Cancer is the collective term used to describe a diverse group of diseases that share certain hallmarks, which in turn enables the affected cells to sustain an uncontrolled cell growth. Despite the increasing efforts and advances in cancer therapies, cancers are still responsible for approximately 10% of all the deaths worldwide. Furthermore, the increase in the average human lifespan will further contribute to the cancer incidences. This brings the necessity to focus our efforts on early detection and effective diagnosis methods. With the advances in high-throughput genomics technologies, gene expression signatures have gained attention as a novel method in cancer diagnostics. These signatures are identified by simply comparing the expression levels of genes in tumor and control samples. Here, we propose an alternative method based on the probe expression level measurement of 3&rsquo / UTR of candidate genes. We chose breast cancer as a model and performed an in silico analysis on publicly available gene expression datasets of Affymetrix chips to analyse 3&rsquo / UTR shortening during breast cancer situation. Overall, our analysis suggests that shortening of 3&rsquo / UTR is a significant mechanism observed in breast cancer .

Schizophrenia is a complex mental disorder that is commonly characterized as deterioration of intellectual process and emotional responses and affects 1% of any given population. SNPs are single nucleotide changes that take place in DNA sequences and establish the major percentage of genomic variations. In this study, our goal was to identify SNPs as genomic markers that are related with schizophrenia and investigate the genes and pathways that are identified through the analysis of SNPs. Genome wide association studies (GWAS) analyse the whole genome of case and control groups to identify genetic variations and search for related markers, like SNPs. GWASs are the most common method to investigate genetic causes of a complex disease such as v schizophrenia because regular linkage studies are not sufficient. Out of 909,622 SNPs analysis of the dbGAP Schizophrenia genotyping data identified 25,555 SNPs with a p-value 5x10-5. Next, combined p-value approach to identify associated genes and pathways and AHP based prioritization to select biologically relevant SNPs with high statistical association are used through METU-SNP software. 6,000 SNPs had an AHP score above 0.4, which mapped to 2,500 genes suggested to be associated with schizophrenia and related conditions. In addition to previously described neurological pathways, pathway and network analysis showed enrichment of two pathways. Melanogenesis and vascular smooth muscle contraction pathways were found to be highly associated with schizophrenia. We have also shown that these pathways can be organized in one biological network, which might have a role in the molecular etiology of schizophrenia. Overall analysis results revealed two novel candidate genes SOS1 and GUCY1B3 that have a possible relation with schizophrenia.

Scald disease (Rhynchosporium secalis) is one of the major plant diseases causing considerable yield loss in barley (Hordeum vulgare) plantations in Turkey. To develop, scald resistant barley varieties, C.R.I.F.C. of Turkey has a large accumulated collection of barley seed sources in hand, but these samples are difficult to be followed and used in the breeding programs due to lack of genetic studies on them. Thus, the objective of this study was to characterize and fingerprint of eighty barley seed sources, and assess the magnitude and pattern of genetic diversity that could be used to have more efficient scald disease resistant breeding programs in the future. Forty scald disease resistant and 40 scald sensitive Turkish barley seed sources were screened using 6 simple sequence repeats (SSR) primers. Each of barley seed source were represented with four seeds, assuming they are genetically uniform since barley is a self-pollinated crop. Estimated genetic parameters indicated that scald disease resistant and sensitive barley seed sources still maintain large amount of genetic diversity. For example, expected heterozygosity was 0.62&plusmn / 0.01 and 0.64&plusmn / 0.01 for resistant and sensitive Turkish barley seed sources, respectively. Thirty-nine percent of total genetic variation was between populations for resistant and 46% for sensitive group, while 61% of total variation was within populations for resistant group and 54% for sensitive group. When overall Turkish barley seed sources were considered, genetic distances between scald sensitive seed source S18 and resistant R1 as well as between sensitive S28 and resistant R1 were large. Scald resistant and sensitive barley seed sources were generally located in different clusters in dendrogram. The presence of R25, R39 and S16 barley seed sources with high genetic diversity parameters among studied seed sources, suggests that this diversity could be important drive in future barley breeding program in Turkey. However, further study is needed to illustrate genetic divergence of Turkish barley seed sources with use of more molecular markers.

