Striatal medium spiny neurons (MSNs) are the main output from the striatum, a subcortical part of the forebrain, which is the main input of the basal ganglia (BG) system. 96% of the striatum is composed of MSNs. Huntington’s disease (HD) is caused by a progressive loss of MSNs in the striatum. It is caused by polyglutamine expansion in the Huntingtin protein (HTT). This impairs cerebral cortex function and deregulates several genes that play a role in subpallium development. The identification and use of transcription factors (TFs) to direct the differentiation of stem cells to MSNs is described. Microarray data analysis of MSNs, from data in NCBIs Gene Expression Omnibus (GEO), was performed to detect gene expression profiles involved in telencephalon development and striatum maturation. The genes Dlx2, Gsx2, Mash1, Pax6, Sox4 and Foxp1 were found to play roles in neurogenesis, forebrain neuron fate commitment, cell proliferation, anatomical structure morphology, maturation of MSNs and transcriptional activation and repression. A differentiation protocol was developed in which three TFs, DLX2, GSX2 and MASH1, were selected and cloned into expression vectors, in different combinations,to direct the differentiation of stem cells into naïve rosette neural progenitor cells (nrNPCs). These were then terminally differentiated into striatal MSNs. Expression of DLX2, GSX2 and MASH1 in human embryonic stem cell (hESC) and induced pluripotent stem cell (iPSC) lines successfully directed their differentiationinto nrNPCs. iPSC-derived nrNPCs were successfully terminally differentiated into DARPP-32+ve MSNs. However, only overexpression of DLX2 and MASH1 in iPSCderived nrNPCs yielded functionally active MSNs that expressed DARPP-32, CTIP2, FOXP1, EBF1, DRD1, DRD2, GAD2 and CALBIN-1. It was successful and, therefore, could provide a new cell source for disease modeling in vitro, transplantation studies and drug discovery approaches.

During lytic infection with a number of pathogenic viruses, host cells have been found to exhibit loss of mRNA culminating in loss of cellular proteins, a phenomenon termed host shutoff. In γ-herpesviruses, the precise mechanism by which host shutoff takes place is yet to be fully determined. Initially, host shutoff in Kaposi’s sarcoma-associated herpesvirus (KSHV) was associated with the viral DNA exonuclease SOX (shutoff and exonuclease), which was followed by the identification of host shutoff in Epstein Barr virus (EBV) linked to the SOX homologue BGLF5. Murine γ-herpesvirus-68 (MHV-68) is a γ-herpesvirus that infects murid rodents including laboratory mice, with extensive viral genome homology to KSHV and EBV. MHV-68 was also found to induce host shutoff via the SOX homologue ORF37. The similarities between MHV-68 and the human γ-herpesviruses make the mouse virus an ideal model for studying host shutoff and consequences of host shutoff in disease pathogenesis. In this study, MHV-68 was used to investigate the role of ORF37 in MHV-68 infection in vitro and in vivo and to examine possible mechanisms of mRNA degradation mediated by ORF37. A stop codon mutant of MHV-68 (ORF37Stop) and its corresponding revertant (ORF37StopRev) were engineered. ORF37Stop virus exhibited highly restrictive phenotype in immunocompetent cells. In contrast, lack of ORF37 was tolerated in cells lacking a functional intereferon- α/β receptor (IFNαβRKO). These in vitro observations were replicated, to some extent, in vivo with the mutant virus establishing lytic infection in the lungs of IFNαβRKO mice. Interestingly, host shutoff was found to proceed even in the absence of ORF37, but only where functional type I IFN-mediated responses were lacking. A poly(A) tail assay was used to investigate the possibility of mRNA degradation at the 3’ mRNA. In contrast to a previously published study, the results suggest that cellular poly(A) tails do not appear to be elongated as a result of MHV-68 lytic infection. Data generated during this project have identified a novel function by which ORF37-mediated host shutoff causes mRNA degradation and that a primary role of this viral protein is to overcome the type I interferon-receptor-mediated responses. The observation that host shutoff can proceed in the absence of ORF37 in cells unable to respond to type I IFNs highlight two important possibilities: other viral proteins may mediate host shutoff; and type I IFN effector mechanisms are a key player in host shutoff. These novel observations together with lack of a mechanism by which ORF37 mediates mRNA degradation warrant further studies in this field.

