This work highlights phenotypic heterogeneity and how this may index underlying aetiological heterogeneity. It also indicates the importance of environmental variables and investigating Gene x Environment interactions in molecular genetic studies of ADHD.

Replication of the genome is a central process in cellular life, which must be tightly regulated at the risk of genomic instability. DNA replication has been extensively studied in bacteria and eukaryotes, and recently, in archaea. Universally, DNA replication is started at specific genomic sites termed origins of replication, which are recognised by an initiator factor. While in both bacteria and archaea the initiator factor is a single protein, it is assumed that this role is performed in eukaryotes by a highly conserved six-subunit origin recognition complex (ORC). Recent phylogenetic studies, however, suggest that the presence of a six-subunit ORC might not be as conserved as initially believed. Trypanosoma brucei is a protozoan parasite in which little is known about nuclear DNA replication. To date, initiation of T. brucei DNA replication has been associated with a single factor, TbORC1/CDC6, though highly diverged interacting partners have been identified. To elucidate whether T. brucei possesses a diverged ORC-like complex, TbORC1/CDC6 and its known interacting partners, TbORC1B, TbORC4, Tb7980, Tb3120, and a novel factor, Tb1120, were analysed. First, the protein sequences of these factors were re-analysed, revealing varying degrees of conservation and divergence with other eukaryotes’ ORC proteins. Second, expression silencing by inducible-RNA interference (RNAi) of TbORC1/CDC6, TbORC1B, TbORC4, and Tb3120, in procyclic form (PCF) and bloodstream form (BSF) cells, confirmed their involvement in DNA replication. Third, subcellular localisation and dynamics of TbORC1/CDC6 and its interacting partners during the cell cycle of PCF and BSF cells was investigated by immunofluorescence, revealing TbORC1B to be the sole factor to display an apparent cell cycle-dependent localisation pattern, perhaps suggesting that it might be a DNA replication regulatory factor. Finally, immunoprecipitation and gel filtration assays support the existence of an ORC-like complex, apparently large enough to be composed of TbORC1/CDC6 and known interacting partners, and potentially, additional factors. TbORC1/CDC6-binding sites and origins of replication in T. brucei have been mapped in PCF cells. Like most eukaryotes, no specific sequence elements were found to define TbORC1/CDC6-binding sites or origins of replication, and the TbORC1/CDC6-binding sites outnumbered the mapped origins, which appeared to be activated at different times during S phase. It has been reported in other eukaryotes that different cell types activate different origins or the activation timing differs. Here, origins of replication were mapped in T. brucei PCF and BSF cells, revealing a pronounced inflexibility in origin usage in these two life cycle stages. Only one, notable genome-wide difference was found: in BSF cells, the single active variant surface glycoprotein (VSG) expression site was found to be early replicating, whereas all other silent VSG expression sites were late replicating; in PCF cells however, where all VSG expression sites are silenced, these were all late replicating. These data reveal a locus-specific link between DNA replication and transcription in T. brucei, which may relate to immune evasion. The genomes of T. brucei and related kinetoplastids are highly syntenic. Since most eukaryotic origins of replication are not defined by consensus DNA sequences, but appear instead to be defined by, among other features, chromatin context and status, origins were here mapped in both L. major and L. mexicana promastigotes in order to ask if common features could be found relative to T. brucei. Surprisingly, only a single origin could be found per Leishmania chromosome, in contrast with all eukaryotes examined to date, where each chromosome is replicated from multiple detectable origins. Origin-active loci in Leishmania were found to be distinguishable from related non-origin loci in terms of size, a characteristic not observed in T. brucei, although around 40% of the mapped origins are conserved in location relative to T. brucei. These data reveal pronounced differences in replication dynamics between the two genera, despite the considerable overlap in genome organisation.

