Attention deficit hyperactivity disorder (ADHD) is a common and impairing neurodevelopmental disorder, which frequently co-occurs with autism spectrum disorder (ASD). Both disorders are highly heritable and recent studies report a substantial degree of overlap in genetic risks for ADHD and ASD. The overall objective of this thesis is to examine the clinical co-occurrence and shared genetic susceptibility of these conditions, as well as of related developmental problems. First, the presentation of ASD traits is examined in a clinical sample of children diagnosed with ADHD. This is followed by an assessment of whether the presence of ASD traits in children with ADHD is associated with additional cognitive or developmental difficulties. Lastly, it is investigated whether common genetic risk variants which are associated with clinically-diagnosed ADHD are also associated with ADHD traits, ASD-like social-communication difficulties and neurocognitive abilities (i.e. IQ, working memory, inhibitory control and facial emotion recognition) in children from a general population sample. The results show that ASD traits split into separate, albeit correlated dimensions of social-communication difficulties and restrictive, repetitive behaviours (RRBs) in children with ADHD. They also suggest that there may be some overlap of RRBs and hyperactive-impulsive ADHD symptoms. Increasing levels of ASD traits in children with ADHD are found to index more ADHD symptoms, as well as lower cognitive abilities and a greater likelihood of developmental difficulties. Finally, the results demonstrate that common genetic risk variants relevant to ADHD diagnosis are associated with ADHD and social-communication problems as well as cognitive difficulties (lower IQ and working memory abilities) in children in the general population. This thesis extends our understanding of the clinical importance of assessing ASD in the context of ADHD. Furthermore, the findings demonstrate that common genetic risk variants for childhood ADHD are also relevant to other neurodevelopmental and cognitive outcomes in the general population.

No description available.

iv Abstract Low Copy Number Quantification of DNA Utilising Loop - mediated Amplification (LAMP) with Biolumines cent Assay in Real - Time (BART) Reporter Real time quantitative PCR is the benchmark technology of molecular diagnostics in a wide range of fields including forensic science, clinical diagnosis and the detection of genetically modified (GM) c rops. T here is a requirement for rapid, cheap and simple portable quantitative and specific diagnostics . Quantitative PCR is limited by a number of factors in this regard: t he complex hardware is often expensive and largely laboratory limited. Bioluminescent Assay in Re al Time (BART) is a nucleic acid amplification detection system that converts inorganic pyrophosphate (PPi) , a by - product of DNA synthesis , into light output. The pyrophosphate is converted into ATP which is utilised by a thermostable luciferase to convert luciferin to oxyluciferin with the emission of light. The development of isothermal amplification techniques that use the strand displacement properties of certain DNA polymerases enables the BART detection to be utilised in simple and cheap hardware at a single temperature. Loop - mediated amplification (LAMP) is an isothermal amplification method which is highly specific to the DNA target sequence and produ ces high concentrations of PPi.

The okapi (Okapia& johnstoni) is an endangered, evolutionarily distinct giraffid, endemic to the Democratic Republic of Congo (DRC). The okapi is a flagship species for the DRC,a country that contains some of the greatest biodiversity in the world. The okapi is currently under major threat from habitat fragmentation, human encroachment and poaching, yet to date, very little is known about the species in the wild, and no genetic study in the wild or captivity has ever been carried out. This thesis aims to use genetics to aid conservation efforts of okapi,a species that due to its elusive nature, is highly challenging to study using alternative methods.

