Failure of chemotherapy in cancer patients because of development of drug resistance is a major problem. Alterations of DNA repair mechanisms and drug targets are among the important resistance mechanisms which are developed against topoisomerase II inhibitors etoposide and doxorubicin. Modifications in the expression levels of mismatch repair (MMR) genes due to resistance to topoisomerase II inhibitors are involved in breast cancer. In this study, etoposide resistant sublines were developed from MCF7 breast cancer cell line (MCF7/S) and the expression levels of TOP2A and two important MMR genes MSH2 and MLH1 were examined by real time qPCR. Previously developed doxorubicin resistant cells were also studied for comparison. Etoposide resistant sublines MCF7/1000E, MCF7/1250E and MCF7/2000E were approximately 2, 3 and 4 fold resistant relative to parental MCF7/S cells, respectively. MLH1, MSH2 and TOP2A expressions decreased in both etoposide and doxorubicin resistant sublines relative to MCF7/S cells. Expression levels of TOP2A in resistant sublines differ between 10-95 percent of the expression levels in the parental cells. In the sublines MCF7/200E, MCF7/500E, MCF7/750E and MCF7/1000E a decrease in TOP2A gene expression was determined. In sublines MCF7/1250E and MCF7/2000E fluctuations in the expression levels were observed. Among the doxorubicin resistant sublines (MCF7/600D and MCF7/1000D), in MCF7/1000D which is more resistant to doxorubicin, TOP2A expression level was higher. Expression levels of MSH2 decreased regularly as the resistance increased. However, in MCF7/1250E significant increase relative to MCF7/1000E was observed. In MCF7/2000E, expression levels of MSH2 again significantly decreased to 41 percent of the levels in parental cell line. Expression levels of MLH1 decreased significantly (18-58 percent) in etoposide resistant sublines relative to MCF7/S cells. In doxorubicin resistant sublines, a decrease in MLH1 gene expression was observed in MCF7/1000D. It can be concluded from the results that decrease in the expression levels of TOP2A, MSH2 and MLH1 genes may contribute to resistance together. Above a certain resistance level, sublines may develop new strategies for acquiring higher resistance. Whenever a strategy becomes limited, new strategies emerge. New approaches developed to overcome resistance in cancer chemotherapy should consider the molecular basis of resistance in different stages of the disease.

Osteosarcoma (OS) is a type of cancer that starts in the bone. It generally occurs in the cells called osteoblasts which form matrix of the bone. It is the most common malignant tumor of bone with an incidence rate of 19% among all cancer types. The vast majority of OS patients have pulmonary metastases at the time they are diagnosed, and about half develop lung disease later. Moreover, pulmonary metastatic tumors lead to poor prognosis and increased death rate. Although mutations in the genes coding for p53, Rb, fos and myc were detected in pulmonary metastatic tumors of OS, there is no unique genetic pathway identified for progression of pulmonary metastasis. In this research, a genome wide association study (GWAS) using FFPE samples from lung tissue of 9 patients with pulmonary metastatic OS was performed. Among 358 associated SNPs, rs6499861, rs10884554 and rs12154602 were found to be associated with metastatic OS most significantly. Moreover, second wave analysis of GWAS results provided the significant genes and pathways associated with metastatic OS. A methodology for copy number aberration and LOH analysis of SNP array data of a FFPE sample was generated using R-aroma package. Results were obtained by three different methods, namely, CalMaTe, TumorBoost and Virtual Normal algorithm. Among these, CalMaTe was found to produce less noisy data than VN Algorithm during total copy number segmentation. LOH analysis could only be performed for one sample with the second method due to poor data quality of the other samples. According to the results of copy number aberration and LOH analysis of one tumor sample T8, copy number gains in 1p31.1, 6p21.32, 7p14.3, 11q22.1, 12p12.1, and 18q12.1 chromosomal regions and copy number losses in 2p16.2, 8q24.13, 17q23.3 and 17q21.31 chromosomal regions have been found. Moreover, LOH events were observed in 2q14.3, 11q13.4, 18p11.21, 19q12, 20p13 and 23q21.1 chromosomal regions. Identification associated SNPs and significant copy number changes may be helpful in investigation of potential diagnostic and prognostic markers in metastatic osteosarcoma.

