Cationic lipids are commonly used and relatively safe vectors for plasmid (pDNA) delivery. In search for an optimal lipid-based formulation for siRNA delivery to the lungs, cationic lipid-based carriers were investigated for the delivery of siRNA in comparison with pDNA delivery. The aim was to determine whether the factors that influence cationic-lipid mediated pDNA delivery would similarly affect siRNA delivery. Plasmid DNA encoding for luciferase and GAPDH siRNA were complexed using three model cationic lipid-based systems; DOTAP:DOPE, DOTAP:DOPE:DMPE-PEG5000, DOTAP:DOPE:protamine. Cationic lipid/pDNA (+/-) charge ratio of 2 or greater complexed pDNA most efficiently, while much higher ratios (≥ 10) were still less efficient in complexing siRNA. pDNA complexes were larger (up to 4 μm) and formed aggregates in physiological buffer, compared to water, whereas siRNA complexes remained small (<300 nm). Gene silencing in bronchial and alveolar cell lines Calu-3 and A549 revealed a dependency on high lipid/siRNA (+/-) charge ratio (> 8) compared to the delivery of pDNA complexes (0.5-1). Confocal microscopy and endocytic inhibitors studies indicated that the cellular uptake of siRNA/lipid complexes was via a temperature-dependent pathway, which lead to the vesicular localisation of siRNA in the peri-nuclear region. Gene silencing activity was not dependent on the endocytosis-mediated uptake of the complexes. In conclusion, important differences in the factors that affect of siRNA versus pDNA delivery were revealed: the optimal properties of gene silencing were different depending on the nucleic acid and the lipid used. DOTAP:DOPE:DMPE PEG5000 and DOTAP:DOPE:protamine at charge ratios 8-10 delivered siRNA most effectively.

Statistically predicting the origin of a human DNA sample has proven effective in forensic casework, and is of broad population genetic interest, but many facets of the problem remain unexplored. This thesis consolidates several pieces of work on the genetic prediction of origin; furthering its theory and practice. I begin by examining the performance of straightforward predictive methods when the objective is to correctly determine an individual's origin from one of several closely related genetic groups, e.g. countries within Europe. Of particular interest are the volume of data required to make useful predictions, and the negative impact of combining data from independently collected convenience samples. Depending on these factors, I show that it is possible to predict origin from either Great Britain or Ireland with good accuracy. The same approach was applied to a unique dataset, provided by Dr. Jim Wilson and colleagues, in which individuals were ascertained based on their self-reported village of origin from sets of neighbouring villages. Highly accurate predictions for village of origin were attained, demonstrating the detailed geographic resolution at which this branch of statistical methodology may succeed. Origin has innumerable aspects, many of which may be tractable to genetic prediction. Two in particular are the topics of consideration throughout the remainder of the thesis. First, I develop models for predicting separate ancestral components in each parent of a genotyped 'target' individual (Crouch and Weale, 2012), providing a more detailed profile than models of lone personal ancestry. Accuracy is high when genome-wide data are available and the modelled populations are relatively genetically dissimilar e.g. West Africa, Europe and East Asia. Second, I develop a method for predicting geographic coordinates for target individuals, constituting an estimate of their biogeographic origin in continuous space, and compare performance within Europe against existing approaches. While one alternative (Hoggart et al., 2012) displayed greater accuracy, the merits of each method are discussed in full.