Visual perception is highly variable across healthy individuals and increasing evidence suggests that this inter-individual variation could be due to differences at the genetic, neurochemical, structural and neurophysiological level. Specifically, variation in the GAD1 gene (responsible for synthesising the majority of cortical GABA) has been associated with differences in the level of the inhibitory neurotransmitter GABA. In addition, differences in GABA and cortical structural parameters (surface area and thickness) have been shown to predict differences in neural gamma oscillations. However, these findings have not been replicated in large independent studies. Hence, Chapter 3 and 4 of this thesis combines the non-invasive neuroimaging tools MRI, MRS and MEG with genetic data to investigate the relationship between variations in genes, GABA, structure and gamma oscillations in the visual cortex of a large cohort of healthy individuals. Group differences in GABA, structure and gamma oscillations have also been reported between psychiatric populations (schizophrenia and bipolar disorder) and healthy individuals. However, differences in the direction of effect (increase or decrease) and no group differences have been found. Thus, Chapter 5 aims to further study these inconsistent findings by exploring group differences in GABA, structure and gamma oscillations between a healthy group and a schizoaffective bipolar disorder group. Lastly, inter- individual variation is also present in auditory perception but has received much less attention into the factors driving this variation. As in the visual system, similar links between neurochemical, structural and neurophysiological measures could be present in the auditory domain. Chapter 6 investigates the association between auditory gamma oscillations and auditory structural parameters in a healthy cohort.

The traditional paradigm relying on drug discovery to treat and heal the body is changing. Medicine for the 21st century is moving towards using the body’s internal language of DNA and RNA to cure disease and repair injuries to the body. We now appreciate the complexity of signalling through the genome and its transcribed RNA. The role of micro RNAs and short interfering RNAs are gaining much interest as potential therapeutics. This interest has been sparked by the discovery that the dysregulation of micro RNAs is the origin for a spectrum of diseases from cancer through to osteoporosis. Small regulatory RNAs have been shown to influence stem cell maintenance, proliferation and differentiation, offering the potential to produce new tissue by manipulating RNA levels. However delivery of these molecules is fraught with difficulties. Without protection these molecules are quickly degraded in vivo and in vitro before reaching their intended target. With this in mind, this thesis aims to investigate the potential role for gold nanoparticles to deliver small regulatory RNAs and in turn produce a non-toxic and physiologically significant effect upon the cells. Initial investigations revealed the importance of PEG density and AuNP concentration; with lower PEG densities, allowing attached therapeutic siRNA against C-Myc to reduce C-Myc protein levels and cell proliferation. Subsequently we determined that modulating the expression of osteo-suppressive miRNA, with a nucleic antagonist sequence was able to influence osteogenesis in two cell models (MG63s and hMSCs). This thesis has shown that AuNPs can be used to effectively deliver therapeutically active small molecules to cells in vitro.

Epigenetic modifications are being increasingly recognised to contribute to colorectal cancer formation. Emerging evidence has demonstrated that HP1γ (coded by the gene Cbx3) has an important role in carcinogenesis by regulating several mechanisms, such as heterochromatin formation, gene silencing and DNA replication and repair. The aim of this study was to characterise the role of HP1γ in normal intestinal homeostasis and cancer progression. The Cbx3 gene was conditionally deleted in the murine small intestinal epithelium under normal intestinal homeostasis and in the context of aberrant Wnt signalling using transgenic mouse models. Furthermore, the long-term consequence of Cbx3 loss in Wnt-driven tumorigenesis was examined using a mouse model possessing a heterozygous mutation of the Apc gene. The results presented in this thesis demonstrate that Cbx3 has subtle effects on the histone epigenome, although these do not confer any noticeable manifestation in intestinal homeostasis. Upon aberrant Wnt-activation, Cbx3 loss resulted in prolonged survival potentially via attenuation of Wnt activation at the receptor level and RNA splicing defects. Furthermore, the long-term Cbx3 deficiency accelerates both Wnt-driven tumour development and progression, and reduces survival of male mice with heterozygous loss of Apc. Additionally, marked epigenetic alternations were observed in association patterns of HP1γ and the heterochromatin markers (H3K9me3 and H4K20me3) between wild-type and Apc-deficient intestines, suggesting the multifaceted roles of HP1γ in maintaining a gene-repressive environment in heterochromatin for normal intestinal homeostasis, and providing a gene permissive environment in euchromatin for carcinogenesis. The findings of this study demonstrate for the first time the functional versatility of HP1γ in intestinal homeostasis and tumorigenesis. Cbx3 plays a novel role in positive regulation of Wnt signaling and RNA splicing in the aberrant Wnt-activated intestine and a role in heterochromatin formation and gene silencing in long-term Wnt-activated tumorigenesis. These data contribute to the body of knowledge how colorectal cancer progresses and provide a critical foundation for further investigation of the role of HP1γ in Wnt signaling, RNA splicing, tumour progression, and its interaction with histone marks in mouse models of colorectal cancer.