Protein aggregations of β-Amyloid (Aβ) and Tau are alone not sufficient to account for all the symptoms and progression of Alzheimer’s Disease (AD), as such there is much emphasis upon the immune component of the disease. The cells of the brain that are capable of initiating an immune response are microglia; these are derivatives of hematopoietic cells and they have the capacity to phagocytose and clear Aβ aggregates or release cytokines such as TNF-a, in a neurotoxic role, promoting apoptosis of the surrounding neurons and hence aiding disease progression. To uncover the precise role of microglia in terms of AD we propose a protocol to enable differentiation of microglia from human embryonic stem cells (hESCs). The protocol we propose is a two-step differentiation procedure i) hESCs to monocytes followed by ii) ES-derived monocytes to microglia. Chapter 3: Exogenous over-expression of PU.1, a transcription factor vital in both the onset of haematopoiesis and the terminal differentiation of monocytes and microglia, was revealed to enable differentiation to a hematopoietic fate. Chapter 4: hESCS were differentiated to monocyte-like cells (CD45+/CD11b+) through culture in the presence of the hematopoietic growth factors; M-CSF and IL-3. Chapter 5: focuses on the differentiation of ES-derived monocytes to microglia. It shows that monocytes cultured in astrocyte conditioned medium give rise to cells that are IBA-1+/Glut5+/CD45low/NG2low/CD80+/CD11c+ and have a ramified microglial phenotype, which upon stimulation with Aβ(1-42) can become activated to the amoeboid phenotype. The development of the protocol for the generation of microglia holds great importance in terms of creating in vitro models for AD research as a whole and can be extended to the differentiation of patient IPSCs that contain mutations in genes associated with innate immunity or SNPs associated with AD risk, disease onset and progression.

Plants as sessile organisms have adapted highly sophisticated cellular processes to cope with environmental stress conditions, which include the initiation of complex transcriptional regulatory circuits. The heat shock transcription factors (HSFs) have been shown to be central regulators of plant responses to abiotic and biotic stress conditions. However, the extremely high multiplicity in plant HSF families compared to those of other kingdoms and their unique expression patterns and structures suggest that some of them might have evolved to become major regulators of other non-stress related processes. Arabidopsis thaliana HSFA1b (AtHSFA1b) has been shown to be a major regulator of various forms of plant responses to abiotic and biotic stresses. However, it has also been suggested that overexpression of AtHSFA1b results in a subtle developmental effect in Arabidopsis thaliana and Brassica napus in the form of increased seed yield and harvest index. Through genome-wide mapping of the AtHSFA1b binding profile in the Arabidopsis thaliana genome, monitoring changes in the AtHSFA1b-regulated-transcriptome, and functional analysis of AtHSFA1b in Saccharomyces cerevisiae under non-stress and heat stress conditions, this study provides evidence of the association of AtHSFA1b with plant general developmental processes. Furthermore, the outcome of this research shows that AtHSFA1b controls a transcriptional regulatory network operating in a hierarchical manner. However, in an agreement with a previously suggested model, the results from this study demonstrate that the involvement of AtHSFA1b in the regulation of heat stress response in Arabidopsis thaliana is possibly limited to the immediate and very early phases of heat stress response which also results in a collapse in its transcriptional network which seems to be accompanied by a general shutdown in plant growth and development.

ZNF804A was (at the time this work started) one of only a few robustly implicated schizophrenia susceptibility genes, due to replicated genome-wide significant evidence for association between a polymorphism in the gene and schizophrenia. Determining the function of the ZNF804A protein, which is currently unknown, may provide a way of elucidating the pathophysiology of this relatively common, complex disorder. Based on the hypothesis that the ZNF804A protein regulates gene expression or splicing, the aim of this thesis was to identify genes that exhibit altered expression or splicing in brain tissue from mice in which the orthologue Zfp804a carries a nonsense mutation. No robust evidence was obtained that showed the effects of the mutation on differential expression in individual genes. Although this finding does not support the hypothesis that ZNF804A acts directly to regulate gene expression, the results may reflect the possibility that effects on gene expression may be too subtle to be detected using the methods applied. Evidence was obtained to show the mutation affected the alternative splicing of a number of individual genes, which could suggest a role for ZNF804A in the direct or indirect regulation of alternative splicing. Through RNA sequencing, I identified a novel transcript in Zfp804a with an alternative exon upstream of the Refseq exon 1. I also showed that a proportion of the significant splicing differences identified in mutants were artefacts of strain differences in gene sequences that are likely to affect the efficiency of hybridisation on the exon array. Genes identified as differentially spliced between mutants and wildtypes were enriched in axon guidance and cell adhesion pathways, both thought to be important during development. The findings of this thesis suggest the novel hypothesis that ZNF804A effects risk for schizophrenia via aberrant splicing in the above pathways that are critical to normal brain development. Further studies with increased power are required to understand the effects on gene expression.