Resistance to various chemotherapeutic agents is a major problem in success of cancer chemotherapy. One of the primary reasons of development of multidrug resistance (MDR) is the overexpression of ATP binding cassette (ABC) transporter proteins. Breast cancer resistance protein (BCRP) belongs to ABC transporter family and encoded by ABCG2 gene. BCRP is mainly expressed in MDR1 (P-glycoprotein) lacking breast cancer cells. Overexpression of BCRP leads to efflux of chemotherapeutic agents at higher rates, therefore, decreased levels of intracellular drug accumulation. Despite the fact that several chemical modulators claim to restore BCRP-mediated increased drug efflux, these modulators were shown to display various side effects, precluding their clinical use. Therefore, to reverse BCRPmediated MDR phenotype by a modulator with minimum cytotoxicity may increase clinical benefits and minimize side effects.

Human telomerase is a reverse transcriptase which synthesizes telomeric repeat sequences at the ends of chromosomes. The telomerase enzyme has two essential subunits to be functional which are called telomerase reverse transcriptase (hTERT) and human telomerase RNA (hTR). Telomerase uses its RNA subunit as a template for the addition of hexameric repeats at the ends of chromosomes. The activity of telomerase has been detected in immortal cells but not in most normal somatic cells. Therefore, its activity could serve as diagnostic or prognostic marker in malignancies. Telomeres are heterochromatic DNA sequences bound by a number of telomere binding proteins in order to maintain the stability of chromosomes. Protection of telomere 1(POT1) is a single stranded telomere binding protein which is thought to have significant role in the recruitment of telomerase to telomeres. The objective of the current study to investigate telomerase activity and gene expression of hTERT and hPOT1 in human colorectal cancer tissues. The activity of telomerase was examined in colorectal tumors and normal adjacent specimens by and improved telomeric repeat amplification protocol (TRAP)-Silver Staining Assay. The expression levels of hTERT and hPOT1 genes was analysed by qPCR. The results showed that colorectal cancer tumors showed significantly high telomerase activity whereas normal adjacent tissues were found to be telomerase negative. Among clinicopathological parameters / the stage, histological type, distant metastasis and lymph node metastasis status of tumors were found to show significant differences in terms of telomerase activity. Moreover, the expression of human telomerase reverse transcriptase (hTERT) was found to be overexpressed in tumor tissues compared to normal adjacent tissues. Likewise, colorectal tumors expressed high level of hPOT1 compared to normal tissues. Both the expression of hTERT and hPOT1 correlated with telomerase activity. It can be concluded from the results of the current study that high telomerase activity and overexpression of hTERT and hPOT1, may indicate that they could serve as diagnostic or prognostic indicators in colorectal cancer.

Although the use of atypical antipsychotic drugs is successful in the treatment of schizophrenia, they cause complications in the long term use that is mainly weight gain. In this study, circulating levels of hypothalamic neuropeptides/hormones, which are related to appetite regulation / neuropeptide Y (NPY), alpha melanocyte stimulating hormone (&alpha / -MSH), cocaine and amphethamine regulated transcript (CART) and plus leptin in male schizophrenic patients who were treated with an atypical antipsychotic drug, risperidone, which is a serotonin antagonist, for 4 weeks was investigated. Based on the hypothesis that the risperidone treatment might alter the circulating levels of those neuropeptides through the serotonergic antagonism, it results in the weight gain. Leptin plasma levels were increased in the risperidone treated patients accompanying by weight gain vs controls and NPY, &alpha / -MSH, CART levels were decreased in the patients before the treatment but they were not changed after treatment. To determine alterations of those candidate genes mRNA expression levels, male Wistar rats were orally administered with risperidone for 4-weeks. Rat studies show that the mRNA expression and plasma levels of POMC, AgRP, and NPY were decreased but CART mRNA levels were increased while their plasma levels were decreased unexpectedly. In conclusion, the serotonergic antagonism of risperidone on POMC neurons may cause increase in appetite / and hence, increased weight gain and leptin levels, even in a short term trial.

