Characterized by a wide histological spectrum, myxofibrosarcoma ranges from deceptively bland-appearing lesions to frankly pleomorphic sarcomas, representing a suitable model to elucidate the molecular aberrations in multistep disease progression. Using genome-wide array comparative genomic hybridization, we have profiled DNA copy number alterations in myxofibrosarcoma samples and cell lines in coupled with expression profiling data and identified prominent SKP2 amplification on 5p, frequent homozygous deletion of MTAP on 9p, as well as loss expression of ASS1, a candidate tumor suppressor. As a predominant driving mechanism, SKP2 gene amplification was detected in one-third of cases in independent cohort validation and associated with SKP2 immunohistochemical expression, adverse prognosticators, and worse patient survival. Besides the classical attribute in promoting cell proliferation and tumor growth, we have confirmed the pro-metastatic oncogenic function of SKP2 and identified differentially expressed motility-promoting genes as its potential mediators, including ITGB2, ACTN1, IGF1, and ENAH. Since MTAP and ASS1 are key enzymes in either salvage pathway of nucleologenesis and amino acid biosynthesis, their deficiency suggests alterations involving metabolic homeostasis is important in sarcomagenesis. We in our studies not only validated their deletion and/or epigenetic silencing in myxofibrosaroma, evaluating the therapeutic responses of L-alanosine and ADI-PEG20 in MTAP or ASS1 deficient cells in vitro and in vivo, but also confirmed their tumor suppressor functions with special focus on tumor angiogenesis. Our study provides further insight into the molecular pathogenesis in tumor progression and highlights the prognostic, biological, and potential therapeutic relevance in myxofibrosarcoma.

Abstract Lung cancer is one of the most common cancer death in Taiwan. The dead population of lung cancer are over five thousands per year. High mortality and bad prognosis displayed the severity of the lung cancer. RAPD (Random Amplified Polymorphic DNA), a simple technique for the detection of genomic instability, has been used in this research. We inquire into genetic variation in carcinogenesis, and find out genes association with NSCLC. Three or more than three DNA fragment patterns of normal and lung cancer samples exhibited by RAPD from the seven arbitrary were classified as genomic instability. Four out of seven arbitrary primers have been used for lung cancer RAPD analysis and three of them were newly designed in this investigation. Analysis of genomic instability with these seven random primers in twenty-seven NSCLC patients revealed that 81.48% of NSCLC exhibited genomic instability. The RAPD reproducibilities of primer 6 and primer 7 were the best among the seven primers used in this study. Therefore, the variable DNA fragments of primer 6 and primer 7 in RAPD analysis were subcloned and sequenced for the study of the possible mutated genes in NSCLC. Results showed that DNA gains or losses were found in chromosomes 2, 4, 6, 14 and 22. After bioinformatic searching and alignments with human Genebank, some oncogenes (such as RABL2B, c-fos, n-myc and mas1) and tumor suppressor gene (AR) were found located nearby the locus of these subclones. Genomic instability was investigated in relation with the clinical-pathological features such as age, stage, tumor size, metastasis, differential status, survival days and cancer types. Results, evaluated by the X2 test, were not significant except tumor stage.

Summary While not negating the seriousness of tuberculosis and the need to prevent and combat the disease effectively, the large percentage of infected, apparently healthy individuals who harbour latent infections warrants consideration whether an endosymbiotic relationship is being established between mycobacteria and man. By means of a gene decay process eliminating their most metabolically important pathogenic genes associated with an increasing need for host gene products during prolonged intracellular survival, mycobacteria appears to be undergoing a process of establishing a less dangerous relationship with its host. To have tolerated this relationship over time, humans must have benefited. This is suggested to have occurred via changes in DNA higher order structure altering combinatorially regulated gene expression allowing increased cerebrodiversity. It can be expected that, beyond a certain threshold, negative effects ensued, leading to neuropathology and increased susceptibility for certain psychiatric disorders. These processes have probably been happening since the earliest contact with mycobacteria, but recently may have become modified by the emergence of epidemic tuberculosis and waves of increased oxidative stress following the circumstances associated with the Industrial Revolution and the more recent AIDS pandemic. The organism seems to have uniquely exploited the normal stress reaction of the host. Genomic stresses include changes associated with glucocorticoid effects as well as upregulated reactive oxygen species and stress/(heat shock) protein production, the latter two of which result in host cell cycle delay. Subsequently replication dependent chromosomal fragile sites appear in the host genome and together with upregulated chaperonins and mobile element activation, the scene is set for sequence exchange between the organism and host. If proven, these events raise the possibility of modifying chromatin epigenetically to retain the proposed advantages while silencing pathogenicity factors.