Epilepsy effects up to 1% of the population, and up to 30% of people with epilepsy are pharmacoresistant—they continue to experience seizures despite treatment with maximal doses of multiple antiepileptic drugs. The causes of drug resistance in epilepsy remain poorly understood. In this work, I have used genetic and genomic analysis techniques to explore the causes of epilepsy pharmacoresistance. It has been reported that epilepsy pharmacoresistance results from impaired drug penetration into the epileptic focus secondary to a localized dysregulation of drug transporters. Solute carrier (SLC) transporters form the largest superfamily of multidrug transporters. I used novel in silico and stringent ex vivo strategies for identifying the SLCs that are dysregulated in the pharmacoresistant epileptic human hippocampus. I discovered that the SLCs dysregulated in the pharmacoresistant epileptic human hippocampus are either small metal ion exchangers or transporters of neurotransmitters, not antiepileptic drug transporters, and most likely contribute to pharmacoresistance by enhancing the intrinsic severity of epilepsy. This finding supports the newly-proposed and intuitive ‘intrinsic severity hypothesis’ of epilepsy pharmacoresistance. According to the intrinsic severity hypothesis, pharmacoresistance in epilepsy results from the increased dysfunction of the biological pathways which underlie epilepsy. Hence, I proceeded to perform genome-wide genetic and genomic analyses in order to find the most important pathways underlying epilepsy and pharmacoresistance in epilepsy. I performed an integrative analysis of previously published large-scale gene expression profiling studies on brain tissue from epilepsy surgery; the largest and most robust microarray analysis of brain tissue from surgery for pharmacoresistant mesial temporal lobe epilepsy; and the first-ever genome-wide association study (GWAS) of pharmacoresistant focal epilepsy. By integrating the results of the genetic and genomic studies, I was able to show that pharmacoresistance is the result of accumulation of deleterious genetic variants of increasing severity and/or numbers within the genes that constitute the core pathways underlying epilepsy. I also found that the pathways disrupted in pharmacoresistant epilepsy, at both the genetic and genomic levels, belong to many different diverse and disparate functional domains, for example ‘axon guidance’, ‘transmembrane transport of small molecules’ and ‘cell death signalling via NRAGE, NRIF and NADE’. However, using network analysis techniques, I showed that these seemingly unrelated pathways form a coherent highly interconnected network, and it can be expected that changes in one pathway in this network will have a cascading effect on the rest of the network. The most important pathways in these networks are the central ‘hub’ pathways, which I identified using betweenness centrality network analysis. I then performed the first-ever genetical genomics study in epilepsy using hippocampal samples from resective surgery for refractory mesial temporal lobe epilepsy. By integrating genome-wide genetic, genetical genomic and genomic studies, and then performing pathway, network and centrality analysis, I identified the most important putative central causal pathways underlying epilepsy pharmacoresistance: 'transmembrane transport of small molecules' and 'Deleted in colorectal cancer (DCC) mediated attractive signalling'. In conclusion, by performing genome-wide genetic, genetical genomic and genomic studies, followed by integrative analysis, pathway construction and network mapping, I have identified most important putative central causal pathways underlying epilepsy pharmacoresistance.