Genetic variation in the gene ZNF804A has been shown to be related to risk for psychopathology, especially schizophrenia and bipolar disorder. The main aim of this thesis was to characterise the behavioural effects of Zfp804a, the mouse orthologue of ZNF804A, in order to understand more about how this gene influences brain and psychological functioning, and hence provide clues as to its possible role in mediating risk for mental disorders. Prior to this work, two ENU-mutant mouse lines had been generated from a DNA library of ENU mutagenised mice with two non-synonymous mutations selected as viable candidates for further investigation. The C59X mutation encodes a premature stop codon in exon 2, thought to lead to a functional null of the gene, and the C417Y mutation is missense, substituting cysteine for tyrosine in exon 4. A first series of experiments examined the early development of the ENU-mutant lines and showed no gross developmental abnormalities, although the C59X mutants weighed significantly less than their WT littermate controls at weaning and during adulthood. A comprehensive series of behavioural tests then assayed aspects of emotion, motivation, hedonia, sensorimotor gating and response control. In general, the C59X mutants showed the greatest effects, displaying reduced anxiety, anhedonia, and sensorimotor gating deficits, together with evidence of enhanced response inhibition. The C417Y mutants only showed selective effects in terms of enhanced motivation. The data dissociate between the effects of the two ENU-induced mutations of Zfp804a. Furthermore, the findings with the C59X mutants would suggest, a priori, that genetic variance leading to alterations in ZNF804A expression may be an important mechanism contributing to risk for psychopathology.

Parasite life history theory predicts that lifetime reproductive success evolves through differential allocation of energy to life history traits constrained by trade-offs. These life history traits govern the characteristics of parasites such as their virulence, transmission and infection phenotypes, so understanding their evolution is a key concern for infectious disease prediction and management. This thesis uses the powerful tool of experimental evolution to gain a fuller understanding of the factors and constraints involved in parasite life history evolution, using bacteriophage Φ2 as a model. I found that the evolution of life history in this phage is sensitive to spatial structure, UV-C exposure and coparasitism with plasmids, and evolution can be mediated by co-evolution with the host. The high levels of variance I observed here suggest that evolution of parasite life history is more complex than a single trajectory towards a predicted optimum, and likely involves some degree of epistasis or pleiotropy with genes elsewhere on the genome. There was some degree of independent evolution of individual life-history traits, indicating that simple direct trade-offs were not in operation. I demonstrated that co-evolution with the host provided additional mutational input, resulting in a greater degree of evolution in co-evolved populations than those evolved to a static host. Furthermore, I note that co-parasitism with phage and plasmid may provide the necessary conditions for plasmid persistence under fluctuating selection for plasmid-encoded traits, and that the efficacy and suitability of phage as therapeutic agents against plasmid-encoded antibiotic resistance is complicated. No direct link between mutation and phenotype could be elucidated in this study, suggesting that evolution in life history is either governed by genes not examined in this thesis, or involves epistasis and pleiotropy with genes elsewhere on the genome. I concluded that it is important to consider the specific ecology of the focal parasite, its host and any co-occuring symbionts in order to make informed predictions of life history evolution, and general predictions may not be achievable.

