Salmonella species can cause wide-ranging disease from mild food-poisoning enteritis to a systemic, sometimes fatal typhoid infection. These bacteria have evolved to survive in different environments within and outside the host and do so through the regulation of differential gene expression following activation of certain stress response systems. In gram negative bacteria such as Salmonella, envelope stress responses (ESR) are response systems that target stresses affecting components of the cell envelope such as the periplasm and outer membrane proteins. The two best characterised ESRs are the RpoE stress response system and the CpxAR two-component signal transduction system. Two further ESRs, the BaeSR response and the phage shock response have also recently been identified. The intention of this thesis was to characterise the ESR systems of S. Typhimurium to widen our current knowledge of genes involved in these systems and their role in the pathogenesis of S. Typhimurium with the ultimate aim of identifying possible candidate vaccine genes that may be used in future therapeutics against Salmonella infection. Firstly, extensive mutagenesis and phenotypic analysis studies were undertaken to characterise genes thought to be members of the RpoE regulon. Study of the phage shock response was initiated through mutagenesis, characterisation and regulation studies. A microarray experiment was designed in collaboration with colleagues at the Sanger Centre to identify members of the S. Typhimurium CpxAR regulon, with several members of this regulon being characterised further. The structural components of HtrA, an important ESR protein in S. Typhimurium, were analysed and finally work within this thesis was involved in the investigation of potential overlaps between both the RpoE and CpxAR systems. This led to the establishment of preliminary studies to investigate the vaccine potential of the tol - pal genes in S. Typhimurium.

The vascular endothelium contributes to the maintenance of vascular health by regulating vascular tone and leukocyte adhesion, amongst others. The vasoregulatory actions of the endothelium are mediated through coordinated release of vasodilators such as nitric oxide (NO) and prostacyclin, and vasoconstrictors such as endothelin-1 and thromboxane A2. Endothelial NO is the principal vasodilator in the vasculature and is produced by endothelial nitric oxide synthase (eNOS). Insulin is a vasoactive hormone that exerts its vasodilatory effects through eNOS-mediated NO production. Endothelial function is impaired in a number of disorders, including insulin resistance, diabetes and atherosclerosis, leading to dysregulated vasodilation as well as increased monocyte adhesion and plaque formation (atherosclerosis). The underlying molecular mechanisms leading to endothelial dysfunction are still in question. The work presented in this thesis addressed this question by investigating how insulin signalling and eNOS-mediated NO and superoxide production in human vascular endothelial cells are affected under experimental hyperinsulinaemia (chapter 3) and experimental hyperglycaemia (chapter 4). Atherogenic processes in human aortic endothelial cells (HAEC) were investigated by assessing monocyte adhesion under experimental hyperinsulinaemia (chapter 3), and by determining the contribution of NO and AMP-dependent kinase (AMPK) activity to the regulation of endothelial chemokine production (chapter 6). The potential of insulin to modify the subcellular distribution of eNOS was investigated in chapter 5. Clinical hyperinsulinaemia correlates with attenuated NO-mediated vasodilation, but it is not clear how hyperinsulinaemia impairs eNOS-mediated NO production. The present study modelled hyperinsulinaemia in HAEC and demonstrated a blunted response of hyperinsulinaemic cells to Ca2+-stimulated, but not insulin-stimulated eNOS-mediated NO synthesis. To address the underlying mechanisms responsible, the protein expression levels of components of the metabolic and mitogenic insulin signalling pathways, and of the metabolic energy sensor, AMPK, were quantified. Experimental hyperinsulinaemia slightly and non-significantly increased basal and insulin-stimulated eNOSS1177 phosphorylation in a time-dependent manner, and the levels of eNOST495 increased following acute insulin stimulation under these conditions. No marked dysregulation of individual insulin signalling pathway components was identified as a potential cause, but increased activating AMPKT172 phosphorylation was found to be a potential cause of increased unstimulated eNOSS1177 phosphorylation under experimental hyperinsulinaemia. Monocyte adhesion to hyperinsulinaemic HAEC did not differ from control HAEC, indicating that experimental hyperinsulinaemia did not act as a proatherogenic factor in the present study. Overt diabetes was simulated by experimental hyperglycaemia in human umbilical