M.Sc. / Dense non-aqueous phase liquids (DNAPLs) are groups of chemicals often found beneath the water surface when chemical contamination of water occurs and they are called groundwater contaminants. Their improper storage and extensive use in industries as well as their slow degradation provide a long term source for of low level contamination of ground- and river water. Evidence from both human and animal studies suggests that volatile organic and organochlorinated compounds (specific types of DNAPLs), may increase host susceptibility to microbial infection, induce alterations in the maturation of effector immune cells and compromise immune surveillance mechanisms. These effects of DNAPLs hold special relevance for people living with HIV/AIDS. In light of this, the present study investigated the in vitro immunological effects of the two most common DNAPLs contaminants, Trichloroethylene (TCE) and Aroclor-1254 (ARO) in peripheral blood mononuclear cells (PBMCs) of immunocompromised and healthy donors. TCE and ARO were successfully dissolved in cell culture medium and added to freshly isolated PBMCs in a 1:1 ratio. Following incubation, cell functionality and cytotoxicity (or immunotoxicity) were assessed using MTT and LDH. Viability was confirmed and/or cell death analyzed by flow cytometry. Culture supernatants were used to assess NO and cytokine production as well as for quantification of viral replication. TCE and ARO induced a significant (p<0.05) decrease in cell viability/functionality in a dose-dependent manner. Flow cytometric analysis of cell death pathways indicated that TCE and ARO induced apoptosis. These chemicals also induced the secretion of both NO and proinflammatory cytokines suggesting that they may induce apoptosis via an inflammatory pathway, which may explain the mitochondrial dysfunction as determined by the MTT assay. ARO effects were more prominent than those of TCE, and both were more detrimental to HIV positive PBMCs compared to uninfected cells. The viral p24 levels increased in a dose-dependent fashion suggesting an effect for TCE and ARO on viral replication. This research concludes that DNAPL-contamination is detrimental to especially immuno-compromised systems.

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The clonal selection/defence paradigm appears unable to reconcile immune function with homeostatic activity whereas organismic homeostasis is central to immune function in the network/autopoiesis paradigm. The aim of this investigation, therefore, was to test the proposition that immune function, that is not clonally driven (central immune system activity), contributes to organismic homeostasis in collaboration with psychoneural responses. In one experiment sheep were confined, either in groups or individually, and the time course of changes in cortisol levels, behaviour and T lymphocyte numbers were monitored. In another study, soldiers were monitored during the stressful experience of recruit training. The combined results suggest that, at least when the immune response is not clonally driven, the psychoneural system and the central immune system may not be operating independently of each other but rather as sub-networks of the organismic network. Consequently, homeostasis is properly characterised as a property of the whole organism. In autopoietic terms, then, homeostasis could be defined as the maintenance of network stability. / Doctor of Philosophy (PhD)

The majority of human papillomavirus (HPV) types cause cutaneous and mucosal disease. Persistent infection with high-risk HPV types is the primary risk factor for the development of cervical cancer. The ability of the virus to persist is contributed to by numerous immune evasion mechanisms. We previously demonstrated that the HPV type 16 (HPV16) E6 protein, down-regulates epithelial (E)-cadherin expression and that the associated Langerhans cells (LC) depletion may contribute to impaired immune recognition by the host. The aims of this study were firstly to establish if E6 down-regulation of E-cadherin is conserved amongst all HPV types, secondly to determine if the reduced E-cadherin expression correlates with reduced LC density in HPV-infected tissues, thirdly, to identify a region of E6 responsible in E-cadherin regulation and fourthly to establish if down-regulation of cell surface E-cadherin also occurs in another DNA tumour virus, adenovirus (Ad). E6 protein from a range of HPV types representing the α, β and γ genera was expressed in HCT116 cells and the effect on cell surface E-cadherin expression was measured by flow cytometry. In addition, a series of tissues infected with HPV types representative of HPV of α, β, [nu] and γ genera were stained to confirm E-cadherin regulation in vivo and to determine the functional significance of E-cadherin expression in relation to LC localisation. In order to identify the region of the E6 protein that was important for E-cadherin regulation, a series of HPV16 E6 mutants were tested for their ability to regulate E-cadherin. Finally, the effects of Ad on cell surface E-cadherin were examined by measuring E-cadherin expression in Ad infected HCT116 cells. E6 down-regulation of E-cadherin was conserved in α, [nu] and γ genera but was lost in β-HPV types, correlating with the ability of the virus to persist. In vivo analysis of patient tissues confirmed this pattern of E-cadherin regulation by E6 types and showed a direct association between loss of E-cadherin and LC depletion, suggesting that E-cadherin regulation by E6 is the cause of depletion of LC in infected tissue. Mutational analysis of HPV16 E6 led to the identification of a putative E-cadherin regulatory region with a conserved motif, H/L/V-[phi]-X-X-X-X-R. A potential mechanism used by E6 to regulate cell surface E-cadherin involved down-regulation of p21[waf1/cip1] (p21) via a p53-independent pathway. Finally, study of Ad showed a similar ability of the virus to regulate E-cadherin, indicating conservation in another DNA tumour virus. This research shows that E-cadherin regulation by E6 is directly associated with LC depletion and viral persistence. The data presented here suggest that LC depletion by HPV is widely conserved in HPV types that cause persistent disease. E-cadherin regulation contributes to this effect through a specific regulatory region of the protein and manipulation of levels of cellular p21. These data may provide a foundation for the development of therapeutics for HPV that aim to overcome immune evasion by the virus.

