability on the 8th grade students&rsquo / understanding of ecological concepts and attitude toward science. All 8th grade students from public elementary school in Tosya participated in the study. Students&rsquo / understanding, attitude toward science and reasoning ability were also measured by means of the Test of Ecology Concept (TEC), the Attitude Scale toward Science (ASTS) and the Test of Logical Thinking (TOLT) respectively. In order to investigate students&rsquo / understanding deeply, interview was conducted. Results of the TEC and interview show that students have many misconceptions concerning ecosystem, population, community, decomposers, food chain, food web, energy pyramid and energy flow. Students&rsquo / understanding for the first tier (M= 55.8), combination of first two tiers (M= 27) and combination of all three tiers (M= 21.2) were calculated according to TEC results. Multivariate Analysis of Covariance (MANCOVA) conducted to determine the effect of gender on students&rsquo / understanding of ecological concepts and attitude towards science when reasoning ability was controlled. The results indicated that there was significant gender difference in favor of girls with respect to students&rsquo / understanding of ecological concepts and attitude towards science when reasoning ability was controlled (Wilks&rsquo / Lambda=0.97 / p=.00).

In the present study Fourier Transform Infrared (FTIR) and Attenuated Total Reflectance FTIR (ATR-FTIR) Spectroscopy were used to examine the effects of simvastatin on structure, composition and function of macromolecules of three different rat skeletal muscles EDL (Extensor Digitorium Longus), DIA (Diaphragm) and SOL (Soleus) containing different amount of slow and fast twitch fibers, at molecular level. Simvastatin, a lipophilic statin, is widely used in the treatment of hypercholesterolemia and cardiovascular diseases due to its higher efficacy. However, long term usage of statins give rise to many adverse effects on different tissues and organs. Skeletal muscle accounts for around 45 % of the total body weight and has a high metabolic rate and blood flow. As a consequence, it is highly exposed to drugs within the circulation. Therefore, it is one of the target tissues of statins. The two main types of muscle fibers are type I (slow-twitch) and type II (fast-twitch) fibers / having different structural organization and metabolic features. EDL is a fast twitch muscle while SOL is slow twitch muscle. On the other hand, DIA shows intermediate properties between slow and fast twitch muscle. The results of ATR-FTIR and FTIR spectra revealed a considerable decrease in protein and lipid content of simvastatin treated skeletal muscles, indicating protein breakdown or decreased protein synthesis and increased lipolysis. Moreover changes in protein structure and conformation were observed. In simvastatin treatment, muscle membrane lipids were more ordered and the amount of unsaturated lipids was decreased possibly due to lipid peroxidation. The drug treatment caused a decrease in the content of nucleic acids, especially RNA, and hydrogen and non-hydrogen bonded phospholipids in the membrane structures of skeletal muscles. In all of the spectral parameters investigated EDL muscle was more severely affected from statin treatments while SOL was less affected from the drug treatments. Thus, FTIR and ATR-FTIR spectroscopy appear to be useful methods to evaluate the effects of statin on skeletal muscle tissues at molecular level.

In the present study, haplogroup composition of five native Turkish sheep breeds, (Karayaka, Akkaraman, G&ouml / k&ccedil / eada, Dagli&ccedil / , Morkaraman) and two sheep breeds from neighboring countries (Herik from Iran, samples from Azerbaijan) were determined by single strand length polymorphism (SSCP) analysis of mitochondrial DNA (mtDNA) NADH dehydrogenase subunit 4 (ND4) region and restriction fragment length polymorphism (RFLP) analysis of mtDNA control (CR) region. Results of the SSCP and RFLP approaches were found to be 96,82% consistent. Most of the 3,18% inconsistency was due to unidentified band patterns of 9 individuals. SSCP method could identify haplogroups A, B and C, but not D and E. Similarly RFLP method could identify haplogroup A, B and possibly D, but not E and C. Data of the present study were compared with those of the previous studies to test the robustness of results under different samplings and were found to be homogenous with a previous study with similar sampling strategy. Neighbor joining tree, principal component analysis (PCA), Delaunay network analysis and analysis of molecular variance (AMOVA) were employed to analyze the haplogroup frequencies and breeds were separated in four groups according to the genetic barriers between breeds from different geographical locations. Strongest differentiation was present between two groups which were eastern breeds (Morkaraman, Herik-Iran and Azerbaijan) and western breeds (G&ouml / k&ccedil / eada, Akkaraman, Karayaka and Dagli&ccedil / ). Additionally, Azerbaijan was proposed as the entrance point of the haplogroup A and the Iran was proposed as the entrance point of haplogroup C to Anatolia with the Spearman rank correlation test.