Tuberous sclerosis is an autosomal dominant genetic disorder characterised by the development of benign tumours in multiple organs. It is caused by mutations in the TSC1 or TSC2 tumour suppressor gene, leading to hyperactivation of mTOR signalling in affected tissues. Rapamycin and its analogues are mTOR inhibitors and have been used to treat tuberous sclerosis in both pre-clinical and clinical trials. However, tumours usually relapse after drug withdrawal. The aims of this project were to identify novel agents and strategies for prevention and therapy of tuberous sclerosis using mouse models. First T2 weighted MRI was evaluated for assessment of renal lesions in Tsc1+/- and Tsc2+/- mouse models. MRI identified all types of Tsc-associated renal lesions in both Tsc mouse models. The smallest detectable lesions were <0.1 mm3. Eighty five percent of all renal lesions detected in a first scan at 12 months of age were re-identified in a second scan 2 months later. Between the two scans, MRI revealed a significant increase in the total number and volume of lesions in 9 untreated mice. Compared to histological analysis, MRI detected most cysts and papillary tumours (64%) but only a minority of solid tumours (30%). Metformin is a mild inhibitor of mTOR. The therapeutic effect of metformin on renal lesions in Tsc1+/- mice was investigated using T2 weighted MRI and histological analysis. Metformin treatment for 9 months had no significant effect on renal lesions in these mice. Finally, the preventive effects on renal lesions in Tsc2+/- mice of rapamycin, metformin or both agents in combination were assessed using histological analysis. Treatment started from one month of age and continued for 7 to 9 months. Rapamycin or rapamycin plus metformin but not metformin alone effectively blocked the development of renal lesions including cysts, adenomas and carcinomas through the inhibition of mTOR signalling. These findings suggest that mTOR inhibition may be an effective strategy for preventing emergence of disease manifestations in tuberous sclerosis.

Over the past few decades stem cells have been extensively investigated due to their potentially invaluable therapeutic use. Embryonic stem cells (ESCs) have wide-ranging therapeutic applications in tissue repair and regeneration due to their pluripotent properties and their ability to self-renew indefinitely. However, ethical concerns surround their use and hence alternatives are sought. Adult stem cells (ASCs) have been isolated from various adult tissues including the oral mucosa lamina propria (OMLP). This study aims to isolate ASCs from the OMLP, reprogram these cells to induced pluripotent stem cells (iPSCs) and determine the potential for both to differentiate into functional neurons due to the limited regeneration of neurons in the central nervous system. Such investigations into strategies for the treatment of neural damage are invaluable and timely due to current limitations in the availability of human-derived cells for potential autologous or allogeneic tissue repair. OMLP-PCs represent an ideal cell source for use in regenerative medicine given their ease of isolation, proliferative potential, multipotent properties and immunosuppressive activities. Work in this Thesis has now demonstrated that these oral progenitors expressed numerous pluripotency markers and for the first time, that they could be reprogrammed to iPSCs utilising safer, non-integrating plasmids, thus increasing their potential for use in clinical applications. OMLP-iPSCs were positive for a number of pluripotent stem cell markers including SSEA-4, SSEA-5, TRA-1-60, TRA-1-81, Oct-4 and Sox-2. Moreover, their expression of early stage germ layer markers indicated their potential to differentiate into cell types of the mesoderm, endoderm and ectoderm. OMLP-PCs were also demonstrated within this Thesis to differentiate down an early neural lineage as evidenced by the presence of typical neural markers (Nestin, βIII tubulin, MAP-2 and NF-M). The presence of both ligand-gated and voltage-sensitive calcium channels indicated some limited potential functional phenotype. Unfortunately, utilising the same neural differentiation methodology, OMLP-iPSCs were not able to be similarly driven down a neural pathway. None-the-less this data suggests that OMLP-PCs and OMLP-iPSCs may hold great promise for a wide range of regenerative medicine applications