vein endothelial cells (HUVEC) and its effect on insulin-stimulated eNOS phosphorylation and endothelial superoxide production assessed. Insulin tended to stimulate phosphorylation of eNOSS615 and eNOSS1177, and decrease phosphorylation of eNOSS114, eNOST495 and eNOSS633 under control conditions. Experimental hyperglycaemia slightly reduced basal phosphorylation of Ser633 and significantly reduced insulin-stimulated phosphorylation of Ser114, but mildly increased basal Ser615 phosphorylation, indicating some dysregulation of eNOS phosphorylation. The upstream components of the metabolic insulin signalling pathway were not impaired in hyperglycaemic conditions. The subcellular localisation of eNOS is thought to have implications for its function. This study showed that eNOS localises to the plasma membrane, the nucleus, the cytoplasm and, primarily, the perinuclear area of HAEC. Insulin stimulation did not affect this distribution. Phospho-eNOS species were found primarily at the plasma membrane, and insulin may modulate the abundance of an intracellular eNOST495 species. Previous work in our laboratory on AMPK-mediated reduction of adhesion molecule expression has lead to the investigation of AMPK- and NO-mediated regulation of chemokine production in the present study. Inhibition of NO synthesis increased the production of monocyte chemoattractant protein (MCP)-1 in HAEC. AMPK activity downregulated TNFα-stimulated MCP-1 expression, and this was NO-dependent in the short-term, but may be NO-independent during prolonged AMPK activation. These data implicate NO and AMPK as antiatherogenic mediators in vascular endothelial cells in vitro. Taken together, the data in this thesis provide further insight into some of the molecular mechanisms involved in endothelial function and their response to hyperinsulinaemia, hyperglycaemia and proatherogenic stimulation.

Schizophrenia is a severe, chronic and debilitating psychiatric disorder. Current therapies have no efficacy in treating the cognitive impairments which are largely responsible for the poor quality of life of schizophrenia patients and contribute to the massive economic burden that is associated with the disorder. Although it is known that schizophrenia is highly heritable, the underlying genetic basis is still poorly understood due to the complex polygenetic nature of the disorder. Several candidate genes which are thought to increase risk for the incidence of schizophrenia have been identified. Two such schizophrenia candidate genes are neuregulin 1 (NRG1) and v-erb-a erythroblastic leukaemia viral oncogene homolog 4 (ERBB4). As well as the genetic evidence from genetic association studies, studies of animal models and the endogenous biological functions of NRG1 and ERBB4 in the CNS suggest that these genes may play an important role in the pathophysiology of schizophrenia. However, very little is known about the functions of these genes in specific brain regions in adulthood with respect to cognition. To address this, I have utilised recombinant adeno-associated viral particles (rAAVs) as a vehicle to mediate knockdown of the expression of Erbb4 specifically within the medial prefrontal (mPFC) cortex of adult rats. This allows for a spatially and temporally controlled investigation of the role that Erbb4 signalling may play in prefrontal cortex-dependent behaviours in adulthood. Following initial in vitro and in vivo validation of the functionality of the rAAVs, further in vivo studies confirmed that, five weeks after stereotaxic injection of rAAVs encoding a short hairpin sequence corresponding to Erbb4 (shErbb4.rAAV), into the mPFC of rats, there was significant Erbb4 protein knockdown, as analysed by ELISA. Subsequent western blot analysis revealed that Erbb4 knockdown consequently increased the level of Nrg1 expression and decreased the activity of Akt signalling, but had no effect on Erk signalling. Erbb4 knockdown specifically within the mPFC increased performance accuracy in the 5-choice serial reaction time task at 5 weeks post-surgery. Furthermore, viral mediated Erbb4 knockdown specifically within the mPFC heightened the II sensitivity to the locomotor inducing effects of amphetamine. There were, however, no effects of Erbb4 knockdown on pre-pulse inhibition at any time points assessed. These results indicate that Nrg1-Erbb4 signalling in the PFC modulates cognitive performance but not sensorimotor gating, and that dopaminergic transmission may be regulated by Nrg1-Erbb4 signalling. In conclusion, this study highlights the ability of viral mediated gene manipulation to investigate regionally specific roles of schizophrenia candidate genes in adulthood in terms of cognition and downstream signalling pathways. This may translate to a better understanding of how these genes may exert potentially pathophysiological effects in patients and ultimately lead to improved treatments.