Nature and biological systems have provided the basis for many computational models and systems, such as neural computing and evolutionary computation. This thesis examines the vertebrate immune system which formed the original basis for Artificial Immune Systems (AIS). The vertebrate immune system is highly complex and, for the most part, is successful at providing us with protection from harmful stimuli. Such a system is attractive as a model to inspire a computational system as it exhibits many desirable behaviours: adaptability, diversity, robustness, efficiency and multiple layers of detection. There has been an increasing volume of research in the field of artificial immune systems. However, as the field has expanded, immunological analogies have been reduced at the expense of problem specific optimisation. Hence, this thesis takes a different approach and returns to the immune system which initially inspired research in this field. In this thesis a set of key immunological properties based on immune system concepts and mechanisms are formalised in a model for an artificial immune system. This leads to an AIS framework that is more closely aligned with the immune system, and incorporates both innate and adaptive immune concepts. In particular, antigen presenting cells (APCs), major histocompatibility complex (MHC) molecules and T-cells are modelled within the framework. The differential signalling hypothesis is explored as a model for T-cell development, and provides a novel method for T-cell generation within an AIS. Extensive empirical analysis is performed at an individual level to examine the behaviour of the AIS framework components. These results show that the artificial immune system components exhibit similar properties to the real immune components that inspired them. However, the MHC component of the AIS is found to be of limited value within an individual AIS. The AIS framework is subsequently extended to model a population of artificial immune systems. Further empirical analysis is performed at a population level, and MHC is found to improve the adaptability of an evolving population of artificial immune systems within a dynamic environment. Such a model of immune system function is likely to be useful for immunologists, as it could provide a method of examining immune behaviour under various conditions in a cheaper and more rapid manner than in-vivo or in-vitro. Indeed, it may also provide a solution for examining properties that are unable to be tested using these traditional methods. Finally, the results of these empirical findings are discussed in terms of the relevance and applicability of immunological principles with regard to artificial immune systems for real world problems.

Borrowing ideas from natural immunity, Artificial Immune Systems (AIS) offer a novel approach to solving many diagnosis, optimization and control problems. In the course of this research this paradigm was applied to the problem of optimizing urban traffic. The traffic was micro-simulated with each car on a two junction road system modeled individually. The cars themselves were programmed with 'personalities' to better simulate real traffic. A novel AIS was developed to detect, predict, and control anomalous traffic conditions. It was also used to optimize the flow of traffic through the road network. Benchmarking was performed against the well accepted TRANSYT traffic control system. Though the TRANSYT system performed better initially, the AIS control showed marked improvement over time as it adapted better to changing traffic conditions. This change was expected as TRANSYT is optimized for specific initial conditions unlike the AIS system which adapts to changes.

Alcohol abuse has been commonly associated with enhanced susceptibility to pathogens. Studies on the effects of ethanol on the immune system are complicated by a lack of consensus on whether ethanol activates, inhibits or has no effect on immune cells. We present data showing that acute exposure of T cells to ethanol elicits responses that broadly parallel responses seen in normally stimulated T cells such as the formation of the immune synapse, polarization of the microtubule organizing center (MTOC) to the synapse and tyrosine phosphorylation of signaling proteins as seen when the T cell Receptor (TcR) engages antigen-MHC. However, incomplete activation of the T cell signaling program leads to unresponsive or anergic T cells. Our data suggests the hypothesis that ethanol can activate T cells in a manner that leads to anergy. We have found that ethanol triggers calcium signaling and this has provided one of the primary tools for analyzing the effects of ethanol on T cells. Ethanol induced calcium transients are dose-dependent and are comparable to those triggered by low doses of anti-TcR antibody. This is important because it allows us to compare ethanol dependent signaling to that normally triggered through stimulation of the T cell receptors. Analysis of the calcium signaling pathway indicates that ethanol-stimulated calcium transients depend on calcium entry and are likely due to opening of CRAC type calcium channels. The observed calcium transients go a long way towards explaining how ethanol may stimulate T cells and provides a mechanism for immune suppression through the observed translocation of NF-AT in ethanol pulsed cells. The translocation of NF-AT is particularly important because of reports that it plays a crucial role in triggering anergy and immunosuppression. Taken together, these data can help explain how ethanol can both activate T cells and cause immunosuppression.

This thesis investigates the use of an Artificial Immune System as a method for dynamically creating computer game strategies in a non deterministic environment

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