In 1988 an article from the Harvard School of Public Health sparked the 'directed mutation' debate. Its authors claimed that bacteria starved of their accustomed food were able to specifically control their genetic mutations and so adapt directly to use alternative food sources. Apparently, they were not at the mercy of random mutation to achieve adaptation as Neo-Darwinian theory demands. Rather, the authors claimed the bacteria chose their mutations and participated in their evolution as Lamarckian theorists had previously supposed. The controversy that followed was comprised of two sub-debates. The first concerned negotiation of this molecular genetic anomaly in bacteria. The second concerned the broader debate between Lamarckians and Darwinians, and contributed a new episode to a considerable historical legacy of similar dissent. The directed mutation debate has been protracted. I argue that it has been prolonged by active factors, which I refer to as 'perpetuating forces'. These include: the historical and cultural context of the controversy, the influence of scientific dogma on the evaluation of the anomalies, the role of defamation by association in Lamarckian resurrections, the interdisciplinary contest for authority and participants' styles of advocacy. I also analyse the role of the Internet in the protraction of this debate and provide quantitative analysis of the scale change caused by uptake of the debate in what I term 'the Internet forum'. To enable this analysis I apply boundary theory and the cartographic metaphor. I extend that theory in line with its architects recommendations addressing the concept of 'old maps', and identifying boundary work at the interdisciplinary boundaries within the territory 'science'.

Ageing is a major factor contributing to human morbidity and disease, including cancer. To study the possible involvement of epigenetic changes in ageing, murine haematopoiesis was used as a model system. The key cells determining ageing in this system are thought to be lower side population (LSP) cells of the bone marrow, which are enriched for long-term reconstituting haematopoietic stem cells (LT-HSCs). In this thesis, rare primary LT-HSCs from young, middle-aged and old mice were isolated and phenotyped. A protocol, termed Nano-MeDIP-seq was developed for methylome analyses on such rare cells. DNA and RNA from these cells were then subjected to comprehensive methylome (MeDIP-seq) and transcriptome (RNA-seq) analysis. Age-related changes in the LT-HSC methylome and transcriptome were observed in this study, many at genes associated with migration/adhesion and not previously implicated in ageing. These changes also include directional (young to old) global loss of DNA methylation of approximately 5%, 111 significantly (FDR < 0.2) age differentially methylated regions (aDMR) and more than one thousand significantly (FDR < 0.05) differentially expressed transcripts. Ingenuity pathway analysis (IPA) identified significant (p << 0.0001) age-related decline in B-Cell development and significant (p << 0.0001) alterations in pathways and functions associated with cell movement. A number of genes were identified to be significantly age differentially methylated and differentially expressed. One aDMR, associated with the Serum deprivation protein response (Sdpr) gene, was functionally validated to demonstrate a negative correlation between promoter methylation and differential expression. The findings in this thesis support a model involving an epigenetic dysregulation of the genes that control the interaction between LT-HSCs and their regulatory niche, during physiological ageing.

No description available.

The principal aim of the study was to develop methods for the measurement of potential urinary biomarkers of oxidative stress using liquid chromatography/tandem mass spectrometry with minimum sample preparation to avoid artefact formation. Initially the development of an assay to measure the urinary concentrations of isoprostanes (8- isoPGF2α) was attempted but this did not prove to be sufficiently sensitive and gave nonreproducible results. An assay to measure the intact sulphate and glucuronide conjugates of urinary metabolites of vitamin E [α-tocopheronolactone (α-TLHQ) and α-carboxy-ethylhydroxychroman (α-CEHC)] was then developed, as it has been suggested that α-TLHQ with an oxidised chroman ring might be an indicator of oxidative stress. A novel method was also developed to quantitate urinary amino acids associated with NO• metabolism (Larginine - precursor, L-citrulline - product, L-ADMA –inhibitor of nitric oxide synthase and L-homocysteine – reduces bioavailability of nitric oxide). This method was extended to quantitate seven additional amino acids. The latter two methods were applied to 32 children with type 1 diabetes and compared with age and sex matched controls. The mean concentrations of all the α-THLQ conjugates were highly significantly increased in the diabetic subjects (p<0.002). The concentrations of the α-CEHC conjugates were also increased but not to the same degree of significance (p<0.05). When the diabetic children were divided into those who were poorly (n=24) and adequately (n=8) controlled, the α- THLQ conjugates remained highly significantly increased (p<0.002) in the poorly controlled group compared to controls. However, the concentrations of the α-CEHC conjugates were not significantly different. The diabetic subjects had a highly significantly increased concentration (p<0.0001) of all the urinary amino acids studied compared to controls. These results suggest that the measurement of urinary α-TLHQ conjugates may provide a useful biomarker of oxidative stress. The clinical relevance of the increased concentrations of urinary amino acids in children with type 1 diabetes requires further investigation.

