Characterization of the Transcriptional Properties of Equine Infectious Anemia Virus.

The transcriptional properties of equine infectious anemia virus were examined in two distinct equine cell lines in which the virus establishes either a persistent or a cytopathic infection. Northern hybridization analyses were performed to determine the number, sizes, and relative levels of the EIAV transcripts encoded during persistent or cytopathic infections. Three species of viral mRNA were detected in infected cells: an 8.2-kb full-length genomic mRNA, a 3.5-kb single-spliced mRNA, and a low abundance 1.5-kb mRNA, presumably formed by a double-splicing event of the full-length mRNA. Analysis of the levels of EIAV-specific RNA\u27s present during persistent and cytopathic infections has revealed that quantitative differences characterize the transcriptional patterns of EIAV in these two infections. In persistently infected FEK cells the 8.2- and 3.5-kb mRNA\u27s are the predominant viral transcripts and are detected in approximately equal concentrations, while the 1.5-kb mRNA is detected at very low levels. During the cytopathic infection of FDD cells, however, the 3.5-kb mRNA is the predominant viral transcript, comprising nearly 75% of the total viral mRNA, while the 8.2- and 1.5-kb mRNA\u27s constitute the remaining 25% of viral transcripts. Moreover, the cytopathic infection is characterized by almost a thirty-fold higher level of viral transcripts than those detected during the persistent infection. The splicing patterns of the full-length EIAV mRNA during the cytopathic infection were determined by cDNA cloning and sequencing, Northern hybridization analyses using splice donor-specific oligonucleotide probes, and S1 nuclease mapping of RNA from virus-infected cells. The results have identified the splice donor and acceptor sites used to generate the spliced mRNA\u27s of EIAV in infected cells. The expression of a putative regulatory protein of EIAV from a structural viral gene was investigated by analysis of in vitro and in vivo expression products. In vitro transcription and translation along with in vivo expression in transfected COS-7 cells were used to analyze the expression of the viral env gene. Based on the results of these studies, a potential mechanism for co-expression of two separate proteins from the env mRNA is proposed

Self-adaptive containers: interoperability extensions and cloud integration

Driven by an ever-increasing diversity of application contexts, execution environments and scalability requirements, modern software is faced with the challenge of frequent code refactoring. To address this, we have proposed an STL-like self-adaptive container library, which dynamically changes its data structures and resource usage to meet programmer-specified Service Level Objectives relating to performance, reliability and primary memory use. A prototype of this library has been implemented and utilised in two case studies to prove its viability. In the present work, we explore a low-cost means to extend our library to satisfy wider classes of Service Level Objectives. This is achieved through the integration of third-party container frameworks, which exploit parallelism to boost performance and disk-based data offloading to reduce primary memory consumption, and the integration of cloud storage services, which offer cost-effective location-free storage. We demonstrate our library's application in a state-space exploration case study. With very low programmer overhead, experimental results show that our library can improve performance with a 76% reduction in insertion time and an 86% reduction in search time, and can also exploit out-of-core storage, including cloud storage

Characterisation of Endogenous Retroviral Elements in Canine DNA

Dogs suffer from a variety of neoplastic, immunological, degenerative and proliferative disorders for which a retroviral aetiology is suspected. Despite decades of effort, the significance of retroviruses in canine disease remains obscure. This murky subject has been clouded further by a dearth of information concerning canine endogenous retroviral complement and expression. Previous reports have described retroviral expression in both normal and neoplastic canine tissues; but have not defined the origin of the expression. It was considered of interest to determine whether such expression reflected the presence of endogenous retroviral elements in canine genomic DNA. If such elements were found, their aetiological significance in canine diseases would be investigated

Genes of the ovine major histocompatibility complex class II region

The major histocompatibility complex (MHC) is a multi-gene family encoding proteins which play important roles in the immune response to antigenic challenge. Three distinct regions designated class 1, 11, and 111 have been defined in the MHC of mouse and man. This thesis focuses on the genes of the class 11 region of the sheep. The products of the class 11 alpha (A) and beta (B) genes are heterodimeric glycoproteins whose physiological function is to present exogenous peptides to helper T cells. The recognition of MHC class 11/peptide complexes by the T cell receptor signals the release of a cytokine cascade resulting in T and B cell proliferation, macrophage activation and B cell differentiation with the production of increased amounts of pathogen-specific antibody.Much is known about the detailed structure and function of the MHC of man and mouse. However, when this project began little was known about the detailed structure of the MHC of the ungulates, the economically important group of animals which contains cattle, pigs, horses and sheep. As part of a study investigating fundamental cellular immunology in the sheep, this thesis describes the characterisation and expression of the genes of the sheep MHC class 11 region.Cosmid libraries prepared from DNA from three unrelated sheep were screened with probes from the DP, DQ and DR sub-regions of the human and mouse MHC class 11 regions. Cosmids were used because they facilitate the cloning of relatively large genomic inserts. Restriction maps of the cosmids have been produced showing that some of the clones overlapped. The MHC A and B genes within the clones have been sequenced and assigned to a specific sub-type. Functional genes have been identified by the reaction of their products with anti-sheep class 11 monoclonal antibodies following DNA-mediated transfection into the mouse L cell, a fibroblast cell line which does not express endogenous mouse class 11 genes. Transcription of some of the genes has been demonstrated by Northern blots and reverse transcription polymerase chain reaction.A restriction map of the sheep class 11 DQ sub-region has been constructed and shown to contain two distinct DQA loci with associated DQB genes. The DQ1 A/B gene pair was expressed in the mouse L cell. The sheep class 11 DQ1 product at the cell surface reacted with a sub-set of the available anti-sheep class 11 monoclonal antibodies. The DQ2 genes were transcribed and some evidence for their cell surface expression was obtained, although this was not formally proved.A previous study demonstrated the expression of a putative sheep DRA gene when co-transfected with a sequenced DRB gene. The sequence of the sheep DRA gene is described here together with sequence data from a number of DRB genes or pseudogenes which show that, depending on haplotype, the sheep DR sub-region may contain up to five DRB genes.The cloning and sequencing of ruminant orthologues of the HLA-DNA and -DOB genes is described for the first time. Although evidence was obtained for the transcription of the sheep DNA gene, its DOB gene partner is transcriptionally silent.A class 11 locus designated DY which is not found in mouse or man is described from the sheep MHC. Cloning and sequencing has shown that it contains a class 11 A/B gene pair like that of the expressed DQ1 locus, however, it appears to be transcriptionally silent