Replication fork stalling is a source of potentially tumourigenic genomic instability. The RecQ family helicase, Rqh1, is critical for the prevention of replication fork collapse and the formation of potentially deleterious recombination intermediates following fork stalling. Previous work in our lab with Schizosaccharomyces pombe (fission yeast) has shown that rqh10/rqh10 diploids are inherently unstable and show rapid reversion to the haploid state. The current work exploits this characteristic of fission yeast rqh10 mutants in a screen for genes that normally promote replication associated genomic instability. The rqh10rad30 mutant strains employed in this work incorporate the checkpoint deficiency caused by a lack of Rad3, so as to exacerbate the genomically unstable nature of this model. The current work describes the lithium acetate transformation based random mutagenesis by non-homologous integration of the ura4+ selectable marker into the rqh10rad30 fission yeast strains. This random mutagenesis generated extensive (24,500 – 50,000) mutant libraries. The quality of the libraries was assessed by can1 mutant assay, confirming an adequately extensive mutagenesis for the proposed screen. The process to be employed in the screen would involve the crossing of the mutant libraries, with the hope of generating diploids that will have two mutant copies of the same gene. Some of these diploids would appear unusually stable, showing a normal sporulation phenotype. This would indicate the mutation of a gene that normally promotes genomic instability following replication fork stalling. The practicality of the proposed screen of a vast number of diploids was assessed and described in detail in the current work. A technique involving inverse PCR (IPCR) adopted from previous work to identify mutants of interest, was also investigated. The investigation of this technique, and the work of others, suggests that transformation using such selectable marker fragments results in most apparent transformants containing extrachromosomal ura4+ fragments. These fragments are thought to provide the predominant template for IPCR, rendering the process unsuccessful at identifying the mutation in the current screen. However, with the mutant libraries generated, and the screen procedure detailed, the stage is set to conduct the screen once a more appropriate mutation location technique is identified. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-05-31 22:25:14.009

No description available.

Previous experimentation using differential immunological screening identified Tp0326, a protein predicted to be located in the outer membrane (OM) of the bacterium Treponema pallidum. This protein is homologous to BamA members of the β-barrel assembly machinery (BAM) family of proteins, which are conserved throughout pathogenic Gram-negative bacteria. In Escherichia coli the BAM proteins are found as a complex composed of five proteins: BamA, which is an integral membrane protein, and four accessory lipoproteins, BamB - BamE, which localize to the inner leaflet of the outer membrane. In E. coli BamA has been shown to mediate the insertion and assembly of proteins in the OM via interaction with the BAM complex and periplasmic chaperones (SurA, Skp, and DegP). We hypothesize that a similar OMP translocation complex exists within T. pallidum and that this complex is responsible for ushering T. pallidum OMPs to the bacterial surface. Characterization of the putative T. pallidum OMP transport machinery was performed by bioinformatic analyses and protein-protein interaction studies. Protein-protein interaction studies included screening a T. pallidum Lambda genomic expression library with recombinant T. pallidum protein Tp0326 and Far-Western blotting techniques. Using bioinformatic analyses we have identified putative T. pallidum homologues of the E. coli lipoproteins BamD (Tp0622) and BamB (Tp0133) as well as putative homologues of the E. coli chaperone proteins Skp (Tp0327) and DegP (Tp0773). The T. pallidum Lambda genomic expression library screen identified the putative E. coli BamD homologue (Tp0622), which was originally discovered through bioinformatic analyses. The expression library screen also identified two putative T. pallidum OMPs (Tp0750 and Tp0751) as potential interaction partners of Tp0326. Combined bioinformatic analyses and protein-protein interaction studies provide evidence a BAM complex may exist within T. pallidum, and similar to E. coli, this complex may be involved in ushering T. pallidum OMPs to the bacterial surface. / Graduate

The intestinal spirochaete Brachyspira hyodysenteriae is the causative agent of swine dysentery (SD), a diarrhoeal disease of pigs which has significant economic impact worldwide. Controlling SD remains problematic, particularly as there is no effective vaccine and there are few definitive diagnostic methods available. In this study, a partial genomic sequence of B. hyodysenteriae was screened in silico. A total of 19 putative open reading frames (ORFs) encoding outer-membrane proteins then were selected and these were subjected to a laboratory screening process. To select potential universal vaccines, a preliminary study was conducted using PCR to determine the distribution of the putative genes in 23 strains of B. hyodysenteriae. A total of 17 of the 19 ORFs were considered to be suitable for further testing as they were found to be present in the majority of strains investigated. After molecular cloning and protein expression and purification, of 19 cloned candidate molecules derived from 17 genes (one large gene was divided into two parts encoding N and C terminal proteins, respectively), 14 were expressed in E. coli and the recombinant proteins were successfully produced. A variety of sera from pigs naturally and experimentally infected with B. hyodysenteriae were tested for reactivity with the 14 recombinant proteins in an immunoblotting assay. Seven molecules from six genes reacted strongly with the tested sera, and therefore were selected and used to immunize mice. All these proteins generated a specific antibody response. Post-immunization sera raised against each recombinant protein had the capacity to agglutinate B. hyodysenteriae cells, and also recognized the cognate proteins of B. hyodysenteriae in cell extracts. Further sequencing analysis demonstrated that these molecules were highly conserved in the genomes of different B.hyodysenteriae strains. Therefore, from the genomic based study, the products of six genes were identified as promising candidates for vaccines or as diagnostic targets. Four genes were expressed on a large scale, the product (NAV-H7, NAV-H17 Cterminal, NAV-H34 and NAV-H42) were combined into one vaccine, and then this preparation was used to immunise pigs that subsequently were challenged with B. hyodysenteriae. These antigens generate systemic and colonic antibody responses, and vaccination tended both to delay the onset of clinical signs and attenuate lesion development. Hence these recombinant proteins showed promise as components for further SD vaccines. Recombinant proteins from the selected genes also were used as antigens in class-specific ELISAs used as serological assays for SD. Three antigens (NAV-H8, NAV-H42 and Bhlp29.7) were selected as good indicators of seroconversion in IgM ELISAs, and these were evaluated further using a large range of serum samples. The NAV-H8 IgM ELISA using a cut-off value 2.5 times the mean value of all negative pigs could be used as a herd test for SD, and both the NAV-H8 and NAV-H42 IgM ELISAs had potential for detecting exposure to B. hyodysenteriae at the pig level.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2006. / Vita. Includes bibliographical references.