The work reported in this thesis is concerned with understanding aspects of temporal behaviour. A large part of the thesis is based on analytical studies of temporal properties and interface and architectural concerns. The main areas covered include: i. analysing long-term human processes and the impact of interruptions and delays ii. investigating how infrastructures can be designed to support synchronous fast pace activity iii.design of the Getting-to-Know (GtK) experimental notification server The work is motivated by the failure of many collaborative systems to effectively manage the temporal behaviour at the interface level, as they often assume that the interaction is taking place over fast, reliable local area networks. However, the Web has challenged this assumption and users are faced with frequent network-related delays. The nature of cooperative work increases the importance of timing issues. Collaborative users require both rapid feedback of their own actions and timely feedthrough of other actions. Although it may appear that software architectures are about the internals of system design and not a necessary concern for the user interface, internal details do show up at the surface in non-functional aspects, such as timing. The focus of this work is on understanding the behavioural aspects and how they are influenced by the infrastructure. The thesis has contributed to several areas of research: (a)the study of long-term work processes generated a trigger analysis technique for task decomposition in HCI (b)the analysis of architectures was later applied to investigate architectural options for mobile interfaces (c)the framework for notification servers commenced a design vocabulary in CSCW for the implementation of notification services, with the aim of improving design (d)the impedance matching framework facilitate both goal-directed feedthrough and awareness In particular, (c) and (d) have been exercised in the development of the GtK separable notification server.

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The aims of this European Commission funded project, carried out at the University of Glasgow, were to develop an approach for the inclusion of ethics in a science undergraduate curriculum and to evaluate the success of that approach. The moral nature of science as an academic discipline and as a professional career justifies the resources spent on novel ethics teaching within a science course. Choices in science - allocation of research funds, selection of research topics, interaction with research subjects (animals, environment, other humans) etc. - often, if not always, include some elements of morality. The dilemmas involved require decision-making which cannot, and should not, be made without reflection on the values that govern science and society at large. From the student perspective, the ethics curriculum aims to promote and accelerate moral development. In the context of ethics teaching in a science curriculum, moral development consists of two components: moral sensitivity and moral cognitive skills. Moral sensitivity is an ability to understand that moral aspects are as valid as factual data, and to distinguish between the two. Moral cognitive skills consist of an ability to 1) analyse the moral aspects of a situation, 2) differentiate the significant from the insignificant, 3) foresee the moral consequences of actions, and 4) to make moral decisions, in particular when it is necessary to choose between two or more incompatible values. The minimal ethics teaching intervention used in this study was a success as it captured students' motivation and interest and supported moral sensitivity development, which is the first step of moral development. The results show that ethics education is needed to support students' search for adequate moral decision-making tools and their ability to include moral considerations in their general decision-making.

