Mixed genotype Hepatitis C virus infections: incidence in Scotland and methods for detection

The diagnosis of mixed genotype hepatitis C virus (HCV) infection is rare and information on incidence in the UK, where genotypes 1a and 3 are the most prevalent, is sparse. Considerable variations in the efficacies of direct-acting antivirals (DAAs) for the HCV genotypes have been documented and the ability of DAAs to treat mixed genotype HCV infections remains unclear, with the possibility that genotype switching may occur. In order to estimate the prevalence of mixed genotype 1a/3 infections in Scotland, a cohort of 512 samples was compiled and then screened using a genotype-specific nested PCR assay. Mixed genotype 1a/3 infections were found in 3.8% of samples tested, with a significantly higher prevalence rate of 6.7% (p<0.05) observed in individuals diagnosed with genotype 3 infections than genotype 1a (0.8%). An analysis of the samples using genotypic-specific qPCR assays found that in two-thirds of samples tested, the minor strain contributed <1% of the total viral load. The potential of deep sequencing methods for the diagnosis of mixed genotype infections was assessed using two pan-genotypic PCR assays compatible with the Illumina MiSeq platform that were developed targeting the E1-E2 and NS5B regions of the virus. The E1-E2 assay detected 75% of the mixed genotype infections, proving to be more sensitive than the NS5B assay which identified only 25% of the mixed infections. Studies of sequence data and linked patient records also identified significantly more neurological disorders in genotype 3 patients. Evidence of distinctive dinucleotide expression within the genotypes was also uncovered. Taken together these findings raise interesting questions about the evolutionary history of the virus and indicate that there is still more to understand about the different genotypes. In an era where clinical medicine is frequently more personalised, the development of diagnostic methods for HCV providing increased patient stratification is increasingly important. This project has shown that sequence-based genotyping methods can be highly discriminatory and informative, and their use should be encouraged in diagnostic laboratories. Mixed genotype infections were challenging to identify and current deep sequencing methods were not as sensitive or cost-effective as Sanger-based approaches in this study. More research is needed to evaluate the clinical prognosis of patients with mixed genotype infection and to develop clinical guidelines on their treatment.

Reading Pitscottie's Cronicles: a case study on the history of literacy in Scotland, 1575-1814

This thesis addresses a range of research questions regarding literacy in early modern Scotland. Using the early modern manuscripts and printed editions of Robert Lindsay of Pitscottie’s late sixteenth-century 'Cronicles of Scotland' as a case study on literacy history, this thesis poses the complementary questions of how and why early modern Scottish reading communities were encountering Pitscottie’s 'Cronicles', and how features of the material page can be interpreted as indicators of contemporary literacy practices. The answers to these questions then provide the basis for the thesis to ask broader socio-cultural and theoretical questions regarding the overall literacy environment in Scotland between 1575 and 1814, and how theorists conceptualise the history of literacy. Positioned within the theoretical groundings of historical pragmatics and ‘new philology’ – and the related approach of pragmaphilology – this thesis returns to the earlier philological practice of close textual analysis, and engages with the theoretical concept of mouvance, in order to analyse how the changing ‘form’ of Pitscottie’s 'Cronicles', as it was reproduced in manuscript and print throughout the early modern period, indicates its changing ‘function’. More specifically, it suggests that the punctuation practices and paratextual features of individual witnesses of the text function to aid the highly-nuanced reading practices and purposes of the discrete reading communities for which they were produced. This thesis includes extensive descriptive material which presents previously unrecorded data regarding twenty manuscripts and printed witnesses of Pitscottie’s 'Cronicles', contributing to a gap in Scotland’s literary/historiographical canon. It then analyses this material using a transferable methodological framework which combines the quantitative analysis of micro-data with qualitative analysis of this data within its socio-cultural context, in order to conduct diachronic comparative analysis of copy-specific information. The principal findings of this thesis suggest that Pitscottie’s 'Cronicles' were being read for a combination of devotional and didactic purposes, and that multiple reading communities, employing highly nuanced reading practices, were encountering the text near-contemporaneously. This thesis further suggests that early modern literacy practices, and the specific reading communities which employ them, should be described as existing within a spectrum of available practices (i.e. more or less oral/aural or silent, and intensive or extensive in practice) rather than as dichotomous entities. As such, this thesis argues for the rejection of evolutionary theories of the history of literacy, suggesting that rather than being described antithetically, historical reading practices and purposes must be recognised as complex, coexisting socio-cultural practices, and the multiplicity of reading communities within a single society must be acknowledged and analysed as such, as opposed to being interpreted as universal entities.

