MicroRNAs are small non-coding RNAs which regulate gene expression by binding to 3&rsquo / UTR of their target mRNAs. Deregulated expression of microRNAs is detected in many pathologies including different types of cancers. miR-106b, is a member of miR-106b-25 cluster and overexpressed in many cancers including breast cancer. Based on miR-106b overexpression, we hypothesized that miR-106b may be an oncogene candidate. To explore miR-106b related phenotypes, we used an already miR-106b transfected model cell line system. Stably transfected MCF10A cells were investigated for alterations in cell growth, motility, migration and invasion. Our results showed that miR-106b overexpression caused increased growth motility and migration. On the other hand, based on matrigel invasion assay miR-106b expression caused a reduction in cell invasion. Further studies are needed to be performed to understand the precise role of miR-106b in breast cancer. Studies are underway to detect possible miR-106b targets that may help to explain these phenotypical alterations.

ABSTRACT GENETIC DIFFERENTIATION OF LIQUIDAMBAR ORIENTALIS MILL. VARIETIES WITH RESPECT TO matK REGION OF CHLOROPLAST GENOME &Ouml / ZD&amp / #272 / LEK, Asli M.S., Department of Biology Supervisor: Prof. Dr. Zeki Kaya August 2007, 87 pages Liquidambar L. genus is represented with mainly 4 species in the world and one of these species, Turkish sweet gum (Liquidambar orientalis Mill.) which is a relictendemic species is naturally found in only southwestern Turkey, mainly in Mugla Province. The limited distribution of species with two disputed varieties (var. integriloba Fiori and var. orientalis) and increased anthropogenic threats to its genetic resources signify the importance of studying genetic diversity in the species to have better conservation and management programs. For this purpose, 18 different populations were sampled throughout the species range and matK region of chloroplast DNA (cpDNA) was sequenced to assess the genetic structure of the species. Turkish Liquidambar orientalis populations were evaluated at two categories: variety level and geographic level. Also, two sectors of matK region were examined to assess which part of the region was more variable. All molecular analysis was conducted in this study by using MEGA version 3.1 and Arlequin 2.000 softwares. v Moleculer diversity analysis indicated that the population located in Fethiye- G&uuml / nl&uuml / kbasi district has the highest number of polymorphic sites. This population is also genetically the most distant from the others (average genetic distance 0.0038). Among the studied varieties, the average genetic distance within var. integriloba (0.0016) which also includes population Fethiye-G&uuml / nl&uuml / kbasi was the greatest. Among the geographic regions, Mugla-1 including Fethiye-K&ouml / ycegiz-Aydin district as well as population Fethiye-G&uuml / nl&uuml / kbasi showed the highest average genetic distances within the region with a value of 0.0015. According to the molecular variance results, among varieties and among geographic regions, there was no significant differentiation, but great amount of total variation was found (~86%) within Turkish sweet gum populations. With respect to the Fst values among varieties, the highest genetic differentiation was observed between var. orientalis and unknown group (0.040). Furthermore, based on the results of phylogenetic analysis, Turkish populations of L. orientalis have genetically closer to USA relative (L. styraciflua L.) than Chinese relatives (L. acalycina H.T Chang and L. formosana Hance). In conclusion, 10 Turkish sweet gum populations were found to be important for conservation issues. Furthermore, eight of these located in Mugla province and sixth of them belong to var. integriloba. Especially Fethiye-G&uuml / nl&uuml / kbasi, Marmaris&Ccedil / etibeli and Mugla-Kiyra populations should be included in either insitu or exsitu or in both conservation programs in the future.

