Gram-negative bacteria are the most common cause of the sepsis and lipopolysaccharide (LPS) a major component of Gram-negative bacteria is known to be of major importance in the development of sepsis. Human infection with Salmonella, a Gram-negative bacterium, is associated with a number of cases of sepsis and is particularly important in childhood sepsis. During salmonellosis, monocytes and macrophages produce a number of different pro-inflammatory mediators such as TNF-α, IL-1α, IL-12, IL-18, IFN-γ, reactive nitrogen species and oxygen species. Although the production of these inflammatory mediators is required for resolution of bacterial infections, they are contraindicated in diseases such as sepsis. In the initial (acute) phase of sepsis a Systemic Inflammatory Response Syndrome (SIRS) occurs in which inflammatory mediators are produced in high concentration, which can lead to organ failure and death. The SIRS phase is then replaced by a Compensatory Anti-inflammatory Response Syndrome (CARS) phase which leads to immunosuppression. The CARS phase can lead to secondary infection and subsequent mortality within 28 days of hospital admission. To date, several studies have evaluated the role of vasoactive intestinal peptide (VIP) as an anti-inflammatory agent that may have therapeutic potential in septic patients both in vitro and in vivo. VIP has been shown to inhibit production of inflammatory mediators produced by human monocytes in response to LPS. The aim of the work described in this thesis was to investigate the therapeutic potential of VIP in sepsis using an ex vivo human monocytes model infected with viable Salmonella Typhimurium 4/74 (rather than LPS). The study shows that VIP (10-7 M) stimulates an increase in the numbers of Salmonella recovered from infected human monocytes (MOI = 10). In addition, VIP also increases the survival rate of human monocytes infected with Salmonella. These two results may suggest a detrimental effect of VIP during bacteraemia and sepsis, since monocyte death may be beneficial during sepsis and bacterial overgrowth could lead to further increased LPS (and other antigen) stimulation of the immune system. However, VIP did significantly decrease Salmonella and LPS-induced TNF-α, IL-1β and IL-6 in monocyte supernatants. VIP also had a positive effect on IL-10 production in human monocytes infected with Salmonella or stimulated with LPS. Whether this suggests a possible detrimental effect of VIP is unknown but septic patients with high serum IL-10/ low TNF-α concentration ratio have previously been shown to have a poor prognosis. Higher IL-10 concentrations in infected monocytes (due to VIP) could also increase the CARS phase of disease with increased immunosuppression. Flow cytometry and qPCR analyses showed that of all of the VIP receptors, VPAC1 was expressed most highly during Salmonella infection, or LPS stimulation, of human monocytes. Administration of VIP inhibited VPAC1 has been shown by many studies to be the most important receptor by which VIP inhibits production of inflammatory immune mediators, or increases IL-10 production from murine macrophages. Results in this thesis, therefore, suggested that Salmonella infection may promote VPAC1 expression and so provide a mechanism of inhibiting the production of inflammatory mediators in infected cells. This could then increase intracellular survival of Salmonella and provide a means of greater dissemination of the infection. To ascertain how increased VPAC1 expression on the surface of monocytes may be achieved, analysis of the expression of known intracellular endosomal and exosomal constituents was performed. Confocal laser microscopy, using specific antibodies, showed that VPAC1 on the monocyte cell membrane was internalised within early endosomes (measured by co-localisation of VPAC1 and EEA1) rather than being degraded within lysosomes (measured by immunoreactivity to LAMP1). VPAC1 is then transported via a Rab11A recycling endosome and packaged in the Trans-Golgi network (TGN), shown by co-localisation of VPAC1/Rab11A and the TGN marker (TGN46). VPAC1 was then associated with Rab3a and calmodulin. The function of these latter two proteins in the docking of exosomes to the cell membrane is well known, thus suggesting that Salmonella induced VPAC1 was also recycled to the cell membrane within exosomes. VIP inhibited the expression of both Rab3a and calmodulin but not the co-localisation of VPAC1 with these two proteins. Further studies then showed that a calmodulin agonist (CALP1) increased VPAC1 expression on the surface of monocytes, while a calmodulin antagonist (W-7) decreased expression of VPAC1 on the surface of monocytes. In conclusion, this thesis does present hitherto unknown data regarding Salmonella infection of human monocytes and the effects of VIP on infected monocytes. VIP has potential as an anti-sepsis therapy since it reduces the production of inflammatory mediators by Salmonella-infected and LPS-stimulated monocytes. However, the fact that VIP increases survival of infected human monocyte and increased growth of Salmonella in human monocytes may preclude its use in sepsis.