Ageing is a major cause of diseases in modern society and leads to many age-related diseases. In addition, rising obesity in society increases the likelihood of various diseases. The development of genetic sequencing technologies provides a novel platform to unravel the complexity of ageing and metabolism. The Naked mole-rat (Heterocephalus glaber) is a rodent that is related to mice (Mus musculus) and ages very slowly, and can live for over 30 years without developing cancer. RNAseq analysis of H. glaber and M. musculus livers identified genes that were under- or over-expressed in H. glaber. Relative qPCR analysis was performed to confirm the expression of highly expressed genes in the naked mole-rat. Adenosine-to-Inosine (A-to-I) RNA editing is a post-transcriptional modification of specific bases that can cause an alteration in amino acid and splicing events. Recent studies indicated a gene-specific decline in RNA editing with age in humans in Cyfip2. Using SOLiD RNAseq, we sequenced the transcriptomes of 6-, 12-, and 28-month old rat cerebral cortex. We identify a conserved RNA editing site in Cog3. Upon analysis of known conserved RNA editing targets, no changes in RNA editing were observed during ageing in the rat. These results highlight the biological differences between rodent model organisms and humans, and their significance in the context of RNA editing and ageing. Dietary restriction of caloric intake is known to increase the lifespan of many species. Physiologically, these animals remain lean and show a later onset of age-related diseases. Through this concept, we were interested in studying Gnasxlm+/p- knockout mice, which remain lean due to hypermetabolism, despite increased food intake. Gnasxlm+/p- mice have a deletion in Gnas, which prevents expression of Xlαs. However, it is not known exactly how the loss of Xlαs exerts this phenotype. Due to its role in homeostasis and localisation of Xlαs expression, RNA from whole hypothalami of wildtype and Gnasxlm+/p- mice were analysed by Illumina RNAseq in order to identify expression changes that may help to explain the physiological symptoms. The glial cell marker Gfap was found to be downregulated 2-fold in Gnasxlm+/p- mice, which was confirmed by qPCR. Furthermore, in analysing the localisation of GFAP within the hypothalamus, we identified a distinct loss of GFAP expression in the arcuate nucleus, and suprachiasmatic nucleus. GFAP was not found to be decreased in postnatal (P)1 and P5 mice, indicating that the decrease in GFAP is more likely to be an adaptation to chronic undernutrition as a result of hypermetabolism, than a developmental problem in the mice.

Elite athletic performance is widely acknowledged to result from the exposure of a favourable genetic endowment to a favourable combination of environmental factors including culture, diet, training regime and socioeconomic status. Athletes from West African descendant populations in North America and Western Europe have long been prominent in elite sprint running, constituting 63% of the top 100 performers in each sprint discipline, outperforming athletes from Europe (23%), West Africa (8%) and the rest of the world (6%). These members of the West African diaspora are genetically admixed, resulting in detectable levels of both African and European ancestry because of gene flow between African slaves and Europeans during chattel slavery in the 16th to 19th centuries. The overall aim of this thesis was to investigate the effect that ancestral genetic composition may have had on the likelihood of becoming a top-class sprint athlete amongst samples of African-Americans and Jamaicans. It was hoped that these findings would add to the existing research in attempting to understand the unique combination of factors that produce elite sprint athletes. Ancestrally informative genetic data from elite African-American and Jamaican sprint athletes and ethnically-matched controls were used to explore how genetic ancestry affects sprint athlete status in these populations. These data are also vital when investigating the putative origins of an admixed population, and relatively little research has investigated the genetic ancestry of modern Jamaicans when compared to African-Americans. To bring the two groups to comparable levels of insight, the population history of the Jamaican people was estimated by comparing the observed matrilineal gene pool to the gene pools of known source regions of Africa. By simulating a stable population with the observed population dynamics from slave-era Jamaica, it was possible to draw conclusions about selection acting on the Jamaican slave population from the colonisation of the island by England in 1655 until the abolition of the slave trade in 1807. In addition to the Jamaican maternal lineages already genotyped, paternal lineages in both African-Americans and Jamaicans, as well as maternal lineages in African-Americans were genotyped to assess any association these lineages had with elite sprint athlete status. These lineages were also compared between the cohorts to assess any differences in lineage composition across both groups of athletes and controls. Finally, locus-specific genetic ancestry was calculated to map loci associated with elite athlete status to regions of the genome with a greater amount of African or European ancestry than would be expected under the null hypothesis