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Recent years have seen a huge proliferation in the use of content in distributed applications. This observation has been exploited by researchers to construct a new paradigm called content-centric networking. Within this paradigm, applications interact with the network using a simple content request/reply abstraction. The network is then responsible for routing this request towards the 'nearest' provider that can offer the content. This process therefore exploits the observation that applications rarely have a vested interest in where their content is obtained from. However, it currently ignores the fact that many applications similarly have no interest in how their content is obtained, as long as it falls within certain requirement bounds (e.g. performance, security etc.). Consequently, existing content-centric interfaces offer no support for stipulating such abstract requirements. This thesis therefore proposes an extension of the content-centric abstraction to include the concept of delivery-centricity. A delivery-centric abstraction is one that allows applications to associate (high-level) delivery requirements with content requests. The purpose of this is to offer access to content in a specialised and adaptable way by exploiting an application’s ambivalence towards the underlying means by which it is acquired. Through this, applications can simply issue abstract requirements that are satisfied by the underlying system. These requirements can range from performance needs to more diverse aspects such as overheads, anonymity, monetary cost and the ethical policies of providers. Using the above principles, this thesis proposes the design of a new system that can offer a delivery-centric abstraction. This process is guided by key design goals, which dictate a solution should be interoperable with existing sources, highly deployable and extensible. Specifically, a middleware approach is taken, which results in the development of the Juno middleware. Juno operates by using configurable protocol plug-ins that can interact with various third party providers to discover and access content. Using these plug-ins, Juno can dynamically (re-)configure itself to deliver content from the sources that are most conducive with the application’s requirements. The thesis is evaluated using a number of techniques; first, a detailed study of real-world delivery protocols is performed to motivate and quantify the benefits of using delivery-centricity. Alongside this, Juno’s functional aspects (discovery and delivery) are also evaluated using both simulations and a prototype deployment to understand the performance, overheads and feasibility of using a delivery-centric abstraction. Throughout the thesis, performance is focussed on as the primary delivery requirement. It is shown that utilising a delivery-centric abstraction can dramatically increase the ability to satisfy this requirement, and that Juno’s approach fully supports such improvements. It is then concluded that placing delivery-centricity in the middleware-layer is a highly effective approach, and that it can be performed in a feasible manner to ensure that delivery requirements are met. The key contributions of this thesis are therefore, (i) the introduction of a new delivery-centric abstraction, (ii) the design and implementation of a middleware solution to realise this abstraction, and (iii) the development of novel technologies to enable content-centric interoperation with existing (and future) content providers.

The healthy intestinal mucosa is home to the largest population of macrophages in body. Like all tissue macrophages, intestinal macrophages play vital roles in maintaining tissue homeostasis by removing apoptotic cells and any other cellular debris. In addition they maintain the integrity of the epithelial barrier and support the differentiation and maintenance of regulatory T cells in the mucosa. By virtue of their high phagocytic and bactericidal activity, these macrophages are also vital members of the innate immune system and are strategically positioned adjacent to the epithelium so that they can capture and eliminate any invading organism(s). However unlike other tissue macrophages, those found in the normal gut have several functional adaptations, such as hyporesponsiveness to toll-like receptor (TLR) ligands, which allow them to function without provoking overt inflammation. Macrophages are also abundant during intestinal inflammation, where they show increased TLR responsiveness, pro- inflammatory cytokine and chemokine production and enhanced phagocytic ability. Under these conditions, macrophages perpetuate inflammation. It remains unclear whether these distinct roles in healthy and inflamed intestine roles are carried out by discrete populations of macrophages, or if the resident macrophages alter their behaviour and become pro-inflammatory. One of the main obstacles to gaining a better understanding of the immunobiology of intestinal macrophages during steady state and inflammatory conditions is discriminating them from other mononuclear phagocytes (MP) in the mucosa, such as dendritic cells (DC). At the time of starting my project, it was becoming clear that markers such as F4/80 and CD11c were insufficient for distinguishing between macrophages and DC when used in isolation. Therefore, the aims of this thesis were to first establish reliable multi-parameter flow cytometry staining protocols to allow precise phenotypic and functional characterisation of macrophages in the healthy and inflamed mouse colon, and secondly, to explore the origins of