This research investigates Examination Timetabling or Scheduling, with the aim of producing good quality, feasible timetables that satisfy hard constraints and various soft constraints. A novel approach to scheduling, that of transformation of the problem space, has been developed and evaluated for its effectiveness. The examination scheduling problem involves many constraints due to many relationships between students and exams, making it complex and expensive in terms of time and resources. Despite the extensive research in this area, it has been observed that most of the published methods do not produce good quality timetables consistently due to the utilisation of random-search. In this research we have avoided random-search and instead have proposed a systematic, deterministic approach to solving the examination scheduling problem. We pre-process data and constraints to generate more meaningful aggregated data constructs with better expressive power that minimise the need for cross-referencing original student and exam data at a later stage. Using such aggregated data and custom-designed mechanisms, the timetable construction is done systematically, while assuring its feasibility. Later, the timetable is optimized to improve the quality, focusing on maximizing the gap between consecutive exams. Our solution is always reproducible and displays a deterministic optimization pattern on all benchmark datasets. Transformation of the problem space into new aggregated data constructs through pre-processing represents the key novel contribution of this research.

This thesis addresses a problem that faces developers of applications for mobile devices. There is an ever-increasing number of mobile platforms and form factors in the world, and mobile developers have to build applications that can be used on as many of these as possible while still retaining usability. Furthermore, because of constraints put on the development process by the companies that develop the mobile platforms, there is an absolute requirement that the applications produced by the tool conform to the user interface guidelines for each platform. To address this problem, this thesis uses the concept of “case”, which is a phe- nomenon from natural language. In natural languages, case has many functions and plays a part in many systems. This thesis engages with case in one of these functions: it permits flexible word ordering. Case is used here to allow flexible or- dering of elements within the user’s dialogue with the machine. Case may either be useful because of some analogical process in the developer’s head or because of deeper ideas in linguistic theory. To evaluate this idea, a suitable case system was embedded in a tool and this tool was used in three distinct contexts. First, applications were built for three external companies. Second, a workshop study was done with external developers. Third, more external developers were given the tool for a longer period to produce an application of their choosing. These three contexts gave an excellent view into the use of the case system during the development of applications. This evaluation showed that the kinds of functions that case describes are rel- evant to describing user interfaces; that it is possible to implement a plausible case system usefully in a software tool, at least for mobile development; that the case system when embedded within the tool can be used to build useful applications; and that case can be used and understood by developers other than the author.