Tissue engineered human oral mucosa has the potential to fill tissue deficits caused by facial trauma or malignant lesion surgery. It can also help elucidate the biology of oral mucosa and serve as an alternative to in vivo testing of oral care products. The aim of this thesis was to construct a tissue engineered full-thickness human oral mucosa closely mimicking the native tissue. To this end, the feasibility of the concept was tested by co-culturing fibroblasts and epithelial cells isolated from normal human oral mucosa biopsies in a collagen-glycosaminoglycan-chitosan scaffold, developed in our laboratory to construct a skin equivalent. An oral mucosal equivalent closely mimicking the native one was obtained and characterized by histology, immunohistochemistry and transmission electron microscopy. Using the same model, the influence of mesenchymal cells on oral epithelial development was investigated by culturing epithelial cells on lamina propria, corneal stroma and dermal equivalents. They were found to significantly influence the thickness and the ultrastructure of the epithelium. Finally, in order to improve the adhesiveness of conventional scaffolds, an elastin-like recombinamer (ELR) containing the cell adhesion tripeptide, RGD, was used in the production of novel bilayer scaffolds employing lyophilization and electrospinning. These scaffolds were characterized by mercury porosimetry, scanning electron microscopy and mechanical testing. In vitro tests revealed positive contribution of ELR on the proliferation of both fibroblasts and epithelial cells. It was thus possible to construct a viable oral mucosa equivalent using the principles of tissue engineering.

Type I Diabetes is a multisystem disease having both biochemical and structural consequences. It causes alterations in carbohydrate, protein, and fat metabolisms due to hyperglycemia. Type I diabetes is also correlated with increased formation of free radicals and decreased levels of antioxidant potential. Lower endogeneous antioxidant amounts and elevated lipid peroxidation levels in diabetes constitute the basis of risk factors for the development of diabetic complications. These complications lead to irreversible damages in nearly all vital organs and systems. Since the antioxidant capacity lowered in diabetic conditions, it becomes important to be able to use some common antioxidants, as a complementary treatment strategy for diabetes. The effect of type I diabetes and the recovery role of Vitamin C on the structure, composition and function of the macromolecular content of testicular tissue is still unknown. Therefore, in the current study, it was aimed to investigate the alterations in the macromolecules of rat testes due to Streptozotocin (STZ)-induced type I diabetes using Attenuated Total Reflectance (ATR)-Fourier Transform Infrared (FTIR) spectroscopic and FTIR microspectroscopic techniques. Furthermore it was iv aimed to gain useful information about the recovery role of Vitamin C, as an antioxidant, against the diabetic complications. The detailed spectral analysis revealed that, the macromolecular structure and composition of rat testes are highly affected due to the development of diabetes. The lipid and protein content of diabetic rat testes were shown to decrease considerably, indicating an increase in lipolysis and proteolysis processes. Diabetes was also shown to lead to a decrease in the content of fatty acids and nucleic acids. In addition to the compositional alterations, protein conformation, and protein secondary structural components were also found to alter in diabetic state. Besides, lipid peroxidation levels were found to increase, and the elevated levels of lipid peroxidation products end up with increased levels of unsaturation, and also end up with increased levels of disorderness in diabetic conditions. On the other hand, with the administration of Vitamin C, the diabetes-induced alterations were found to be partially recovered, indicating that after more confirmative researches, Vitamin C may have a chance to be used as a complementary therapy in the treatment of diabetes.