of no association with ancestry. Assuming a difference in the likelihood of sprint athletes originating from either Africa or Europe, detected associations between locus-specific ancestry and sprint status may indicate specific genomic regions of interest. The main findings of this thesis are: a) Modern Jamaicans are mostly descended from slaves originating from the Gold Coast of Africa, despite large influxes of slaves from the Bight of Biafra and West-central Africa before the end of the slave trade. b) There appears to have been selective pressure acting on the slave population of Jamaica. Differences between the presumptive origins of the observed lineages and the outcome of the stable population model suggested varying levels of mortality and fecundity within the slave population, consistent with earlier ethnographic and linguistic studies. c) The distribution of maternal lineages in the African-American athletes were significantly different from that of African-American controls. Maternal lineage distributions between Jamaican athletes and Jamaican controls were not significantly different. There was insufficient statistical power to infer any differences between the paternal lineages of African-American athletes and controls or the Jamaican athletes and controls. This suggests that either maternal ancestry may be a factor in elite sprint athlete status for African-Americans or it could simply be a false positive, inherent to the methodology used. Jamaican maternal lineages are homogeneous with regards to elite sprint athlete status. There was insufficient statistical power to arrive at similar conclusions regarding the paternal lineages of athletes and controls in either group. d) The maternal lineages of African-American athletes and Jamaican athletes were significantly different, although there was insufficient statistical power to determine if there were any differences between the paternal lineages of African-American athletes and Jamaican athletes. This suggests that the same maternal lineage distribution is not associated with sprint athlete status in the two populations, while there is insufficient evidence to make a similar claim regarding paternal lineages. e) The maternal lineages of African-American controls and Jamaican controls were also significantly different, although there was insufficient statistical power to conclude whether significant difference exists in the paternal lineages of African-American controls and Jamaican controls. These results suggest that there is some evidence that the population histories of African-Americans and Jamaicans are significantly different despite the lack of evidence from the paternal lineages. f) The proportion of genome-wide African ancestry did not differ significantly between either African-American athletes and controls or Jamaican athletes and controls. This suggests that environmental factors typically associated with higher levels of African ancestry in these populations (e.g. lower socioeconomic status, diminished access to healthcare) are not directly linked with elite athlete status. g) The estimated number of generations since admixture occurred did not differ significantly between athletes and controls for either African-Americans or Jamaicans. This suggests that athletes were not more likely than controls to have had European ancestors in the recent past, thereby potentially having greater access to resources. h) Admixture mapping was used to detect an enrichment of European ancestry at chromosome 4q13.1 significantly associated with athlete status in African-Americans. There were no significant loci associated with athlete status in Jamaicans. This suggests that the regions of the genome influencing sprint athlete status may be different in the two populations, although there was insufficient statistical power to draw any meaningful conclusions from the Jamaican data. This thesis has potential implications for future work not only explaining the disproportionate success of West African descendant sprint athletes but also for advancing the basic understanding of the genetic influences on the limits of human performance.

This thesis aims to develop and implement population genetic models that are directly interpretable in terms of events such as population fission and admixture. Two competing methods of approximating the Wright--Fisher model of genetic drift are critically examined, one due to Balding and Nichols and another to Nicholson and colleagues. The model of population structure consisting of all present-day subpopulations arising from a common ancestral population at a single fission event (first described by Nicholson et al.) is reimplemented and applied to single-nucleotide polymorphism data from the HapMap project. This Bayesian hierarchical model is then elaborated to allow general phylogenetic representations of the genetic heritage of present-day subpopulations and the performance of this model is assessed on simulated and HapMap data. The drift model of Balding and Nichols is found to be problematic for use in this context as the need for allele fixation to be modelled becomes apparent. The model is then further developed to allow the inclusion of admixture events. This new model is, again, demonstrated using HapMap data and its performance compared to that of the TreeMix model of Pickrell and Pritchard, which is also critically evaluated.