The carbon and energy metabolism of the human pathogen Clostridium difficile is poorly understood. Amino acid metabolism by the Stickland reactions has previously been described as a primary source of energy in a number of Clostridium species, especially when grown in a medium containing only amino acids. Deeper insights may be gained by metabolic analyses using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) platforms, but such experiments are best performed in well-defined growth media. A number of C. difficile strains have been successfully cultivated on defined media but these media provide an excess of nutrients, particularly in terms of amino acid provision, resulting in undesirable background growth in the absence of glucose. To overcome these challenges, three variants of a defined medium were developed that contain the essential nutrients that support the growth of this bacterium, in the presence of a carbon and energy source such as glucose together with an LC-MS/MS method that will simultaneously measure all twenty amino acids. Since the focus in this study was amino acid utilisation, a comprehensive and most effective technique that will provide as much information as possible for understanding the metabolic requirements of eight Clostridium difficile strains (CD630∆erm, DH196, R20291, EK15, EK28, R12801, L26, O17 Serotype F) and two transposon mutants (CRG-2979, CRG-3887) was required. Due to high water solubility and the range of ionic characteristics of amino acids, an aqueous normal phase chromatographic method was considered to simultaneously separate a mixture of amino acids without derivatisation. Aqueous normal phase chromatographic method represents an important new technology with a capability of the silica-hydride-base stationary phases, offering a distinct advantage for practical application with a high degree of reproducibility and long-term stability of polar and non-polar compounds. The developed methods were subsequently adapted to study amino acid utilisation in the presence or absence of a fermentable carbohydrate and/or selenium. OD determinations were performed by measuring absorbance at 600 nm (or OD600) using a Biomate 3 spectrophotometer. The concentration of individual amino acids remaining in the spent C. difficile culture media was measured after 24 h and/or 48 h using the developed LC-MS/MS method in order to determine which amino acids were being utilised by these organisms and in which order. This analysis should give further insight on the importance of amino acids for the survival of this bacterium in the gut, which may possibly lead to discovery of novel fermentable products and metabolic pathways and/or eventually aid in the successful control of the disease. Data obtained for CD630∆erm, DH196, R20291, EK15, EK28, R12801, L26, O17 Serotype F strains showed that they all grew on the fully defined medium, with different growth profiles in terms of lag phase, growth rate, and maximum OD reached. The LC-MS/MS data generated suggest that cysteine, glutamine, isoleucine, leucine, serine, threonine and tyrosine are preferentially utilised, both in the presence and absence of glucose. Several other amino acids, including asparagine, glycine, phenylalanine, proline and valine were also utilised but to a lesser extent. Notable in this study was the lack of glutamate utilisation, except by strain L26, and the excretion of alanine after its initial uptake by most of the tested stains. The excretion of alanine may be due to the use of pyruvate as an amino acceptor during the degradation of preferentially fermented amino acids, whereas, glutamate is not a substrate for most C. difficile strains. Thus, LC-MS/MS profiling confirmed that these organisms derive most of their carbon and energy from the fermentation of a selected range of amino acids. Given the importance of selenium-dependent Stickland reactions to the growth of this bacterium, further studies were undertaken to evaluate the metabolism of amino acids by two different C. difficile strains (630∆erm, R20291) and two transposon mutants CRG-2979 (defective in hadB, encoding one of the two subunits of hydroxyisocaproyl-CoA dehydratase required for reductive degradation of leucine) and CRG-3887 (defective in selA encoding selenocysteine synthase) in the presence or absence of glucose/selenium. LC-MS/MS data reveal that amino acid utilisation was affected by the presence of selenite, notably proline utilisation, which could be explained by the presence of the enzyme proline reductase and the lack of glycine consumption, known to be selenium-dependent. The non-utilisation of glycine could be explained by the presence of proline which represses the formation of the necessary enzyme systems required for glycine degradation. Data generated for CRG-2979 reveals that this mutant could only thrive in the presence of selenium when glucose is present, possibly due to the presence of proline replacing leucine as the major Stickland acceptor. The results of the transposon mutant CRG-3887, were much of a surprise too, because this mutant was predicted to be deficient in proline and glycine breakdown which is also reliant on selenoenzymes. This suggests the presence of selenium independent proline fermentation pathway although this hypothesis is not supported by the existing literature. Experimental data provided further evidence about the ability of this bacterium to obtain its carbon and energy in the absence of a fermentable carbohydrate; by Stickland reactions and that the presence or absence of certain amino acids could repress the utilisation or biosynthesis of other amino acids.