Epilobium hirsutum L. (Onagraceae), a medicinal plant known as hairy willow herb, has been used by people all around the world for treatment or prevention of inflammation, adenoma, rectal bleeding, menstrual disorders, constipates, and prostate. It contains polyphenolics including steroids, tannins such as gallic, ellagic, and p-coumaric acids and flavonoids such as myricetin, isomyricetin, and quercetin. Polyphenols have been known for their multiple biological health benefits, including antioxidant activities. Viscum album L. (Loranthaceae), a species of mistletoe, contains lectins, polypeptides, mucilage, sugar alcohols, flavonoids, lignans, triterpenes, and phenylallyl alcohols. The leaves and twigs of Viscum album L., taken as tea, have been traditionally used for hypertension, stomachache, diarrhea, diabetes, dysuria and also as analgesic and cardiotonic agent in Anatolia, Turkey. In addition, in Europe, sterile extracts of Viscum album L. are among the most common herbal extracts applied in cancer treatment and have been used as prescription drugs, while in US, considered as dietary supplement. Flavin-containing monooxygenases are FAD-containing phase I enzymes responsible for the oxidation of wide-range of nucleophilic nitrogen, sulfur, phosphorus, and selenium heteroatom-containing drugs such as tamoxifen, v methimazole and imipramine, pesticides, neurotoxins, and other chemicals using NADPH as cofactor. The aim of this study was to determine the in vivo effects of Epilobium hirsutum L. and Viscum album L. (subspecies growing on pine trees-subsp. austriacum (Wiesb.) Vollmann) on FMO activity, mRNA and protein expressions in rat liver. The water extracts of Epilobium hirsutum L. (37.5 mg/kg body weight) and Viscum album L. (10 mg/kg body weight) were injected intraperitonally (i.p) into Wistar albino rats for 9 consecutive days. Following the decapitation, the livers were removed and microsomal fractions were prepared by differential centrifugation. Rat liver microsomal FMO activity using methimazole as substrate, mRNA expression by quantitative Real-Time PCR, and protein expression by Western Blot were determined. The results showed that water extract of Epilobium hirsutum L. has no significant effect on FMO activity / however, it decreased significantly (p&lt / 0.05) FMO3 protein and mRNA expression 27.71% and 1.41 fold, respectively, compared as controls. Water extract of Viscum album L. decreased mRNA (2.56 fold), and protein expressions (27.66%) as well as enzyme activity (19%) of FMO with respect to controls. In conclusion, our current data suggest that the metabolism of xenobiotics including drug molecules by FMO-catalyzed reactions may be altered due to the changes in FMO expression and activity by medicinal plants Epilobium hirsutum L. and Viscum album L.

The flavin-containing monooxygenases (FMO / E.C.1.14.13.8) are microsomal NADPH and oxygen-dependent flavoprotein enzymes that catalyze the oxidation of a wide variety of xenobiotics, including drugs and environmental toxicants. Nucleophiles containing nitrogen, sulfur, phosphorus and selenium heteroatoms are the substrates of FMO. Bovine liver microsomal FMO enzyme activity was characterized using methimazole as substrate, which is a highly specific substrate for FMO. From 12 different bovine liver samples, microsomes were prepared and the average specific activity of bovine liver microsomal FMO was found to be 2.37 &amp / #61617 / 0.30 nmol/min/mg (Mean &amp / #61617 / SE, n=12). The rate of reaction was linear up to 0.5 mg of bovine liver microsomal protein. The maximum FMO enzyme activity was detected at 37 &amp / #61616 / C and at pH 8.0. Effects of detergents / Triton X-100 and Emulgen 913, on FMO activity were determined and found that enzyme activity increased by the addition of either detergent at all concentrations (0.1%-1.0%). The apparent Vmax and Km values of bovine liver microsomal FMO for methimazole substrate were found as 1.23 nmol/min/mg and 0.11 mM, respectively. Thermostability of bovine liver microsomal FMO was studied at four different temperatures / 24 &amp / #61616 / C, 37 &amp / #61616 / C, 50 &amp / #61616 / C and 65 &amp / #61616 / C. The incubation time required for the complete loss of enzyme activity was 5 minutes at 65 &amp / #61616 / C, 10 minutes at 50 &amp / #61616 / C and 6.5 hours at 37 &amp / #61616 / C. 68 % of the activity was still detectable at the end of 53 hours at 24 &amp / #61616 / C. Bovine liver microsomal activity towards two drug substrates, imipramine and chlorpromazine, was also determined and found to be 3.73 and 3.75 nmol NADPH oxidized/min/mg, respectively. Effects of two drug substrates, imipramine and chlorpromazine, on bovine liver microsomal FMO-catalyzed methimazole oxidation activity was also studied and found that they inhibit FMO activity at all concentrations studied. Modulation of bovine liver microsomal FMO activity was studied using three different heavy metal ions / Ni+2, Cd+2 and Hg+2. At all other concentrations studied for each heavy metal ion and at all substrate methimazole concentrations (0.1, 0.2, 0.5, 1.0 mM), FMO-catalyzed methimazole oxidation activity decreased compared to control activity. KI values for Ni+2, Cd+2 and Hg+2 were found to be 0.5 mM, 0.085 mM, 4.6 &amp / #61549 / M, respectively. From the Dixon plot, the pattern of inhibition for three heavy metal ions was observed to be noncompetitive.