RNA molecules play a major role in cellular processes such as replication, transcription, and translation. As a result, RNA-based engineering methods have emerged as important tools in biotechnology. However, the structure and function of RNA depends on global interactions, which often prevents the use of a modular design strategy, particularly with allosteric conformations. Using RNA secondary structure prediction tools, computational methods can successfully design RNA switches that work in E. coli. The overarching aim of my research is to develop synthetic RNA switches that could be used for regulation and sensing of molecules in living cells. We have engineered RNA based synthetic signal transduction cascade consisting of a single RNA molecule (regazyme, an RNA chimera of an aptazyme with a riboregulator) that upon sensing a ligand (theophylline) self cleaves and releases a riboregulating small RNA. This small RNA binds to a cis-repressed mRNA allowing translation of a reporter protein. This system can be adapted to be induced by other ligands and can be used as a biosensor. I have also integrated a riboregulated RNA switch into the E. coli genome to study its behaviour at single-cell level. This reduces the transcriptional and translational noise in data collection to inform more accurate computational design of RNA regulatory units. We used computational design to engineer higher-order RNA-triggered riboregulators organized as a hierarchical toehold activation cascade. This has been studied in a single cell as well as in a population of E. coli cells. These RNA riboregulators can be used for construction of new, complex and portable synthetic gene circuits. In addition, I have engineered sense and antisense riboregulators consisting of the small RNA reverse complement of a known riboregulator. This riboregulator can transcribe two small RNAs from the same DNA template depending on the direction of transcription. These two small RNAs independently trans-activate translation of their cognate target genes and both RNAs also silence each other by antisense interaction. We have also engineered an RNA-based tunable antiterminator, a TNA-derived adaptor that acts as a signal converter in a genetic circuit, converting a translation signal to a transcription signal (unpublished). I have engineered a minimum alphabet riboregulator that has only three nucleotides (GCU) that currently validating (unpublished). In order to explore the use of directed evolution for the engineering of RNA switches, I am developing an evolution-based system for generation and selection of new biomolecules. These evolved new biomolecules could be used in future medical applications such as molecular sensing. I have been using T7 and P2 bacteriophages as the basis for this evolutionary system. I have engineered the genome of the T7 phage with (regazyme, Riboswitch and riboregulators) using homologous recombination with marker-based selection. These engineered phages can be used to evolve new biomolecules such as other regazymes, riboswitches and riboregulators.

Functional genomics technologies, in which thousands of mRNAs, proteins, or metabolites can be measured in single experiments, have contributed to reshape biological investigations. One of the most important issues in the analysis of the generated large datasets is the selection of relatively small sub-sets of variables that are predictive of the physiological state of a cell or tissue. In this thesis, a truly multivariate variable selection framework using diverse functional genomics data has been developed, characterized, and tested. This framework has also been used to prove that it is possible to predict the physiological state of the tumour from the molecular state of adjacent normal cells. This allows us to identify novel genes involved in cell to cell communication. Then, using a network inference technique networks representing cell-cell communication in prostate cancer have been inferred. The analysis of these networks has revealed interesting properties that suggests a crucial role of directional signals in controlling the interplay between normal and tumour cell to cell communication. Experimental verification performed in our laboratory has provided evidence that one of the identified genes could be a novel tumour suppressor gene. In conclusion, the findings and methods reported in this thesis have contributed to further understanding of cell to cell interaction and multivariate variable selection not only by applying and extending previous work, but also by proposing novel approaches that can be applied to any functional genomics data.

LncRNAs are increasingly being recognised as functionally important for regulation of biological processes. We have identified an lncRNA which we believe is integral in lineage commitment during haematopoiesis. Here, we report that CCDC26 lncRNA is a regulator of β-globin and c-MYC gene expression. Indeed, CCDC26 silencing in erythroleukemic K562 cells led to several gene expression changes. Upon further investigation of genes linked to erythropoiesis, we observed the upregulation of β-globin expression. Our results suggest that CCDC26 regulates expression of β-globin and other genes by modulating epigenetic changes which could be important in linage-commitment. We have results to suggest the expression of β-globin and c-MYC genes may be synergistic and promotes differentiation of K562. Finally, RNAseq data further supports upregulation of differentiation specific genes further underpinning our hypothesis that this lncRNA may play an important role in lineage commitment.