Context-Aware applications make use of sensed and gathered information about a user’s state to better tailor their behaviour to the user’s needs. There are many streams of information that can be employed as context; these elements have a variety of structures and not all of them are static or fully known to developers at runtime, this can make it challenging to add new streams of context to an application and keep those streams whose structure frequently changes updated. Heterogeneity of sensor technology and information sources means two users may generate information about the same aspect of their state, their location for example, in two different formats. Although there are examples of services that can relate a value from one location sensing technology to a value from another, we lack a general service for building and reasoning about these relationships between any and all representations of context. Moreover, due to their frequent use of sensed physical information, context-aware applications also generate seams, which may cause uncertainty, error and unexpected behaviours. Developers need a simple way to think about context; one which supports the many heterogeneous types of information it comprises, allows them to update the structures of those types as they change or are discovered, gives them the means to reason about relationships between users whose devices sense the same sort of context using different technologies, and which provides them with both opportunity and means to address and respond to some of the seams arising in their applications. This work presents a spatial model for reasoning about context using presence and awareness that attempts to address the requirements above. The model is realised in a platform comprised of an API and a Context-Broker, which provides context-reasoning and relation services for applications that communicate with it via a dataspace. Using the platform to implement a location-aware game raises several issues concerning the usability and presentation of its paradigms and a seam encountered by a user playing the game. We use this as an opportunity to explore methods for analysing how underlying models, platforms and applications create meaning in pervasive and Mixed-Reality context-aware applications, and how seams operate within them. We present changes to our model and platform based on the conclusions drawn from this analysis. Our analysis of the user’s experience leads us to conclude that model paradigms must be carefully structured to support the multiple ways in which context is used, that both spatial and temporal discontinuity can create user confusion and that when mixing virtual and physical spaces it is important to communicate fully all the structures imposed upon them. Our analysis of seams leads us to the conclusion that responses to them can be usefully positioned as simple processes of disparity resolution involving the restructuring of one or more spaces or milieu involved in the application.

This thesis addresses the linguistic phenomenon of vague language (VL) and its effect on the creation of identity in the emerging and developing field of human-agent interaction (HAI). Current research on VL has focused on human interaction, while similar existing literature on language in HAI has focused on politeness theory and facework. This thesis brings the two research fields together and uses them as a focusing lens to investigate the issue of identity in agents – software with varying degrees of autonomy and intelligence. Agents are increasingly common in our everyday lives, particularly in the role of an instructor. Intelligent personal assistants are a frequent feature on smartphones, automated checkout systems pervade supermarkets both large and small, and satellite navigation systems have been a mainstay for over a decade now. Despite their frequency, there is relatively little research into the communication challenges surrounding HAI. Much like other people, the language and voice of agents have the ability to affect our perceptions and of them, and shape the way in which we create their identities. Instruction giving, amongst other facets of talk, in human communication can be mitigated through the use of VL. This can reduce the imposition we have on interaction partners, pay respect to a listener’s face, and establish and maintain a positive rapport with our interlocutors. This can have a profound effect on the desire to interact with someone again. Furthermore, agents that use speech to communicate are assigned one of two varieties of voice – synthesised or pre-recorded human speech, both of which have documented benefits and drawbacks. Given the rise of agents in the modern world, it is in the best interest of all parties to understand the salient variables that affect our perceptions of agents, and what effect VL and other variables such as voice in language and voice may have in our interactions with them. This thesis provides a novel approach to investigating both VL and voice in HAI. A general framework is presented with the use of a specific VL model to apply in the interactions, which is designed around verbal agents giving people instructions on how to construct Lego models. The first study compares the effects of a vague and non-vague verbal agent in this context, while the second study focuses on the comparative use of synthesised text-to-speech voices and professional human recordings in the same context. The results from the investigation reveal key findings regarding the use of VL in a verbal agent instructive context. The first study indicated that a synthesised agent voice is better suited to using non-vague instructions, while the second study revealed that a professional voice actor is a preferable candidate for using VL in comparison to two different synthesised voices. These findings discuss the issue of identities in HAI. They reveal that, when an agent instructor is perceived to have a voice that is non-human and machinelike, it is more likely that its use of VL will be received less positively. This is often because the combination of voice and language do not mix, but is also a result of a clash of perceived group identities between agent and human speech. As agents are typically direct, the use of “humanlike” VL can create a large disparity between a person’s expectations of agent speech and the reality of the interaction. Similarly, if an agent’s voice has more of a humanlike feel to it, then its use of VL will create less disparity and has the potential to bridge the gap between these two group identities. This poses discussions on the nature of agent identity and how it compares to those in humans. The thesis concludes with reflection on the findings in light of existing linguistic theories, and how further research into this field may assist agent designers, researchers, and agent users alike. A suggestion of employing a corpus linguistics approach to HAI is proposed, which may pave the way for future success in this area.