In this study, mechanisms of resistance to Imatinib-induced apoptosis in human K562 and Meg-1 chronic myeloid leukemia (CML) cells were examined. Continuous exposure of cells to step-wise increasing concentrations of Imatinib resulted in the selection of 0.2 and 1 &amp / #956 / M imatinib resistant cells. Measurement of endogenous ceramide levels showed that treatment with Imatinib increased the generation of C18-ceramide significantly, which is mainly synthesized by the human longevity assurance gene 1 (hLASS1), in sensitive, but not in resistant cells. Mechanistically, analysis of mRNA and enzyme activity levels of hLASS1 in the absence or presence of Imatinib did not show any significant differences in the resistant cells when compared to its sensitive counterparts, suggesting that accumulation and/or metabolism, but not the synthesis of ceramide, might be altered in resistant cells. iv Indeed, further studies demonstrated that expression levels, and enzyme activity of sphingosine kinase-1 (SK-1), increased significantly in resistant K562 or Meg-1 cells. The expression levels of glucosyl ceramide synthase (GCS) also increased in resistant cells, comparing to the sensitive counterparts, which indicates conversion of pro-apoptotic ceramide to glucosyl ceramide. Expression analyses of BCR-ABL gene demonstrated that expression levels of BCR-ABL gene increased gradually as the cells acquired the resistance. However, Nucleotide sequence analyses of ABL kinase gene revealed that there was no mutation in Imatinib binding region of the gene in resistant cells. There was also an increase in expression levels of MDR1 gene in resistant cells, which transport the toxic substances outside of cells. In conclusion, these data show, for the first time, a role for endogenous ceramide synthesis via hLASS1 in Imatinib-induced apoptosis, and those alterations of the balance between the levels of ceramide and S1P. Mainly the overexpression of SK-1 seems to result in resistance to Imatinib in K562 cells. The cellular resistance may also result from conversion of ceramide to glucosyl ceramide, from overexpression of BCR-ABL and MDR1 genes but not due to mutations in Imatinib binding site of ABL kinase.

ABSTRACT TRANSCRIPTION PATTERN COMPARISON OF TWO UBIQUITIN SPECIFIC PROTEASES (USP6 AND USP32) Akhavan Tabasi, Shiva M.Sc., Department of Biology Supervisor: Assist. Prof. Dr. A. Elif Erson August 2007, 93 pages Breast cancer is the most common type of cancer among women worldwide. The incidence of breast cancer is 1 in 8 among women. Usually loss of tumor suppressor genes and overexpression of proto-oncogenes are known to be involved during mammary tumorigenesis. USP32 (Ubiquitin Specific Protease 32) gene is located on chromosomal band 17q23, a region of amplification in breast cancer. Gene amplification is known to be a common mechanism in breast cancer cells, through which proto-oncogenes are overexpressed and contribute to tumor progression. Presence of multiple oncogene candidates on 17q23 requires individual characterization of these genes. USPs (Ubiquitin Specific Protease), have various roles in protein degradation pathways (e.g / by editing the ubiquitin chains, recycling of ubiquitin, v deubiquitinating the target proteins and inhibiting their degradation by the proteasome). Deregulated expression of USPs is likely to interfere with the degradation of many key regulatory proteins in the cell. Therefore, USP32 becomes an interesting oncogene candidate that may have roles in protein degradation pathways based on the fact that it is located on an amplicon region and that it is overexpressed in breast tumors. On the other hand, USP6 (Ubiquitin Specific Protease 6), a known oncogene on 17p13, is also a deubiquitinating enzyme, with conserved histidine and cysteine domains, which are also shared by USP32. Interestingly there is a 97% sequence similarity between bases 3,197 to 7,831 of USP6 and 2,390 to 7,024 of USP32 gene. In this study, we aimed to investigate the expression patterns of USP32 and USP6 (including alternative transcripts) in breast tissue to avoid any possibility of overlapping functions of two enzymes due to their high sequence similarity. In addition, we sub-cloned USP32 gene into TOPO-TA vector, so that further functional studies (e.g / localization and overexpression) can be performed. Further characterizations of Ubiquitin Specific Protease 32, may help us understand its importance in the protein degradation pathway during breast tumorigenesis.