Clathrin is a well-studied molecule yet its assembly properties have been characterised only in fairly simple biochemical terms to date. When clathrin is isolated for study in vitro, certain important aspects of its in vivo functionality are conserved: • purified clathrin units can be induced to assemble, reversibly, into cage-like forms that exhibit an array of polygonal binding motifs, similar to those observed in a vesicle's coat complex; • the artificial clathrin cages so produced are found to be heterogeneous, resulting in particles of different size according to the number of units each incorporates. Laboratory methods that investigate mechanisms of cage disassembly have become integral to the supply of knowledge about clathrin's structural biology. Yet there has been little progress towards a quantitative approach for exploring the assembly mechanisms that might explain clathrin cage dispersity; indeed, for some biophysical techniques the latter is an unfortunate property that might even preclude a meaningful analysis. This thesis shows that when purified clathrin is made to assemble in vitro by an overnight dialysis method (x2.8.3), the distribution of coalescent particle sizes that results can be predicted according to the pH and composition of the buffer solution and the final concentration of the clathrin sample: • Within the confines of a certain parameter space, variation in the distribution of particle sizes is found to be consistent with the predictions of a Becker-D�oring model for reversible coalescence: the empirical evidence suggests that a dynamic population of supramolecular cage structures provides a mechanism for energy minimisation towards achieving thermodynamic equilibrium. • Addition to, or substitution for, the basic compounds that constitute the assembly buffer solution exerts detectable effects upon the clathrin particle size distribution; the nature and magnitude of these effects may be expressed in terms of the state function Gibbs energy. To demonstrate these, a methodology was developed to focus on the preparation of samples suitable for analysis by the dynamic light scattering (DLS) technique, thereby aiming to provide an extensible and far richer analysis than was a afforded previously by the somewhat erroneous, certainly tedious, task of manually counting and classifying particles by size to construct a histogram from electron microscopy images. DLS determines the frequency profile of fluctuations in the electromagnetic field issued by a bulk sample to measure the diffusion processes of its particles. To then derive a number distribution relies upon exploiting prior knowledge of the physical system or else assumptions for the same. So, the research presented here has involved both a theoretical and an experimental component: over the course of the project there has been a process of iteration between developing experimental methods at the bench and interpreting the data recovered, until the physical picture and the theoretical model that have emerged appear to be consistent. And the results described in this thesis do indeed encourage further work. A number of very promising themes have not been advanced further here due only to the limitations of time: • 3. By developing a quantitative approach that relates the physical properties of the clathrin particle in solution to thermodynamic measures, the empirical results that have been collected over the course of this project are expressed in terms that translate more readily to the language of simulation and dynamical systems modelling. • 4. Analysis of DLS makes a valuable contribution to the project that aims to resolve accessory proteins bound to clathrin cages using the SANS contrast variation technique, with auxilin as a model. • Perhaps the most important conclusion to be drawn, however, is that: 5. The additional insight made available by analysis of DLS allows greater control to be exercised over the directed assembly of clathrin in vitro, which in turn provides fresh opportunities for experimental design. Chapter 1: The scientific questions addressed by this thesis; key concepts; review of the academic literature; project aims and objectives. Chapter 2: Details of the practical methods and materials used to conduct experiments in the laboratory with clathrin; software resources. Chapter 3: Description of the theoretical framework that has been developed over the course of the project; mathematical methods. Chapter 4: The empirical results for clathrin that have been recovered over the course of the project; statistical methods and models. Chapter 5: How the main findings of Part II have been used to solve a problem for clathrin studies when using a high resolution application (SANS). Chapter 6: Discussion of the main findings with respect to the project aims and objectives; directions for further work and unresolved questions. Figure 1: The narrative of this thesis. Part IV consists of appendices: statistical diagnostics, examples of programming and supplementary images.