The genomic variability of sixty-nine populations of the potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis from Europe and South America were analyzed using the RAPD-PCR technique with sixty-six 10-mer primers. Large genomic differences were found between the two PCN species (i.e. 33%). The genomic pool of British G. pallida populations showed considerably less variation than the Peruvian populations, with 73% and 41% similarity between populations respectively. The genomic similarity among populations of G. rostochiensis was 89% for UK populations and 82% when the two continental European populations were included. Nevertheless, between populations within each species and from the same locality, genomic differences were still found. The RAPD-PCR technique proved to be useful for revealing the genomic variability between and within species using DNA extracted from 50 cysts, but it gave variable results when DNA extracted from individual females or cysts was used, suggesting that for evaluating the genomic variability of individuals it is better to use specific primers. RAPD-PCR was also used successfully to distinguish the two PCN species, individuals selected and selected for virulence and even biotypes using individual cysts. Based on the results found when comparing biotypes of Globodera pallida, it is suggested that all the biotypes considered in the International Pathotype Scheme could be grouped into Pa1 and Pa2/3 when classifying European populations, and Pa1A or Pa1B, P4A, P5A and P6A when analyzing South American populations. However, these groupings should be regarded just as a reference, because virulence bioassay results plus the data found using the RAPD-PCR technique suggested that, at least in G. pallida, virulence seems to be a polygenic trait ruled by several genes with additive effects. On the other hand, based on the same sort of data, virulence in G. rostochiensis seems to be ruled only by major genes. Selected and unselected populations of G. pallida, reared on either potato clone Solanum vernei (VTn)2 62.33.3 or a susceptible control, were distinguished using the RAPD-PCR technique and primers Operon A-07, E-06, G-16 and I-05. Three of the fragments that appeared to distinguish the unselected from the selected populations were cloned into an isolate of E. coli and their sequences obtained. Gpalpha, seems to be part of a promoter region of a gene probably related or linked to virulence. The use of differential clones to characterize PCN populations with different proportions of each virulence gene is a valuable tool. Whilst diagnostic probes for routine identification of virulent populations are being developed, the use of the “gene pool similarities” concept involving the DNA patterns of standard populations as genetic virulence types (i.e. virulence biotypes), integrated with information on their response to differential clones bearing genes for resistance, would represent the best approach towards devising a sustainable control strategy to optimize the usefulness of whatever resistance is available.

Genome annotation is an important topic since it provides information for the foundation of downstream genomic and biological research. It is considered as a way of summarizing part of existing knowledge about the genomic characteristics of an organism. Annotating different regions of a genome sequence is known as structural annotation, while identifying functions of these regions is considered as a functional annotation. In silico approaches can facilitate both tasks that otherwise would be difficult and timeconsuming. This study contributes to genome annotation by introducing several novel bioinformatics methods, some based on machine learning (ML) approaches. First, we present Dragon PolyA Spotter (DPS), a method for accurate identification of the polyadenylation signals (PAS) within human genomic DNA sequences. For this, we derived a novel feature-set able to characterize properties of the genomic region surrounding the PAS, enabling development of high accuracy optimized ML predictive models. DPS considerably outperformed the state-of-the-art results. The second contribution concerns developing generic models for structural annotation, i.e., the recognition of different genomic signals and regions (GSR) within eukaryotic DNA. We developed DeepGSR, a systematic framework that facilitates generating ML models to predict GSR with high accuracy. To the best of our knowledge, no available generic and automated method exists for such task that could facilitate the studies of newly sequenced organisms. The prediction module of DeepGSR uses deep learning algorithms to derive highly abstract features that depend mainly on proper data representation and hyperparameters calibration. DeepGSR, which was evaluated on recognition of PAS and translation initiation sites (TIS) in different organisms, yields a simpler and more precise representation of the problem under study, compared to some other hand-tailored models, while producing high accuracy prediction results. Finally, we focus on deriving a model capable of facilitating the functional annotation of prokaryotes. As far as we know, there is no fully automated system for detailed comparison of functional annotations generated by different methods. Hence, we developed BEACON, a method and supporting system that compares gene annotation from various methods to produce a more reliable and comprehensive annotation. Overall, our research contributed to different aspects of the genome annotation.