The evolution of smart devices and software technologies has expanded the domain of computing from workplaces to other areas of our everyday life. This trend has been rapidly advancing towards ubiquitous computing environments, where smart devices play an important role in acting intelligently on behalf of the users. One of the sub fields of the ubiquitous computing is context-aware systems. In context-aware systems research, ontology and agent-based technology have emerged as a new paradigm for conceptualizing, designing, and implementing sophisticated software systems. These systems exhibit complex adaptive behaviors, run in highly decentralized environment and can naturally be implemented as agent-based systems. Usually context-aware systems run on tiny resource-bounded devices including smart phones and sensor nodes and hence face various challenges. The lack of formal frameworks in existing research presents a clear challenge to model and verify such systems. This thesis addresses some of these issues by developing formal logical frameworks for modelling and verifying rule-based context-aware multi-agent systems. Two logical frameworks LOCRS and LDROCS have been developed by extending CTL* with belief and communication modalities, which allow us to describe a set of rule-based context-aware reasoning agents with bound on time, memory and communication. The key idea underlying the logical approach of context-aware systems is to define a formal logic that axiomatizes the set of transition systems, and it is then used to state various qualitative and quantitative properties of the systems. The set of rules which are used to model a desired system is derived from OWL 2 RL ontologies. While LOCRS is based on monotonic reasoning where beliefs of an agent cannot be revised based on some contradictory evidence, the LDROCS logic handles inconsistent context information using non-monotonic reasoning. The modelling and verification of a healthcare case study is illustrated using Protégé IDE and Maude LTL model checker.

This thesis explores a novel framework for implementing and evaluating type-1 (T1) and interval type-2 (IT2) models of Adaptive Network Fuzzy Inference Systems (ANFIS). A fundamental requirement for this research is the capability to reliably and efficiently implement ANFIS models. In the last ten years, many studies have been devoted to creating IT2 ANFIS models. However, a clear architecture for IT2 ANFIS has not yet been presented. This somehow has been an obstacle to the research of IT2 ANFIS and its application to real-world problems. In this thesis, we introduce an extended ANFIS architecture that can be used for both T1 and IT2 models. In conjunction with this, a crucial obstacle to the use of IT2 fuzzy systems in general (and including IT2 ANFIS) is that IT2 models are often more computationally expensive than T1 models. Note that a bottle-neck for IT2 ANFIS is to aggregate the output of each rule produced by the inference process of the Karnik-Mendel (KM) algorithm. Many enhanced algorithms have been proposed to improve the computational efficiency of the KM algorithm. However, all of these algorithms are still based on iterative procedures to determine the switch points required for the lower and upper bounds of defuzzification. This thesis introduces a `direct approach' which can be used to determine these switch points based on derivatives, without the need for multiple iterations. When comparing various models (including T1 and IT2 ANFIS models), it is necessary to conduct fair comparisons. Partly to address this issue, a new accuracy measure is proposed which combines the best features of various alternative measures without having their common drawbacks. Experimental comparisons are made between T1 and IT2 ANFIS using the novel accuracy measure in addition to the commonly used RMSE, on both synthetic and real-world data. Finally, it is shown that IT2 ANFIS models are not easy to optimise from scratch due to difficulties with the output intervals, that are not present in T1 ANFIS models. Detailed experiments are carried out to evaluate the comparative performance of IT2 ANFIS models, including the best method for initialising the IT2 membership functions. In summary, a coherent framework for efficiently implementing IT2 ANFIS models and fairly evaluating their comparative performance is presented. This framework allows the implementation of IT2 ANFIS in any application context, and the resultant performance to be carefully considered, since clear performance improvement compared to T1 ANFIS may not always be found.