these macrophage populations to assess whether they were derived from distinct precursors, or whether a relationship existed between them. Lastly, I examined the potential mechanisms underlying the characteristic TLR hyporesponsiveness that intestinal macrophages exhibit, focusing on the role of the inhibitory CD200R1-CD200 axis. In Chapter 3, I first set out to characterise phenotypically the macrophage populations present in the steady state mouse colon using multi-parameter flow cytometry. These studies revealed that expression of the chemokine receptor CX3CR1 could be used to identify two main populations of myeloid cells, the bigger of which was a homogeneous population of CX3CR1highCD11b+ macrophages that dominated the resting mucosa. A smaller population of CD11b+ cells expressing intermediate levels of CX3CR1 (CX3CR1int) was also present in the steady state mucosa, but this was remarkably heterogeneous, with at least 4 subsets distinguishable on the basis of Ly6C, class II MHC, F4/80 and CD11c expression. These included F4/80+Ly6ChighMHCIIneg CD11cneg cells that were phenotypically indistinguishable from blood monocytes, F4/80+Ly6C+MHCII+CD11c+/neg cells and F4/80+Ly6CnegMHCII+CD11c+/int cells that were phenotypically and morphologically similar to CX3CR1high macrophages except for their lower level of CX3CR1. Finally there was a minor subset of F4/80negLy6Cneg MHCII+CD11chigh cells that expanded markedly in response to in vivo flt3L treatment and appeared to be genuine DC. CX3CR1neg cells were also found within the CD11b+ population in the healthy mucosa, most of which were Siglec F+ eosinophils, together with a few neutrophils. In the second half of Chapter 3, I examined how these populations changed during acute colitis induced by feeding dextran sodium sulphate (DSS). These experiments demonstrated that the CX3CR1int compartment expanded dramatically during acute inflammation, with preferential accumulation of the Ly6Chigh subsets and relative loss of the CX3CR1high population as colitis progressed. Together these studies suggested that CX3CR1high and CX3CR1int cells represent resident and pro-inflammatory macrophages respectively. I next set out to explore the in vivo origin of the CX3CR1int and CX3CR1high populations, to address whether they were derived from independent precursors as would be predicted by current theories of monocyte heterogeneity, or if a relationship existed between them. By using adoptive transfer of purified BM monocytes, the studies described in Chapter 4 show that 'inflammatory' Ly6Chigh, but not Ly6Clow 'resident' monocytes replenished the CX3CR1high resident macrophage population in the steady state mucosa. This appeared to involve local differentiation of Ly6Chigh monocytes through CX3CR1int intermediary stages, which was accompanied by the acquisition of class II MHC, loss of Ly6C and upregulation of F4/80 and CX3CR1. In vivo BrdU incorporation studies supported the idea that the majority of CX3CR1int cells in the resting intestine represented short-lived intermediaries on their way to becoming CX3CR1high macrophages. Together these studies suggested that rather than representing independent macrophage subsets, the CX3CR1int and CX3CR1high cells in the resting colonic mucosa comprise a differentiation continuum from Ly6Chigh monocytes to mature CX3CR1high macrophages. Analysis of BM chimeric mice confirmed that BM-derived monocytes were the source of the vast majority of colonic LP macrophages. These findings were supported by the fact that CCR2 KO mice, in whom Ly6Chigh monocyte egress from the BM is blocked, lack Ly6Chigh colonic monocytes and have markedly reduced numbers of mature colonic macrophages. In Chapter 4, I also explored whether factors present in the normal mucosa, such as colony stimulating factor (CSF)-1, TGFbeta and the chemokine CX3CL1, could direct monocytes to acquire the phenotype of mucosal macrophages. Although initial in vitro studies suggest that none of these were effective on their own, in vivo administration of recombinant CSF-1 appeared to promote in situ monocyte differentiation in the gut. Taken together, the results in this chapter highlight that the CX3CR1high macrophage population is maintained by Ly6Chigh blood monocytes and that their differentiation is controlled by local factors in the mucosa. In Chapter 5, I went on to investigate whether the phenotypically identifiable differentiation of mucosal macrophages was accompanied by alterations in their functional capacity. Intracellular cytokine staining, qRT-PCR and reporter gene expression revealed that as Ly6Chigh monocytes differentiate locally through the CX3CR1int stages into CX3CR1high macrophages, they progressively acquired the ability to produce IL10 and have reduced production of pro-inflammatory mediators. In addition, the maturation of monocytes was accompanied by an increased ability to phagocytose and kill bacteria. Their response to exogenous stimulation by TLR ligands also altered as differentiation proceeded, with the Ly6Chigh monocytes responding robustly to TLR2 and TLR4 ligation in a TNF-alpha dominated manner, whereas the CX3CR1high macrophages responded less vigorously and their TNF-alpha production was balanced by IL10. This pattern was retained during experimental colitis, where the CX3CR1int cells showed enhanced spontaneous TNF-alpha production, whereas IL10 remained the dominant product of CX3CR1high macrophages. Adoptive transfer experiments in Chapter 6 then showed that donor Ly6Chigh monocytes were recruited to the mucosa of colitic mice, but unlike in resting mice, they failed to acquire the CX3CR1high phenotype.