Antigenic variation processes play a central role in parasite invasion and chronic infectious disease, and are likely to respond to host immune mechanisms and epidemiological characteristics. Whether changes in antigenic variation strategies lead to net positive or negative effects for parasite fitness is unclear. To improve our understanding of pathogen evolution, it is important to investigate the mechanisms by which pathogens regulate antigenic variant expression. This involves consideration of the complex interactions that occur between parasites and their hosts, and top-down and bottom-up factors that might drive changes in the genetic architecture of their antigenic archives. Increasing availability of pathogen genomic data offers new opportunities to understand the fundamental mechanisms of immune evasion and pathogen population dynamics during chronic infection. Motivated by the growing knowledge on the antigenic variation system of the sleeping sickness parasite, the African trypanosome, in this thesis, we present different models that analyze antigenic variation of this parasite at different biological scales, ranging from the within-host level, to between-host transmission, and finally the parasite genetics level. First, we describe mechanistically how the structure of the antigenic archive impacts the parasite population dynamics within a single host, and how it interplays with other within-host processes, such as parasite density-dependent differentiation into transmission life-stages and specific host immune responses. Our analysis focuses first on a single parasitaemia peak and then on the dynamics of multiple peaks that rely on stochastic switching between groups of parasite variants. We show that the interplay between the two types of parasite control within the host: specific and general, depends on the modular structure of the parasite antigenic archive. Our modelling reveals that the degree of synchronization in stochastic variant emergence (antigenic block size) determines the relative dominance of general over specific control within a single peak, and can divide infection scenarios into stationary and oscillatory regimes. A requirement for multiple-peak dynamics is a critical switch rate between blocks of antigenic variants, which depends on host characteristics, such as the immune delay, and implies constraints on variant surface glycoprotein (VSG) archive genetic diversification. Secondly, we study the interactions between the structure and function of the antigenic archive at the transmission level. By using nested modelling, we show that the genetic architecture of the archive has important consequences for pathogen fitness within and between hosts. We find host-dependent optimality criteria for the antigenic archive that arise as a result of typical trade-offs between parasite transmission and virulence. Our analysis suggests that different traits of the host population can select for different aspects of the antigenic archive, reinforcing the importance of host heterogeneity in the evolutionary dynamics of parasites. Variant-specific host immune competence is likely to select for larger antigenic block sizes. Parasite tolerance and host life-span are likely to select for whole archive expansion as more archive blocks provide the parasite with a fitness advantage. Within-host carrying capacity, resulting from density-dependent parasite regulation, is likely to impact the evolution of between-block switch rates in the antigenic archive. Our study illustrates the importance of quantifying the links between parasite genetics and within-host dynamics, and suggests that host body size might play a significant role in the evolution of trypanosomes. In Chapters 4 and 5 we consider the genetics behind trypanosome antigenic variation. Antigen switch rates are thought to depend on a range of genetic features, among which, the genetic identity between the switch-off and switch-on gene. The subfamily structure of the VSG archive is important in providing the conditions for this type of switching to occur. We develop a hidden Markov model to describe and estimate evolutionary processes generating clustered patterns of genetic identity between closely related gene sequences. Analysis of alignment data from high-identity VSG genes in the silent antigen gene archive of the African trypanosome identifies two scales of subfamily diversification: local clustering of sequence mismatches, a putative indicator of gene conversion events with other lower-identity donor genes in the archive, and the sparse scale of isolated mismatches, likely to arise from independent point mutations. In addition to quantifying the respective rates of these two processes, our method yields estimates for the gene conversion tract length distribution and the average diversity contributed locally by conversion events. Model fitting is conducted for a range of models using a Bayesian framework. We find that gene conversion events with lower-identity partners are at least 5 times less common than point mutations for VSG pairs, and the average imported conversion tract is short. However, due to the high frequency of mismatches in converted segments, the two processes have almost equal impact on the rate of sequence diversification between VSG sub-family members. We are able to disentangle the most likely locations of point mutations vs. conversions on each aligned gene pair. Finally we model VSG archive diversification at the global scale, as a result of opposing evolutionary forces: point mutation, which induces diversification, and gene conversion, which promotes global homogenization. By adopting stochastic simulation and theoretical approaches such as population genetics and the diffusion approximation, we find how the stationary identity configuration of the archive depends on mutation and conversion parameters. By fitting the theoretical form of the distribution to the current VSG archive configuration, we estimate the global rates of gene conversion and point mutation. The relative dominance of mutation as an evolutionary force quantifies the high divergence propensity of VSG genes in response to host immune pressures. The success of our models in describing realistic infection patterns and making predictions about the fitness consequences of the parasite antigenic archive illustrates the advantage of using integrative approaches that bridge between different biological scales. Even though quantifying the genetic signatures of antigenic variation remains a challenging task, cross-disciplinary analyses and mechanistic modelling of parasite genomic data can help in this direction, to better understand parasite evolution.