Genomic instability is commonly seen in breast cancers. To date, various chromosomal or segmental loss or amplification regions have been detected in primary tumors and cell lines. Hence, an intensive search for potent tumor suppressors or oncogenes located in these regions continues. MicroRNAs (miRNAs) are ~18-24 nt long non-coding RNAs that regulate protein expression either by target mRNA cleavage or translational repression. We hypothesized that miRNAs located in genomic instability regions in breast cancer cells may contribute to the initiation or maintenance of breast tumors. Here, we investigated genomic levels of miRNAs on frequent loss or gain regions of breast cancer cells. First, using bioinformatics resources we mapped known miRNAs and candidate miRNAs to reported genomic instability regions. Our extensive searches resulted with more than 30 known miRNAs and 35 candidate miRNAs. To further confirm loss or amplification of miRNA genes on these chromosomal regions in breast cancer cells, we designed specific primers for the known pre-miRNA DNA regions and performed semi-quantitative PCR in 20 breast cancer cell lines, 2 immortalized mammary cell lines, and 2 control samples. Densitometry results suggested that a striking 61 % (22/36) of selected miRNAs showed either loss or amplification in at least 3 different breast cancer cell lines. Interestingly most of these alterations were found to be amplifications even in regions reported to harbor losses in breast tumors. Genomic fold change results of these microRNAs provide a biologically relevant starting point for further expression and functional experiments of microRNAs in breast cancer studies. Genomic fold change analysis followed expression analysis of two significant microRNAs (hsa-miR-21 and hsa-miR-383) was done by qRT-PCR method. Our data provide a wide screen of genomic instability of 36 microRNA genes in 20 breast cancer cells and normal samples detected by semi-quantitative duplex PCR method as well as expression analysis of two microRNAs. To this date, such an extensive data on genomic status of microRNA genes in breast cancer cells did not exist. Therefore, our results are the first comprehensive investigation of many microRNA genes on genomic instability regions in breast cancers and provide further clues to the potential involvement of these microRNAs in breast tumorigenesis MicroRNA genomic instability may affect their expression and therefore their targets&rsquo / expressions. Understanding how these microRNAs regulate their targets and contribute to the neoplastic events will also contribute to the field by using this information for future diagnostic and threaupetical applications.

The aim of this study was cloning of wheat (Triticum aestivum L. cv. Bayraktar) Trehalose-6-phosphate synthase gene and examining of gene expression pattern of wheat seedlings in response to salt and drought stress conditions using Wheat GeneChip (Affymetrix). In this study, 10-days old wheat seedlings were subjected to the salt (350 mM NaCl) and drought stress (20% PEG) for 24 hours, then root and leaf tissues were used for wheat TPS gene cloning and microarray studies. RACE (Rapid Amplification of cDNA Ends) was used to determine cDNA sequence of wheat TPS gene, TaTPS. The ORF of TaTPS encodes a putative protein of 859 amino acids with a predicted molecular weight (MW) of 96.7 kDa and an isoelectric point (pI) of 5.97. Based on tblastx, TaTPS showed great similarity with other plants TPS genes. In root tissue, expression of TaTPS increased under drought stress while no change was observed under salt stress. In leaf tissue, both salt and drought treatments repressed the expression of TaTPS. Microarray study was used to monitor transcript abundance in salt and drought stressed wheat. Data analyses were determined by using GCOS 1.4 and GeneSpring GX10. The genes encoding ferritin, Lipid transfer protein, LEA/Dehydrin, early nodulin, cold regulated protein and germin like proteins were upregulated at least 10-fold under salt and drought stress conditions. In addition, salt and drought stresses induced the expression of genes identified as DREB, ERF, NAC, MYB, and HSF, suggesting existence of various transcriptional regulatory pathways under salt and drought stresses.

Multidrug resistance phenotype of tumor cells describes resistance to wide range of structurally unrelated anticancer agents and is a serious limitation to effective chemotherapy. It is a multifactor yet not fully elucidated phenomenon by the involvement of diverse cellular pathways. Aim of this study was to investigate the resistance mechanisms developed against docetaxel and doxorubicin that are widely used in the treatment of breast cancer in model cell line MCF-7. Resistant sublines were developed by application of drugs in dose increments and effect of docetaxel and doxorubicin on drug applied cells were investigated by cell viability assays. Expression analysis of P-gp, MRP1, BCRP, Bcl-2, Bax and &amp / #946 / -tubulin isotypes were performed by RT-PCR, qPCR, Western blot and immunocytochemistry. Genome-wide expression analysis was also performed by cDNA microarray. According to cell viability assays, drug applied cells developed varying degree of resistance to docetaxel and doxorubicin. Gene expression analysis demonstrated that de novo expression of P-gp contributed significantly to drug resistance. Expression levels of class II, III and V &amp / #946 / -tubulin isotypes increased in docetaxel resistant sublines. According to microarray analysis, a variety of genes showed significantly altered expression levels particularly drug metabolizing and detoxification enzymes (i.e. increased GPX1 and GSTP1 with decreased POR), survival proteins (e.g. decreased TRAIL together with increased decoy receptors and CD40), extracellular matrix components (e.g. increased integrin signaling), growth factors and cytokines (e.g. EGFR1, FGFR1, CTGF, IL6, IL8 and IL18 overexpression), epithelial-mesenchymal transition proteins (i.e. increased vimentin and N-cadherin with decreased E-cadherin and occludin) and microtubule dynamics related proteins (e.g. increased MAP1B and decreased MAP7). Development of cross-resistance and combined drug effects on resistant sublines were also studied. Results demonstrated that docetaxel and doxorubicin resistant cells developed cross-resistance to paclitaxel, vincristine, ATRA, tamoxifen and irradiation. Finally, modulatory effects of verapamil and promethazine in combined drug applications were investigated and verapamil and promethazine were shown to decrease MDR1 expression level thus reverse the MDR. They also showed synergic and additive effects in combined docetaxel and doxorubicin applications. Identification of resistance mechanisms may personalize chemotherapy potentially increasing efficacy of chemotherapy and life quality of patients.