The generation of induced pluripotent stem (iPS) cells is a useful tool for regenerative medicine. However, the risk of tumor development of the aforementioned cells should be addressed before they can be used for clinical applications. During the reprogramming process a number of signal pathways are activated, which may lead to direct differentiation of specific cell lineages prior to the cells reaching the pluripotent state. In order to test this hypothesis we designed a combined protocol of reprogramming and differentiation in an attempt to achieve direct differentiation of fibroblasts to specific cell lineages. Human fibroblasts were shortly reprogrammed by overexpression of four reprogramming factors (OCT4, SOX2, KLF4 and c-MYC) and maintained in reprogramming media on a gelatin substrate for four days. These cells were defined as partially induced pluripotent stem (PiPS) cells. PiPS cells did not form tumours in vivo and differentiated into smooth muscle cells (SMCs) when seeded on a Collagen IV substrate and maintained in differentiation media (DM). The PiPS-SMCs expressed a panel of SMC markers such as SMA, SM22 and Calponin at mRNA and protein levels. Immunofluorescent staining of PiPS-SMCs showed positive staining for the above markers, demonstrating a typical SMC morphology. These cells displayed a greater potential to differentiate into SMCs than iPS cells. In order to elucidate the mechanism of PiPS cell differentiation into SMCs, data from a series of experiments indicated that the gene DKK3 was involved in SMC differentiation of PiPS cells. DKK3 was expressed in parallel with SMC markers, while its overexpression or stimulation induced SMC marker expression. Furthermore, DKK3 silencing resulted in downregulation of SMC markers on both the mRNA and protein levels. Finally, additional experiments revealed that the upregulation of SMC markers by DKK3 is mediated by activation of Wnt signalling through interaction of DKK3 with the transmembrane receptor Kremen 1. Therefore, we developed a protocol to generate SMCs from PiPS cells through a DKK3 signal pathway. These findings provide a new insight into the mechanisms of SMC differentiation with therapeutic potential to vascular disease.

Language is a unique human ability influenced by genetic, cultural and social factors. Decades of research in the field have identified those brain networks that have made the development of language in humans possible. This PhD thesis introduces three studies that aim to explore the anatomy of the perisylvian language pathways (three segments of the arcuate fasciculus) both in healthy and pathological condition, using dffusion tensor imaging tractography. First study examined typical developmental trajectories of the perisylvian language network in a normative data of 101 subjects (age range: 9-49 years). After observing how these pathways mature across life span, second study investigated how these are influenced by genes and environment in a sample of 43 adult twin pairs (26 monozygotic and 17 dizygotic pairs). The results showed that perisylvian language pathways exhibit distinct maturational patterns and vary in respect to genetic control that guides this process. Familial effects played an important role for those tracts that lateralised early in life (frontal lobe connections), whereas those tracts that continue to remodel throughout adolescence (temporo-parietal connections) were driven more by unique environmental effects. While the first two studies explored anatomy of perisylvian language pathways in healthy population, third study examined neural correlates in a pathological condition that affects language processing. This study included 61 adults with autism spectrum disorder and 61 matched neurotypical controls. Localised abnormalities were identified in the left perisylvian language pathways in people with autism spectrum disorder and an association was found between these white matter abnormalities and severity of past language deficits. In conclusion, these findings may be important to furthering our knowledge of the anatomy of the perisylvian language pathways in healthy population. Also, they may facilitate our understanding of possible biological mechanisms that underpin language dysfunction in psychiatric disorders, and lead to new approaches for early diagnosis and treatment.

T-acute lymphoblastic leukaemia (T-ALL) is a common childhood cancer. Multiple genetic mutations have been identified in T-ALL patients. The most common is the mutation of the NOTCH 1 oncogene occurring in more than 50% of patients. Poor prognosis has often been shown to correlate with the migration and accumulation of T-ALL cells in the tissues. Rap and Rho family GTPases play key roles in T cell adhesion and migration and are often involved in cancer progression. Most of these proteins are post-translationally isoprenylated to facilitate their anchorage to membranes, where they function to stimulate signal transduction processes. In the first part of these studies, statins were used to reduce prenylation of GTPases, and to investigate whether the resulting alteration of GTPase membrane targeting affects T-ALL cell migration. Statins inhibited adhesion, chemotaxis and transendothelial migration of T-ALL cell lines. A similar effect was observed with geranylgeranyl transferase inhibitors and siRNA depletion of Raplb but not Rap la, RhoA, Racl, Rac2 and Cdc42. These results suggest that statins and Raplb depletion could be used to reduce tissue invasion in T-ALL. In contrast to other Rho family proteins, the atypical Rho GTPases RhoU and RhoV are not prenylated but undergo palmitoylation for membrane targeting. They are expressed at higher levels in primary T-ALL samples compared to primary T cells, and RhoU expression was shown to be unregulated by Notch 1. In the second part of these studies downregulation of RhoU and Notch 1 by siRNA reduced adhesion and migration of T-ALL cell lines. Interestingly, similar functional effects were observed upon siRNA depletion of RhoV. RhoU and RhoV partially co-localized and moved dynamically between endosomes and the plasma membrane, suggesting they act together to regulate membrane trafficking. These results indicate that RhoU and RhoV could contribute to T-ALL invasion by regulating T-ALL cell adhesion and migration.