Neutrophils are the major cellular constituent of blood leukocytes and play a central role in the inflammatory response, expressing an array of destructive molecules and antimicrobial processes that characterise the cells as front-line defenders of the innate immune system, thus neutrophils are crucial to host defence. It is now appreciated that neutrophils produce and respond to a variety of inflammatory signals and are able to regulate both the innate and adaptive immune response. The molecular changes that underlie this regulation are poorly defined, yet represent an attractive area of research to fully elucidate the role and regulatory capacity of neutrophils within the immune response. RNA-Seq provides an accurate and robust mechanism for global characterisation of cellular transcripts. Neutrophils were isolated from healthy donors and incubated with or without inflammatory cytokines for 1 h. RNA was extracted and analysed by RNA-Seq using the SOLiD or Illumina platforms. Raw data was quantified using a number of software packages which formed a bioinformatic pipeline for data analysis which was developed during the course of the research. Results were validated by a selection of traditional laboratory functional assays. Priming of neutrophils by GM-CSF and TNFα was found to induce differential gene expression and activation of transcription factors, which led to differential regulation of apoptotic pathways. Stimulation of neutrophils with inflammatory cytokines/chemokines (IL-1β, IL-8, G-CSF, IFNγ) resulted in expression of discrete gene sets and differential activation of signalling pathways. Stimulation of neutrophils with IL-6 did not induce any significant expression of genes but result in activation of STAT signalling. Comparison of gene expression of neutrophils isolated by density gradient and magnetic bead preparation revealed significant differences in gene expression and function, in part attributable to levels of contamination associated with each isolation method. Bead isolation was found to enrich a more heterogeneous neutrophil population including a subpopulation of neutrophils expressing transcripts previously associated with low density granulocytes. Thus, RNA-Seq and bioinformatic analysis has provided a full characterisation of neutrophil gene expression under inflammatory conditions and identified several new areas of research that could lead to targeted drug design for the treatment of inflammatory disease.

FOXC1 encodes a mesenchymal transcription factor that is not normally expressed in haematopoietic cells. However, recent studies from this and another laboratory demonstrated that FOXC1 is inappropriately de-repressed in Acute Myeloid Leukaemia (AML). Through epigenomic profiling of primary AML samples, we showed that FOXC1 was specifically upregulated in the aggressive FLT3-ITD subtype of AMLs, in parallel with the activation of FOX:E-box composite cis-regulatory elements. Furthermore, complementary studies from the Somervaille laboratory demonstrated that FoxC1 expression in AML was leukaemogenic by establishment of a monocyte differentiation block and enhancement of clonogenic potential. Collectively, these data indicated that FoxC1 plays a critical role in leukaemogenesis, but the target genes and mechanisms by which this occurred were not known. To address this, we performed an integrative genome-wide analysis of FoxC1 binding, chromatin accessibility and gene expression in primary AML samples, in vivo models and cell lines. These studies revealed that FoxC1 acts to block normal myeloid differentiation by contributing to the widespread repression of differentiation-specific target genes. Critically, we identify Meis2, a proto-oncogene which collaborates with Hoxa9, as a putative direct target of FoxC1, providing compelling indications of a potential FoxC1-dependent oncogenic mechanism.

For the most part, diagnostic clinical microbiology still relies on 19th century ideas and techniques, particularly microscopy and laboratory culture. In this thesis I investigate the utility of a new approach, whole-genome sequencing (WGS), to tackle current issues in infectious disease. I present four studies. The first demonstrates the utility of WGS in a hospital outbreak of Acinetobacter baumannii. The second study uses WGS to examine the evolution of drug resistance following antibiotic treatment. I then explore the use of WGS prospectively during an international outbreak of food-borne Escherichia coli infection, which caused over 50 deaths. The final study compares the performance of benchtop sequencers applied to the genome of this outbreak strain and touches on the issue of whether WGS is ready for routine use by clinical and public health laboratories. In conclusion, through this programme of work, I provide ample evidence that whole-genome sequencing of bacterial pathogens has great potential in clinical and public health microbiology. However, a number of technical and logistical challenges have yet to be addressed before such approaches can become routine.