The emergence of drug-resistance of tumor cells is a major complication for succesful chemotherapy. In this study, the molecular mechanisms of resistance to prednisone, vincristine and melphalan in multiple myeloma cell lines, RPMI-8226 and U-266 were investigated. Drug resistance was induced by application of the drugs by stepwise dose increments and confirmed by XTT cytotoxicity assay. Gene expression analysis demostrated that MDR1 gene is one of the most important factor causing the multidrug resistance phenotype in prednisone, vincristine and melphalan resistant multiple myeloma cell lines. According to microarray analysis alterations in laminin, integrin and collagen genes were detected. Additionally, upregulation of some oncogenes and growth factors (Rho family of GTPases, YES1, ACT2, TGFBR, EPS15, PDGF) was shown to have a role in MDR in multiple myeloma. Significant downregulation of suppressors of cytokine signalling gene expressions and upregulation of different types of interleukine and interferon gene expressions (IL3 and interferon-gamma receptor) which are related to JAK-STAT signalling pathay was shown. Alterations in expression levels of genes related to ceramide metabolism were shown especially for melphalan resistance in multiple myeloma. The data from vincristine/prednisone and vincristine/melphalan drug combination studies were shown that the usage of vincristine on prednisone and melphalan resistant multiple myeloma cell lines increase the efficacy of the chemotherapy. On the other hand the cross-resistance development of prednisone and melphalan resistant sublines to irradiation was detected. These results may help to understand the molecular mechanisms of prednisone, vincristine and melphalan resistance in multiple myeloma model cell lines RPMI-8226 and U-266.

In this study, the adaptation and response of Aspergillus nidulans and Aspergillus fumigatus wild type and cpcA strains to antifungal compounds were studied using cultural, genetic and proteomic methods. CpcA is the fungal cross pathway control protein which may also have a role in the development of resistance to antifungal that has become a major problem in human and plant fungal diseases and many studies are devoted to address the drug resistance mechanisms. Cell adapts itself to stress when it is subjected to a stress repeatedly. The ancestor of CpcA, ATF4 (CREB2) has recently been found to be important in the survival of tumor cells after starvation and nutrient limitation and these findings are expected to open new insights into the future antifungal therapy. Fungal cross pathway control system conserves similar mechanism with the stress response pathway in humans as a response to amino acid starvation. Fungal adaptation to antifungal agents was studied using the genetic model A. nidulans with the experimentally induced adaptation setup. It was concluded that A. nidulans cells are able to adapt to antifungal. In order to understand how cell becomes resistant to a previously susceptible agent, it is important to investigate the process when the cell encounters the agent for the first time. Fungal cellular response to antifungal drugs was studied using the human opportunistic pathogen A. fumigatus at the protein level. This is the first proteomic study directed to investigate the A. fumigatus response to voriconazole (VRC). The recently developed two dimensional gel electrophoresis approach, Fluorescence 2-D Differential Gel Electrophoresis (DIGE) method was applied to visualize differentially expressed proteins. It was concluded that, about 150 proteins were differentially regulated as a response to stress exerted by azole group antifungal drugs. cpcA strains of A. nidulans and A. fumigatus were compared to wild type strains in terms of susceptibility to various stresses, adaptation potential also at the proteome level. The results obtained in this study showed that CpcA was important in the response of Aspergillus to oxidative, heat stress and in the adaptation of cells to VRC and that its absence drastically changed the cellular response to VRC at the protein level by changing the expression of about 80 proteins. Thus, this protein is a good candidate in future as a potential drug resistance mediator and further characterization is needed to elucidate its mechanism of action on drug resistance.

microRNA dependent gene expression regulation has roles in diverse processes such as differentiation, proliferation and apoptosis. Therefore, deregulated miRNA expression has functional importance for various diseases, including cancer. miR-125b is among the commonly downregulated miRNAs in breast cancer cells . Therefore we aimed to characterize the effects of miR-125b expression in MCF7 breast cancer cell line (BCCL) to better understand its roles in tumorigenesis. Here, we investigated mir-125 family members

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.