17q23 amplicon is one of the many chromosomal regions that undergo amplification in breast tumors. Such amplicons harbor proto-oncogenes that may be overexpressed due to gene amplifications. Copy number analysis in breast cancer cell lines and breast tumors identified several independently amplified regions within the 17q23 amplicon, suggesting that a number of genes are selected for amplification as they may independently contribute to tumor formation and progression. To characterize distinct amplicons on 17q23 and localize putative oncogenes, various studies are done on this region. In order to better understand the role of 17q23 amplification in breast cancer, characterizing unidentified genes or ESTs (Expressed Sequence Tags) on the 17q23 amplicon is crucial. In this study, in silico analysis of human (H.sapiens), chimpanzee (P.troglodytes), and mouse (M.musculus) genomes were performed to examine sequence homology between these 3 species for the purpose of identifying novel genes. The objective of this study was to analyze a 50 kb region between TBX2 and TBX4 genes and characterize the EST T02811 located on that region. Analysis and comparisons of these three genomes were established based on genomic sequences and availability of ESTs with gene prediction programs (BLAST, Pipmaker, Vista, GENSCAN etc.). Based on our results, we prepared a homology map between these 3 species, including positions of ESTs that may indicate novel genes. In this 50 kb region, we found in silico and experimantal evidence for the presence of an unidentified gene. We managed to extend the 313 bp EST T02811 size to 1423 bp which we think is as of yet incomplete. Further studies are needed to characterize this novel gene as a potential oncogene candidate. Characterizing the roles of such candidate oncogenes in amplicons will provide a better understanding of genomic amplicon regions and their roles in tumorigenesis.

Yellow rust disease is one of the most important problems in wheat production. It causes substantial yield losses throughout the world. There are resistant and susceptible wheat varieties to various yellow rust pathotypes. In this thesis genes that are induced in wheat, in virulence and avirulence conditions upon yellow rust inoculations were investigated. Consequently, it was aimed to identify genes that may be playing critical roles in the disease resistance mechanism. The strategy was to construct subtracted cDNA libraries from resistant and susceptible plants and analyse the sequences obtained from these libraries. The subtraction approach in this study differs from the common subtraction designs implicated in plant-pathogen interactions / instead of comparing a compatible or an incompatible interaction with a control, one of the subtractions in this study is done taking a compatible interaction as the tester and an incompatible one as the driver, and the second subtraction, vice versa. Therefore, it was intended to compare the transcriptomes from compatible and incompatible plant-pathogen interactions directly. Suppression Subtractive Hybridization method was used to construct subtracted cDNA libraries. Two subtractions were performed / SSH1 (D-R), taking a compatible interaction as the tester sample and an incompatible one as the driver sample, and SSH2 (R-D), taking an incompatible interaction as the tester sample and a compatible one as the driver. In the end, two subtracted cDNA libraries, SSH1 (D-R) library (1536 clones) and SSH2 (R-D) library (1152 clones) were obtained and the libraries were sequenced. Sequence results were subjected to BlastN and BlastX analysis. We looked for a group of genes that were frequently emphasized in plant disease related studies when we searched within the Blast N homology results of the two libraries. We found out that 19 such genes are present in our libraries. We discussed supposed induction of these genes in the interactions investigated in our study. The fact that these genes were found to be present in our libraries enhances the reliability of our results suggesting that the gene sequences we found indeed belong to genes differentially expressed in the respective comparisons investigated in our study. As such, it also implies that other sequences that were found similar to genes of known functions may represent candidate genes as subjects of further studies investigating wheat-yellow rust interactions.