The aim of this study is to develop a metabolically engineered P. pastoris strain for production of an active extracellular thermostable glucose isomerase (GI) enzyme by using genetic engineering techniques. For this purpose, research program was performed in two sub-programs. In the first sub-program, xylA gene from Thermus thermophilus was amplified and inserted into pPICZ&alpha / -A expression vector. Thereafter, this pPICZ&alpha / -A::xylA vector was cloned into AOX1 locus in P. pastoris genome and expressed under alcohol oxidase promoter which is induced by methanol. After constructing the recombinant P. pastoris strains, the best producing strain was selected according to the specific enzyme activity assay and SDS-PAGE analyses in batch shaker-bioreactor experiments. The selected recombinant P. pastoris clone carrying xylA gene in its genome was named as eP20. Using recombinant P. pastoris eP20 clone, effects of salt and sorbitol concentration on the cell growth and recombinant GI activity were investigated. The data obtained from the experiments showed that the maximum cell and GI activity values were obtained in production medium that contained 30 g L-1 sorbitol, 4.35 g L-1 ammonium sulphate, 0.1 M potassium phosphate buffer (pH 6.0), 14.9 g L-1 MgSO4&bull / 7H2O, 1.17 g L-1 CaSO4 &bull / 2H2O, 1 ml L-1 chloramphenicol and 4.35 ml L-1 PTM1 / where, the maximum biomass and recombinant GI activity were calculated , respectively, as 6.3 g L-1 and 3.21 U L-1. Moreover, the research program related with the effect of initial sorbitol concentration shows that optimum initial sorbitol concentration, CS0 is 50 g L-1 that resulted a cell concentration and recombinant GI activity which are 7.32 g L-1 and 3.6 U L-1, respectively. In the second part of the M.Sc. of the study, a pilot scale bioreactor experiment in a working volume of 1 L was performed in controlled bioreactor system. The variations in the cell growth, recombinant GI activity, AOX activity, total protease activity and organic acid concentrations throughout the fermentation were analyzed whereas the specific growth rates, yield coefficients and specific consumption rates were also calculated. The results showed that a pH and oxygen controlled operation enabled an important increase in recombinant GI activity. In this context, recombinant GI activity was increased as 56.1-fold and resulted in 202.8 U L-1 at t=12 whereas the maximum biomass concentration was obtained as 85.2 g L-1 at t=36. In this study, an active thermostable recombinant GI enzyme was produced extracellularly by a yeast cell, i.e. recombinant P. pastoris, for the first time.

MORPHOMETRIC AND GENETIC DIFFERENTIATION BETWEEN ANATOLIA AND CYPRUS BOMBUS (BOMBUS) TERRESTRIS (L. 1758) POPULATIONS BETON, Damla M. Sc., Department of Biology Supervisor: Prof. Dr. Aykut Kence September 2004, 86 pages Four microsatellite loci were used to investigate differentiation in Bombus terrestris, a bumblebee of interest for its high value crops pollination. Two bumblebee populations, one from Ankara (the capital of Turkey) and one from North Cyprus were analyzed. In these populations, the total number of alleles detected per polymorphic locus ranged from 7 to 12. FST genetic distance between Ankara and North Cyprus B. terrestris populations based on four microsatellite loci was calculated as 0,09351. This applies that there is significant (P&lt / 0,001) differentiation between Anatolian and Cypriot populations. Moreover, statistically significant differences between two populations were found in wing characters studied. According to the potential for local adaptation and individual fitness of bumblebees, microsatellite data calls for protection of Bombus terrestris populations against importation of bumblebees of foreign origin which are used as crop pollinator.

In this study the genetic diversity in Turkish native sheep breeds was investigated based on microsatellite DNA loci. In total, 423 samples from 11 native and crossbreed Turkish sheep breeds (Akkaraman, Morkaraman, Kivircik, ivesi, Dagli&ccedil / , Karayaka, HemSin, Norduz, Kangal, Konya Merinosu, T&uuml / rkgeldi) and one Iraqi breed (Hamdani) were analyzed by sampling from breeding farms and local breeders. After excluding close relatives by Kinship analysis, the genetic variation within breeds was estimated as gene diversities (HE), which ranged between 0.686 and 0.793. The mean number of observed alleles (MNA) ranged between 5.8 and 11.8. The allele frequency distribution across Turkey showed no gradient from east to west expected in accordance with the Neolithic Demic Diffusion model. The differentiation between different samples of Akkaraman, Dagli&ccedil / and Karayaka breeds was tested by FST index. Akkaraman1 sample from the breeding farm was significantly (P&lt / 0.001) different from the other two Akkaraman samples. Deviation from HW expectations observed for Akkaraman1, ivesi, Morkaraman and HemSin breeds. AMOVA analysis revealed that most of the total genetic variation (~90%) was partitioned within the individuals. In parallel to this observation, when factorial correspondence analysis and shared alleles distances were used to analyze the relationship between the individuals of the breeds, there was no clear discrimination between breeds. Moreover, NJ tree constructed based on DA genetic distance, and PC analyses were used to analyze among breed differentiation. Delaunay Network drew 4 genetic boundaries (two of them being parallel to geographic boundaries) between breeds. All the results indicated that Kivircik was the most differentiated breed. Finally, Mantel Test and Bottleneck analysis did not reveal a significant result. Kivircik breed, among all native Turkish breeds, was found to be the genetically closest to the European breeds based on the loci analyzed. The genetic variation in Turkish breeds was not much higher than that of European breeds, which might be a consequence of the recent sharp decrease in sheep number.