A large body of evidence indicates that object recognition memory is dependent upon plastic processes in perirhinal cortex. In particular, a decrement in neuronal responses to stimulus repetition is likely to form the core mechanism of the ability to judge the prior occurrence of objects. Local disruption of various neurotransmitter systems by drug infusion or viral transduction of perirhinal cortex produces an impairment in object recognition memory. However, it has yet to be shown that disruption of neuronal response decrements can produce a concurrent impairment in object recognition memory. This is likely to be due, in part, to the difficulty in demonstrating such changes in the acute preparation. The current study reports the development of a new behavioural-neurophysiological task that permits the presentation of multiple visual stimuli, while performing chronic extracellular recordings from, cells held over a period of days to weeks. This task was then used to investigate the effects of drugs on the activity patterns of single neurons, and on population activity (through analysis of the EEG). Of 103 cells recorded from perirhinal and dorsally adjacent cortex, only seven (7 %) exhibited consistent visually-evoked changes in activity. However, 35 (34 %) exhibited activity changes that related to other components of the task. Reasons for these observations are discussed. When administered systemically, scopolamine, MLA, verapamil and lorazepam disrupt object recognition memory in rats performing a test of spontaneous novel object preference. The effects of these drugs on the firing rates of individual neurons, and on the power and frequency of theta oscillations in perirhinal and adjacent c011ex are investigated with the hope of providing an insight into their behavioural effects.

No description available.

Members of the vertebrate <I>Msx</I> gene family encode homeodomain-containing transcription factors that are expressed in a variety of tissues during mouse development, including the eye. <I>Msx 1</I> and <I>Msx 2</I> are reported to be essential for eye development, but their precise function during the formation of this organ is not clear. Msx function in the eye was investigated by performing a detailed analysis of <I>Msx 1</I> and <I>Msx 2</I> expression in E9.5-E13.5 mouse embryos. The optic vesicle gives rise to the pigmented retina epithelium (PRE) and neural retina. <I>In situ</I> hybridisation showed that <I>Msx 2</I> was expressed in cells of the optic vesicle that are presumed to give rise to the neural retina. Expression was not detected in the PRE. These observations suggest that <I>Msx 2</I> may play a role in specifying neural retina cell fate or in suppressing PRE fate. This function was investigated by ectopically expressing mouse <I>Msx 2</I> in primary cultures of dissociated 5 to 9 day-old chick PRE cells. This resulted in a small proportion of expressing cells acquiring a neural phenotype, but had no effect on non-expressing neighbouring cells. Dedifferentiated PRE, cultured for up to 14 days, remained responsive to Msx 2. The results presented in this thesis suggest that mouse Msx 2 may function in chick PRE cells in one of two ways; either to generate multipotent neural retina precursors or to directly drive the differentiation of individual PRE cells into different neural cell types. <I>Msx 2</I> expression in retinal progenitors of the optic vesicle, but not progenitors within the differentiating neural retina is consistent with Msx 2 functioning in the optic vesicle to recruit neural retina precursors rather than to drive their final differentiation. The <I>Msx </I>genes may play a similar role at other sites of expression.