Extracellular endo-1,4-b-mannanase (EC 3.2.1.78) gene of Aspergillus fumigatus IMI 385708 (formerly known as Thermomyces lanuginosus IMI 158749) was cloned and transformed into Aspergillus sojae (ATCC 11906) and Pichia pastoris GS115. High level of expression was achieved in both expression systems. Attempts to produce heterologous mannanase in Arabidopsis thaliana, suitable for large scale production, were not successful. Comparison of the expression levels of heterologous mannanase reveals that A. sojae is a better expression system than P. pastoris with respect to extracellular mannanase activity. The production of mannanase in A. sojae (AsT1) after 3 days of incubation reached 204 U/ml in YpSs containing 1 % glucose. In P. pastoris (PpT1), highest production was observed after 10 hrs of induction with methanol (61 U/ml). Expressed enzymes were purified and analyzed. Both enzymes have specific activity c. 349 U/mg protein with pH and temperature optimum of c. 4.5 and c. 60 &deg / C for mannanases from AsT1 and c. 5.2-5.6 and c. 45 &deg / C for mannanases from PpT1. A truncated form of mannanase (MAN-S) deleted at amino acids from P291 to P368, which still displayed hydrolytic activity was also isolated and characterized. MAN-S has pH and temperature optimum of c. 6.5-8.0 and c. 60 &deg / C. During incubation of the mannanase on locust bean gum, transglycosylation reactions, in which longer or rare prebiotic oligosaccharides could be produced catalyzed by glycolysis, was detected. The products of hydrolytic activity of the enzyme on various carbohydrates were analyzed by PACE and MALDI-TOF. Accordingly, hexamannose and smaller oligosaccharides were characterized.

In the present study samples from four native Turkish Cattle Breeds / South Anatolian Red (n= 48), East Anatolian Red (n= 34), Anatolian Black (n= 42) and Turkish Grey (n=46) and elite bulls of Holstein (n=21) were genotyped with respect to two milk production enhancer genes, Prolactin (PRL) and Diacylglycerol acyltransferase (DGAT1), and one disease (Complex Vertebral Malformation) causing gene (SLC35A3). A allele frequency for PRL gene, believed to be positively associated with the milk yield in cattle, ranged between 0.5645 (Anatolian Black) - 0.7558 (South Anatolian Red). K allele frequency which is thought to be related with the milk fat content in cattle varied between 0.7794 (East Anatolian Red) - 0.9250 (Anatolian Black). Complex Vertebral Malformation gene was not observed in any of the examined individuals (n= 164), hence, SLC35A3 locus was monomorphic. Pairwise Fst values based on the two polymorphic loci revealed that breeds are not significantly different from each other with respect to these two genes. Correlations, but weak, between the PRL A allele frequency and milk yield and similarly DGAT1 K allele and milk fat content was observed, Principle Component Analysis generated two compound axis based on the two polymorphic loci. Positions of the breeds on the first axis were correlated with the milk fat content of the breeds, perfectly. Again, positions of the breeds on the second axis were correlated with the milk yield of the breeds. Furthermore, PCA revealed that both A of PRL and K of DGAT1 genes seemed to have contributions in milk yield Results are believed to be useful for the management efforts of Turkish native cattle breeds.

Scytalidium thermophilum is a thermophilic fungus with an important role in the composting process of mushroom cultivation. An extracellular phenol oxidase of Scytalidium thermophilum (STEP) with novel features was previously studied in our laboratory. This enzyme later turned out to be a catalase having phenol oxidase activity. The aim of this study was to clone Scytalidium thermophilum bifunctional catalase/phenol oxidase encoding gene and express the gene in Aspergillus sojae for future site directed mutagenesis studies. Scytalidium thermophilum catalase gene was first cloned into E. coli XL1 Blue MRF&rsquo / and then heterologously expressed in Aspergillus sojae ATCC11906. For that aim, the catalase gene was amplified using specific primers, excluding the signal and pro-peptide coding regions and amplified fragment was then cloned into E.coli XL1 Blue MRF&rsquo / and sequenced. It was observed that the cloned gene, named as catpo, was 10 amino acids different from the amino acid sequence of the S.thermophilum catalase gene formerly cloned by Novo Nordisk. The catpo gene encoding a mature protein of 681 amino acids was then ligated onto expression vector pAN52-4 and the recombinant plasmid was transformed into Aspergillus sojae ATCC11906. Heterologous expression was observed under the control of the glyceraldehydes 3-phosphate dehydrogenese promoter of Aspergillus nidulans and the secretion signal of the glucoamylase gene of Aspergillus niger. Catalase activity of the transformants reached at a level of 13206 U/g at the end of the fourth day of cultivation. However, this is still lower than the catalase activity of the gene donor strain of Scytalidium thermophilum.