Cell transplantation is a promising strategy for treating blindness caused by the loss of photoreceptors. Recent work by other members of the lab has demonstrated the restoration of visual function following the transplantation of photoreceptor precursors obtained from early post-natal retinas into adult GNAT1-deficient mice. This thesis has sought to investigate the use of Embryonic Stem (ES) cells as a source of photoreceptors. The studies presented here aim to (1) characterize ES derived retinal progenitors development, (2) determine the stage of development of ES cell- derived rod photoreceptor precursors by comparing it to normal development and to (3) demonstrate the capability of integration and maturation of ES cell derived photoreceptor precursors following transplantation to the adult degenerate retina. To provide a comprehensive understanding of the ES differentiation system in 2D and 3D cultures, we analysed the differentiation of ES cells into retinal progenitor cells (RPCs) and retinal cell types. Photoreceptor genesis was characterized at the transcriptional and protein levels not only to establish if these cultures followed normal development but also to find the appropriated time point for transplantation. Recombinant AAV2/9 viral vector was used to label ES cell-derived retinal cell types not only to enrich for a pure population of retinal cells but also to allow their identification after subretinal transplantation in to models of retinal degeneration. These studies demonstrated that retinal development in vitro resembles normal development, following a conserved order of birth in both differentiation cultures. ES cell-derived photoreceptors are comparable to early post-natal stages of development. Following transplantation, 2D generated retinal cell types did not integrate into the host adult retina, whereas ES cell-derived photoreceptors generated from 3D cultures were able to integrate into the outer nuclear layer and demonstrated mature photoreceptor morphology. Therefore, ES cells can be used as a source of integration competent photoreceptor precursors.

This thesis addresses how demographic processes affect both genetic and cultural variation. Drawing on theory and techniques from both gene-culture coevolution and population genetics, and using both genetic and archaeological data, I present a number of projects covering a wide range of questions on the evolutionary history of our species. Chapter 2 develops a simulation model of the gene-culture coevolution of lactase persistence and dairying in Neolithic Europe. Using approximate Bayesian computation (ABC) to integrate modern genetic and archaeological data, the results demonstrate that this coevolutionary process began —7,500 years ago in central Europe. The inferred origin is closely associated with the emergence of the Linearbandkeramik (LBK), an early cattle-based dairying culture. Chapter 3 extends a previous cultural evolutionary model to show that heterogeneity in subpopulation density causes spatial structuring of culturally inherited skill accumulation. Genetic estimates of regional effective population density demonstrate that the appearance of modern human behaviour during the Late Pleistocene can be explained by demographic factors. Chapter 4 reviews the application of the neutral model in archaeological and other cultural contexts, and develops a novel statistical approach to test for deviation from neutrality. Results show that surprisingly high levels of non-neutrality, in the form of frequency- dependent copying, can go undetected. Chapter 5 develops a novel neutral model of cultural evolution, relaxing some previous common assumptions outlined in Chapter 4. The model allows accurate estimation of population parameters from minimal archaeological data, and is applied to a decorated pottery dataset from the LBK settlements of southwest Germany. Chapter 6 outlines the utility of the coalescent model of population genetics in inferring potentially complex demographic histories using both modern and ancient DNA samples. I provide two examples of my work from collaborative projects on the domestication of Near Eastern cattle and the demographic history of the Hispaniolan hutia.

Congenital melanocytic naevi (CMN) are pigmented birthmarks, known to be associated with an increased risk of neurological abnormalities and malignant melanoma. Current treatments for the cutaneous lesions and complications are limited for severely affected individuals, driving the research within this thesis into the aetiopathogenesis of this condition. The potential offered by studying rare diseases is also relevant to this project, particularly on the background of the rising incidence of melanoma in young people in the UK. The aims of this research were to expand the clinical description of the condition, to look for predisposing genetic alterations in the germline, to identify possible biomarkers for risk of complications and to investigate the lineage of naevus cells. To these ends we studied a large population of affected children to expand the phenotypic associations, investigating facial features, endocrinological status, pigmentary phenotype and growth, we undertook histopathological phenotyping of cutaneous and neurological lesions using immunohistochemistry and electron microscopy and employed array comparative genomic hybridisation and next generation sequencing for screening of germline haplotypes and mutations. Typical facial features were described in the majority of children with CMN, and the term CMN syndrome was proposed to encompass these. Post-natal growth was found to be rapid, leading to significantly increased BMI compared to the current UK population. Skewed anterior pituitary hormone measurements and increased parental thyroid abnormalities were suggestive of central hormonal dysfunction, however hormonal production from the cutaneous lesions could not be excluded. Histological phenotyping inferred that the primary abnormality underlying CMN may affect a population of stem cells, not necessarily destined to be melanocytes. An association between MC1R genotype and presence and severity of CMN was found using a candidate gene approach, and other germline candidates were identified. The results in this thesis have improved our understanding of the clinical and histological phenotype of CMN, and have identified genetic factors involved in the pathogenesis of the condition.