Governing open source software (OSS) communities is defined in the relevant literature as the formal and informal means to control and coordinate the collective efforts towards common objectives (Markus, 2007). OSS communities are not based on a fixed structure. Instead, the structure emerges through collaboration. Participants, technical artefacts, ideas, resources, and interactions are fluid (Faraj et al., 2011) in the sense that they are reconfigured over time, depending on the context of the community. This has raised governance challenges in terms of determining “how open is open enough” (West, 2003). Governing a fluid complex technically-mediated ecosystem, such as OSS communities, requires determining whether to keep the boundaries open to all, which may risk the quality of the deliverables, or restricting the contributions to an elite population, which restrains collaboration (Ferraro and O'Mahony, 2012). In this thesis, I argue that OSS governance is a boundary decision to determine and legitimise the practices that best govern the collective effort in a particular context (Ferraro and O'Mahony, 2012). The current literature focused on two types of boundaries; the external boundary that separates OSS communities from the commercial world; and the role-based boundary that identifies the roles and responsibilities of the individuals (Chen and O’Mahony, 2009). The former boundary has been extensively discussed in the literature by focusing on how firms reap the benefits of OSS products without exploiting the collective effort. The latter boundary focuses on individuals as the main actors of the community. The current views on OSS governance have two main limitations. First, current accounts focus on creating a governance structure that facilitates the collaboration among dispersed individuals, neglecting the issues of fluidity and dynamicity. As a result, scholars continue to build their studies on taken-for-granted assumptions overlooking the transformations that have occurred to the overall settings of the OSS community. One of the overlooked areas is the emergence of vertical (i.e. domain-specific) OSS communities, which is the main interest of this thesis. Second, technology, in the context of OSS, is either considered as an end product or a medium of governance. Current studies failed to address the materiality of technology, where materiality refers to the ways in which the properties of technology are arranged and rearranged in relation to each other to accomplish governance practices in a particular context. The materiality of technology entails different possibilities for governance practices, which is not sufficiently addressed in the literature. Therefore, I argue and demonstrate that any attempt to explain OSS governance without addressing materiality is considered incomplete. In this thesis, I demonstrate that OSS communities are governed through boundary decisions, where decisions refer to delineating the boundaries of the community. This is achieved by identifying the actors, actions, and resources required to control and coordinate the collaborative effort in a particular context. Boundary decisions entail remaining sensitive to the changes that occur to the context and change the boundaries accordingly. I adopt grounded theory approach to conduct a case study on Kuali; a vertical OSS community that develops ERP system for the higher education sector. The research findings contribute to the OSS governance literature by developing a theoretical foundation that explains OSS governance as a boundary decision. The emergent theory explains OSS governance in terms of context, control, resources, and materiality. I illustrate through empirical evidence how these constructs interact with each other to govern the collective effort. The thesis contributes to the OSS literature by bringing to the fore the dynamicity and materiality of OSS governance. The thesis also has implications in the area of boundary management. OSS communities represent a non-traditional organisational settings, and thus provides novel theoretical insights with regards to boundary management.

Clostridium difficile is the leading cause of health care associated diarrhoea and remains a burden for the NHS. Disease symptoms can range from mild diarrhoea through to fulminant pseudomembranous colitis, resulting in mortality for some patients. Recurrence is a major problem and estimates are that 20% of all patients with disease will either relapse (with the same strain) or have a re-infection (with a different strain). Arguably, the main virulence factors are toxins A (TcdA) and toxin B (TcdB) which cause disease symptoms. The genes encoding TcdA and TcdB are located within the pathogenicity locus (PaLoc) along with three accessory genes; tcdR, tcdE and tcdC. The regulatory network has been studied but we aimed to add to this knowledge by using two under investigated strains R20291 a so-called hypervirulent strain and VPI 10463 a strain known to produce higher levels of toxin. Two different methods of investigation were employed during this study to improve our understanding of both the regulation of TcdA / TcdB but also the genetic mechanisms behind clinical relapse. These methods were; using forward and reverse genetic analysis to assess phenotypic differences and using bioinformatics to identify genes and / or single nucleotide variants (SNP) that may play a role. Using a combination these methods we have identified potential regulators of toxin production in both strains. We have also identified unique genes and SNPs that might provide a fitness benefit to strains of C. difficile that were isolated from patients who had suffered relapse episodes.