In an effort to further characterise the role of Stat3 in the self-renewal of ES cells, a screen to identify Stat3 molecules with altered activity was undertaken. This screen identified two Stat3 variants that were capable of maintaining ES cells in the absence of LIF. These molecules are Stat3β, a naturally occurring Stat3 isoform, and Stat3 Δ722, a C-terminal truncation of Stat3α. Both displayed indistinguishable effects on ES cell self-renewal, suggesting that increased Stat3β acts as a truncation of full-length Stat3α. Analysis of LIF-independent ES cell lines expressing increased Stat3β level confirmed the constitutive tyrosine phosphorylation and DNA binding activity of Stat3β. Cellular assays performed in the presence of a LIF receptor antagonist revealed that autocrine LIF signalling is not required for Stat3β-mediated ES cell self-renewal. Furthermore, biochemical analysis of Stat3β transfectants demonstrated that Stat3β acts to promote ES cell self-renewal without detectable Stat3α activity in the absence of LIF stimulation. These data indicate that Stat3β acts independently of Stat3α and is sufficient for ES cell maintenance. Further characterisation of Stat3β transfectants revealed additional effects. Differentiation of ES cells expressing increased Stat3β levels was suppressed <i>in vitro</i> and <i>in vivo</i>. Only when treated with potent pro-differentiation agents or when grafted into the kidney capsule of adult mice did Stat3β transfectants display any significant degree of differentiation. Increased Stat3β expression was also observed to correlate with a down-regulation of endogenous Stat3α expression. Quantitation of expression levels of both Stat3 isoforms in a range of cell lines revealed a consistent correlation, with increasing Stat3β levels associated with decreasing Stat3α levels. This suggests that Stat3β directly effects endogenous Stat3α expression.

<i>De novo</i> generation and physiological expansion of definitive haematopoietic stem cells (dHSCs) occurs exclusively during embryogenesis and is initiated within the dorsal aorta/para-aortic mesenchyme (Ao) of the E11.5 aorta-gonad-mesonephros (AGM) region. The elucidation of the <i>in vivo</i> induction of dHSC emergence and regulation of expansion is likely to be a preparation to the success of generating dHSC <i>in vitro</i> and facilitating an effective and robust expansion of pre-existing dHSCs, thus providing a basis for potentially useful therapeutic translation. Utilising flow cytometry and the <i>in vivo</i> long-term haematopoietic reconstitution assays, we describe the endothelial affiliation of early dHSC as they emerge within the embryonic AGM region and yolk sac, fundamentally defined by a VE-cadherin ⁺CD45⁺ immunophenotype, and the successive restriction to a haematopoietic identity upon hepatic colonisation. Well-defined haematopoietic colony forming unit-culture (CFU-C) and endothelial network forming assays were used to reveal that the early strong endothelio-haematopoietic promiscuity observed in the early embryo is lost in favour of a mutually exclusive lineage commitment. Experiments using a novel liquid suspension culture system reveal that successful <i>in vitro</i> myeloid differentiation of VE-cadherin ⁺CD45⁺dHSCs/multipotent haematopoietic progenitors from the E11.5 AGM region is dependent on an interaction with the endothelial compartment. To directly test the predominating hypothesis of a dorso-ventral polarity in haematopoietic activity of the AGM region I have bisected the Ao from the E11.5 AGM region along the dorso-ventral axis and subjected the resultant ventral (AoV) and dorsal (AoD) aspects to <i>in vivo </i>and <i>in vitro</i> tests. Here I provide the first functional data to support the hypothesis of an anatomical polarity of dHSC distribution. Furthermore, I provide evidence to support an hypothesis that <i>in vivo</i> components of the E10.5-11.5 Ao only permit ventral emergence and expansion of dHSCs. Investigation into the transcriptional activity of the E11.5 AoV and AoD highlight the differential expression of GATA2, GATA3, HoxB4, Runxl, BMP4, Noggin and Chordin. These data demonstrate the conductive nature of the AGM region, which after structural disruption not only supports the <i>ex vivo</i> maintenance of dHSC activity but also exhibits a marked capacity for rapid stem cell expansion. Thus, in our hands we have a powerful system with the potential to allow useful investigation into the fundamental processes that occupy the field of stem cell biology; the mechanisms of stem cell emergence, induction and expansion.