Gaucher disease is a rare inherited lysosomal disorder caused by deficiency of the enzyme glucocerebrosidase. Classically, three forms of the disease are recognised: type I or nonneuronopathic, type 2 or acute neuronopathic, and type 3 or subacute or chronic neuronopathic. Neuronopathic Gaucher disease (NGD) is defined as a confirmed diagnosis of Gaucher disease in the presence of neurological symptoms and signs, for which there is no other cause. Horizontal gaze palsy is the clinical hallmark of NGD. Other neurological manifestations include seizures, cerebellar ataxia and pyramidal tract involvements. However, NGD is very heterogeneous and the neurological features vary greatly from patient to patient, not only in terms of manifestations involved but also in terms of severity. The emergence of enzyme replacement therapy has changed the ‘natural history’ of the disease, and patients are now living longer where previously they would have succumbed to the visceral complications of the disease. New emerging therapies are being developed for NGD, however a suitable surrogate marker to monitor neurological disease is lacking. In this study, three different assessment tools were explored to examine their value and sensitivity to assess neurological involvement in NGD. A Severity Scoring Tool developed specifically for NGD was modified and validated to offer a robust assessment tool, with demonstrated sensitivity to track disease progression and distinguish between phenotypes. Additional assessments utilised were gait analysis and diffusion tensor imaging, both of which were sensitive enough to distinguish between the NGD and Type I cohort studied. This is the largest cohort of NGD patients (recruited across three European countries) to be studied prospectively and systematically. It is also the first study to describe the gait pattern of NGD children, and to provide an in-vivo insight of the Gaucher brain utilising diffusion tensor imaging.

In recent years, population genetic data has been used increasingly to make inferences on population history – particularly concerning the human past. However, any observed genetic data is only one outcome amongst a very large number of possibilities that can arise under the same population history; and reversely, multiple different histories can give rise to the same data. For this reason direct interpretation of patterns in genetic data to recover evolutionary history is highly problematic, and inferring evolutionary histories – including demographic and natural selection-related parameters – in a secure statistical framework, requires the exploration of a range of explicit models. Such models are better informed when conditioned on multiple data sources rather than purely genetic data, such as archaeological, environmental, and behavioural or cultural data. This PhD aims at integrating such data into a single framework in order to examine how well various population history hypotheses can explain observed patterns in various data. The approach I have used is simulation modeling coupled with approximate Bayesian computation (ABC) techniques to investigate three population histories. I have used a forward simulation model coupled with ABC to investigate demographic and evolutionary parameters of (i) the evolution of the ectodysplasin-A receptor (EDAR) derived allele in Southeast Asia, and (ii) the gene-culture co-evolution of lactase persistence (LP) and dairying in Europe. In both cases, the model simulates the allele frequency and the underlying population demography, conditioned on archaeological data for when and where farming starts. Natural selection is inferred to have driven both alleles to high frequencies in their respective regions. However, the reasons why these alleles would have been favored are still unclear. I therefore further apply the simulation / ABC framework to explore various selective hypotheses conditioned on key environmental factors. (iii) I have used a coalescent approach to assess how domestication has affected goat mitochondrial DNA (mtDNA) diversity. I have used the coalescent to simulate genealogies under demographic models informed by archaeological data. I then applied an ABC approach to determine which of those models best explains the observed patterns of mtDNA diversity in goats and to estimate demographic parameters from those models.

Males and females differ in their reproductive roles, and as a consequence each sex is subject to divergent selection pressures to optimise its own reproductive success. Due to the shared genome between males and females these selection pressures frequently act on shared phenotypic traits. Divergent selection can favour the invasion of sexually antagonistic alleles which increase the fitness of one sex at the detriment of the other. Sexual antagonism can be subsequently resolved through the evolution of sex-specific gene expression, allowing the sexes to diverge phenotypically. While sexual dimorphism is common, recent evidence shows that antagonistic genetic variation continues to segregate in populations of many organisms. The basis of sexual antagonism remains poorly understood. I first present empirical data on the interaction between sexual antagonism and genetic drift in small populations that had independently evolved under standardised conditions. I demonstrated that these experimental populations of Drosophila melanogaster had diverged in male and female fitness, with some populations showing increased male but decreased female fitness, while other populations showed the reverse pattern. I also exploited a sample of nine genomes that belonged to three fitness classes (low male/high female, high male/low female, intermediate in both sexes) to test the association between the sexually dimorphic trait wing morphology (size and shape) and fitness in both sexes. I found that wing morphology significantly affected the fitness of both males and females, but to a differing degree in each sex. In males wing shape rather than wing size was especially important. I found evidence that there was appropriate genetic architecture for the existence of sexual antagonism, and for divergent selection on aspects of wing morphology. I place all of my findings in the context of variation in sexually dimorphic traits and sex-specific fitness.