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Thesis (Ed.M.)--Boston Universit

Prediction of RNA secondary structure in hepatitis C and related viruses

The existence and functional importance of RNA secondary structure in the replication of positive-stranded RNA viruses is increasingly recognised. In this thesis several computational methods to detect RNA secondary structure in the coding regions of hepatitis C virus (HCV), hepatitis G virus (HGV)/GB virus C (GBV-C) and related viruses have been used. These include thermodynamic prediction of folding free energies (FFEs), evolutionary conservation of minimum energy structures between virus genotypes, suppression of synonymous variability and analysis of covariant and semi covariant substitutions in thermodynamically favoured structures. Each of the predictive methods provided evidence for conserved RNA secondary structure in the core and NS5B encoding regions of HCV and throughout the entire coding region of HGV/GBV-C.Positions in the HCV genome with predicted RNA structure localise precisely to regions of marked suppression of variability at synonymous sites, indicating that RNA structure constrains sequence change at what are generally regarded as phenotypically neutral sites. Combining these methods, the computational data obtained in this thesis demonstrates the existence of at least ten conserved stem loop structures within the NS5B coding region and three in that coding for the core protein both within the coding region of HCV. Analysis of the NS5B coding region and 3' untranslated region (3'UTR) of HGV/GBV-C indicates an even greater degree of RNA secondary structure. Remarkably, it appears from analysis of FFEs that extensive RNA secondary structure may exist along the entire length of both the HCV and HGV/GBV-C genomes, a finding with considerable implications for future functional studies.The existence of predicted RNA structures in the HCV genome was determined using controlled nuclease mapping of RNA transcripts from the core and NS5B regions under conditions which retained potential long-range RNA interactions. The pattern of cleavage sites of nucleases specific for single and double stranded RNA provided strong experimental support for structures previously predicted in this study. Electron microscopy was also used to directly visualise the RNA folding structure of HGV/GBV-C and provided some evidence for at least four structures within the NS5B coding region and long range RNA folding across the length of the virus genome.The degree of structural conservation between diverse HCV and HGV/GBV-C genotypes and related viruses suggests roles in virus replication, and/or RNA packaging for the discrete structures identified in this thesis. Whilst this role and that of the genome wide structure identified is currently not understood the structures predicted in this work are providing a starting point for such functional studies using the HCV replicon

Adaptive electronic throttle control of road vehicles

Previous work at Loughborough University has clearly demonstrated the gains that can be made in overall performance feel through the manipulation of the engine demand map. In particular the studies have shown the importance of the throttle progression, and the relationship between throttle pedal progression and wide-open throttle performance. These studies concluded with a clear set of design guidelines for the initial set up of a vehicle to achieve optimal performance feel for a population of drivers. These studies also highlighted the wide variation in response from different subjects indicating that further gains in satisfaction could be achieved if the demand map were optimised for each driver. Failing to provide optimum performance feel for the driver can result in reduced satisfaction, in turn making vehicles less saleable and more difficult to drive through the increased concentration needed to drive the vehicle. This thesis attempts to solve the problem of demographic and driver preference variation, by developing an electronic throttle system that adapts to driver preference. [Continues.

A twist in the tail : SHAPE mapping of long-range interactions and structural rearrangements of RNA elements involved in HCV replication

The RNA structure and long-range interactions of the SL9266 cis-acting replication element located within the NS5B coding region of hepatitis C virus (HCV) were determined using selective 2′-hydroxyl acylation analysed by primer extension. Marked differences were found in the long-range interactions of SL9266 when the two widely used genotype 2a JFH-1 (HCVcc) and genotype 1b Con1b sub-genomic replicon systems were compared. In both genomes, there was evidence for interaction of the sub-terminal bulge loop of SL9266 and sequences around nucleotide 9110, though the replication phenotype of genomes bearing mutations that disrupted this interaction was fundamentally different. In contrast, a ‘kissing loop’ interaction between the terminal loop of SL9266 and sequences in the 3′-untranslated X-tail was only detectable in JFH-1-based genomes. In the latter, where both long-range interactions are present, they were independent, implying that SL9266 forms the core of an extended pseudoknot. The presence of the ‘kissing loop’ interaction inhibited the formation of SL9571 in the 3′-X-tail, an RNA structure implicated in genome replication. We propose that, SL9266 may contribute a switch function that modulates the mutually incompatible translation and replication events that must occur for replication of the positive-strand RNA genome of HCV

War and peace in Achaemenid imperial ideology

Military activity played a determinative role in the history of the Achaemenid empire. This chapter considers some ideological dimensions of this fact. It does so through a separate examination of Persian and Greek representations of the role of war and warriors in the imperial setting. The place of war in the elite Persian psyche does remain rather elusive, but the Persian and Greek data-sets, radically different in content and character, are not far apart in their depiction of an ideological environment in which military values played a larger role than is sometimes acknowledged but were less fundamental than one might have expected. What is sometimes called the pax Achaemenica is certainly an artificial construct, but nothing compels us to replace it with the vision of a truly militarist society

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents

The journey of Zika to the developing brain

Zika virus is a mosquito-borne Flavivirus originally isolated from humans in 1952. Following its re-emergence in Brazil in 2015, an increase in the number of babies born with microcephaly to infected mothers was observed. Microcephaly is a neurodevelopmental disorder, characterised phenotypically by a smaller than average head size, and is usually developed in utero. The 2015 outbreak in the Americas led to the World Health Organisation declaring Zika a Public Health Emergency of International Concern. Since then, much research into the effects of Zika has been carried out. Studies have investigated the structure of the virus, its effects on and evasion of the immune response, cellular entry including target receptors, its transmission from infected mother to foetus and its cellular targets. This review discusses current knowledge and novel research into these areas, in hope of developing a further understanding of how exposure of pregnant women to the Zika virus can lead to impaired brain development of their foetus. Although no longer considered an epidemic in the Americas, the mechanism by which Zika acts is still not comprehensively and wholly understood, and this understanding will be crucial in developing effective vaccines and treatments