Applied evolution: An integrated approach to studying life history traits in response to drug selection

The use of chemical control measures to reduce the impact of parasite and pest species has frequently resulted in the development of resistance. Thus, resistance management has become a key concern in human and veterinary medicine, and in agricultural production. Although it is known that factors such as gene flow between susceptible and resistant populations, drug type, application methods, and costs of resistance can affect the rate of resistance evolution, less is known about the impacts of density-dependent eco-evolutionary processes that could be altered by drug-induced mortality. The overall aim of this thesis was to take an experimental evolution approach to assess how life history traits respond to drug selection, using a free-living dioecious worm (Caenorhabditis remanei) as a model. In Chapter 2, I defined the relationship between C. remanei survival and Ivermectin dose over a range of concentrations, in order to control the intensity of selection used in the selection experiment described in Chapter 4. The dose-response data were also used to appraise curve-fitting methods, using Akaike Information Criterion (AIC) model selection to compare a series of nonlinear models. The type of model fitted to the dose response data had a significant effect on the estimates of LD50 and LD99, suggesting that failure to fit an appropriate model could give misleading estimates of resistance status. In addition, simulated data were used to establish that a potential cost of resistance could be predicted by comparing survival at the upper asymptote of dose-response curves for resistant and susceptible populations, even when differences were as low as 4%. This approach to dose-response modeling ensures that the maximum amount of useful information relating to resistance is gathered in one study. In Chapter 3, I asked how simulations could be used to inform important design choices used in selection experiments. Specifically, I focused on the effects of both within- and between-line variation on estimated power, when detecting small, medium and large effect sizes. Using mixed-effect models on simulated data, I demonstrated that commonly used designs with realistic levels of variation could be underpowered for substantial effect sizes. Thus, use of simulation-based power analysis provides an effective way to avoid under or overpowering a study designs incorporating variation due to random effects. In Chapter 4, I 3 investigated how Ivermectin dosage and changes in population density affect the rate of resistance evolution. I exposed replicate lines of C. remanei to two doses of Ivermectin (high and low) to assess relative survival of lines selected in drug-treated environments compared to untreated controls over 10 generations. Additionally, I maintained lines where mortality was imposed randomly to control for differences in density between drug treatments and to distinguish between the evolutionary consequences of drug treatment versus ecological processes affected by changes in density-dependent feedback. Intriguingly, both drug-selected and random-mortality lines showed an increase in survivorship when challenged with Ivermectin; the magnitude of this increase varied with the intensity of selection and life-history stage. The results suggest that interactions between density-dependent processes and life history may mediate evolved changes in susceptibility to control measures, which could result in misleading conclusions about the evolution of heritable resistance following drug treatment. In Chapter 5, I investigated whether the apparent changes in drug susceptibility found in Chapter 4 were related to evolved changes in life-history of C. remanei populations after selection in drug-treated and random-mortality environments. Rapid passage of lines in the drug-free environment had no effect on the measured life-history traits. In the drug-free environment, adult size and fecundity of drug-selected lines increased compared to the controls but drug selection did not affect lifespan. In the treated environment, drug-selected lines showed increased lifespan and fecundity relative to controls. Adult size of randomly culled lines responded in a similar way to drug-selected lines in the drug-free environment, but no change in fecundity or lifespan was observed in either environment. The results suggest that life histories of nematodes can respond to selection as a result of the application of control measures. Failure to take these responses into account when applying control measures could result in adverse outcomes, such as larger and more fecund parasites, as well as over-estimation of the development of genetically controlled resistance. In conclusion, my thesis shows that there may be a complex relationship between drug selection, density-dependent regulatory processes and life history of populations challenged with control measures. This relationship could have implications for how resistance is monitored and managed if life histories of parasitic species show such eco-evolutionary responses to drug application.