Serine proteases are a family of proteases that utilize an activated serine residue in the sub&not / strate-binding pocket to catalytically hydrolyze peptide bonds. Enzymes which belong to this family, with a diverse array of metabolic and regulatory functions, play critical roles in cell physiology and pathology. &lsquo / Clp&rsquo / s are a class of ATP dependent serine proteases which are composed of a protease (ClpP) and an ATPase (ClpA or ClpX) component. Their involvements in degrading proteins are especially implicated under stress conditions. In contrast to members of Bacteria and Eukarya, little is known about the energy-dependent proteolysis and there is no report on Clp family proteases in Archaea. In this study, for the fist time, a periplasmic Clp P-like (PSP) and a membrane bound serine protease (MSP) genes from thermophilic archaeon Thermoplasma volcanium GSS1 were cloned and expressed in E. coli. PCR amplifications at 55 &ordm / C yielded unique fragments of 971 and 1521bp, for PSP and MSP genes, respectively, which were ligated to p-Drive cloning vectors and introduced into E.coli TG1 competent cells. Recombinant clones were screened depending on blue/white colony selection. Putative recombinant plasmids were analyzed by restriction enzyme digestions. Serine protease activities of the three positive clones (E. coli TG-S1, E. coli TG-S4 and E. coli TG-M1) were determined spectrophotometrically by using chromogenic oligopeptide substrates. These results indicated that cloned PSP and MSP genes were successfully expressed in E. coli under the control of their own promoters. Heterologous expression of PSP gene was also attempted by adding 6xHis tag to the 5&acute / end of the PSP gene in pQE 30 expression vector. Competent E.coli TG1 cells were transformed by pQE expression constructs. Positive clones were detected on colony blots using Anti-His HRP conjugates and chromogenic DAB substrate. Plasmids of these colonies were analyzed by restriction digestions to select the true recombinants. Expression of the 6xHis-PSP fusion protein from the recombinant E. coli TG-pQE-S1.7 strain was confirmed by functional analysis and SDS-PAGE. An NCBI domain search and multiple sequence alignment using Clustal W 1.82 program indicated homologies between PSP and MSP of Tp. volcanium and various bacterial ATP dependent ClpPs. Signal peptide search using Signal P 3.0 server predicted a signal peptide sequence in MSP homologous to that of Gram (+) bacteria.

In this study, Scytalidium thermophilum laccase gene was first cloned into E. coli and then heterologously expressed in A. sojae. S. thermophilum is a thermophilic fungus with an important role in determining selectivity of compost produced for growing Agaricus bisporus. S. thermophilum laccase gene was first cloned by Novo Nordisk Bio Tech, Inc. in 1998. This laccase gene (lccS) has an open reading frame of 2092bp. It is composed of five exons punctuated by four small introns. The coding region, excluding intervening sequences is very GC-rich (60.8% G+C) and encodes a preproenzyme of 616 amino acids: a 21 amino acid signal peptide and a 24 amino acid predicted propeptide. lccS gene was amplified using specific primers to exclude the signal and pro-peptide coding regions and ligated to expression vector pAN52-4. The recombinant plasmid was used to transform Aspergillus sojae ATCC11906 (pyrG-). Heterologuos expression was observed in glucose-containing media, under the control of the glyceraldehydes 3-phosphate dehydnogenese promoter and the secretion signal of glucoamylase gene. Laccase gene is an important step towards the high level expression of this enzyme in a GRAS eucaryotic host and for further biotransformation and enzyme engineering studies. In this study also bioinformatic analysis of N-terminal and C-terminal propeptide cleavage sites of fungal proteins including laccases were studied.