The purpose of this research is to determine the information to be collected for aseptic dispensing in NHS hospitals, and its use for management and business purposes in relation to capacity, demand, performance and efficiency. Mixed methodologies were adopted on an exploratory basis. Qualitative methods included: regular expert input; workshops; out-turn questionnaires; Affinity Analysis; surveys; and structured interviews. Quantitative methods included: activity data surveys; targeted surveys; and Delphi methods. The research systematised the collection and collation of the required data and determined novel ways of analysing and manipulating it to aid decision-making. These were used to evaluate the impact of major capital investment and variations in practices between different parts of the country. A benchmarking approach should be applied in utilising the data and statistical indicators. Nomenclature issues can influence data quality. Therefore clear, unambiguous guidance was developed for data collection. Existing pharmaceutical information systems will be the main sources of the data for the foreseeable future. The research focused on the North West of England, with successful application in the West Midlands. Its transferability to non-NHS and foreign hospitals is inferred, as long as similar operational arrangements apply. The research enables: the measurement of progress towards implementing the Breckenridge (1996) recommendations; the evaluation of performance for aseptic production and usage to inform capacity planning; and the presentation of the degree of collaboration between hospitals. The research addresses the absence of set data for an important hospital support service, and applies relevant lessons from other fields and industries. It enables a systematic approach to capacity planning and performance evaluation, at a time when the contribution of the service to support clinical governance is being fully recognised.

The under-reporting of cases of infectious diseases is a substantial impediment to the control and management of infectious diseases in both epidemic and endemic contexts. Information about infectious disease dynamics can be recovered from sequence data using time-varying coalescent approaches, and phylodynamic models have been developed in order to reconstruct demographic changes of the numbers of infected hosts through time. In this study I have demonstrated the general concordance between empirically observed epidemiological incidence data and viral demography inferred through analysis of foot-and-mouth disease virus VP1 coding sequences belonging to the CATHAY topotype over large temporal and spatial scales. However a more precise and robust relationship between the effective population size (N_e) of a virus population and the number of infected hosts (or 'host units') (N) has proven elusive. The detailed epidemiological data from the exhaustively-sampled UK 2001 foot-and-mouth (FMD) epidemic combined with extensive amounts of whole genome sequence data from viral isolates from infected premises presents an excellent opportunity to study this relationship in more detail. Using a combination of real and simulated data from the outbreak I explored the relationship between N_e, as estimated through a Bayesian skyline analysis, and the empirical number of infected cases. I investigated the nature of this scaling defining prevalence according to different possible timings of FMD disease progression, and attempting to account for complex variability in the population structure. I demonstrated that the variability in the number of secondary cases per primary infection R_t and the population structure greatly impact on effective scaling of N_e. I further explored how the demographic signal carried by sequence data becomes imprecise and weaker when reducing the number of samples are described, including how the extent of the size and structure of the sampled dataset impact on the accuracy of a reconstructed viral demography at any level of the transmission process. Methods drawn from phylodynamic inference combine powerful epidemiological and population genetic tools which can provide valuable insights into the dynamics of viral disease. However, the strict and sensitive dependency of the majority of these models on their assumptions makes estimates very fragile when these assumptions are violated. It is therefore essential that for these methods to be applied as reliable tools supporting control programs, more focused theoretical research is undertaken to model the epidemiological dynamics of infected populations using sequence data.

Feline immunodeficiency virus (FIV) is a naturally occurring lentivirus of domestic cats, which shares many similarities with its human counterpart, human immunodeficiency virus (HIV). FIV infects its main target cell, the CD4+ T lymphocyte, via interactions with its primary receptor CD134 (an activation marker expressed on activated CD4+ T lymphocytes), and, the chemokine receptor CXCR4. According to the different ways in which FIV isolates interact with CD134, FIV may be categorised into two groups. The first group contains strains that tend to dominate during the earlier phase of infection, such as GL8 and CPG41. These strains are characterized by their requirement for an additional interaction with the second cysteine rich domain (CRD2) of the CD134 molecule and are classified as “CRD2-dependent” strains. The second group, on the other hand, contains either laboratory-adapted isolates or isolates that emerge after several years of infection, such as PPR or the GL8 variants that emerged in cats 6 years post experimental infection and were studied in this thesis. These isolates are designated “CRD2-independent” as they can infect target cells without interacting with CRD2 of the CD134 molecule. This study provides the first evidence that FIV compartmentalisation is related to FIV-CD134 usage and the tissue availability of CD134+ target cells. In tissue compartments containing high levels of CD134+ cells such as peripheral blood and lymph nodes, CRD2-dependent viruses predominated, whereas CRD2-independent viruses predominated in compartments with fewer CD134+ cells, such as the thymus. The dynamics of CD4+CD134+ T lymphocytes at different stages of FIV infection were also described. The levels of CD4+CD134+ T lymphocytes, which were very high in the early phase, gradually decreased in the later phase of infection. The dynamics of CD4+CD134+ T lymphocyte numbers appeared to correlate with FIV tropism switching, as more CRD2-independent viruses were isolated from cats in the late phase of infection. Moreover, it was observed that pseudotypes bearing Envs of CRD2-dependent variants infected CD134+ target cells more efficiently than pseudotypes bearing Envs of CRD2-independent variants, confirming the selective advantage of CRD2-dependent variants in environments with high levels of CD134+ target cells. In conclusion, this study demonstrated that target cell types and numbers, as well as their dynamics, play important roles in the selection and expansion of FIV variants within the viral quasispecies. Improved understanding of the roles of target cells in FIV transmission and pathogenesis will provide important information required for the development of an improved, more successful protective FIV vaccine and will provide insight into the development of effective vaccines against other lentiviral infections such as HIV.

Infectious diseases pose a significant threat to animal and human health across the globe, with much of the burden falling on low-income countries. Despite efforts to control many of these diseases, very few have ever been eradicated. Their dynamics are often embedded in complex, heterogeneous landscapes defined by interacting population and landscape level processes. As such, landscape heterogeneity plays a key role in driving disease transmission and persistence. Incorporating landscape heterogeneity in studies of pathogen dynamics is challenging but the accessibility of data, particularly next generation sequencing data, has opened new avenues of research. Landscape epidemiology involves using an integrated approach to understand spatial patterns of disease, using methods that combine landscape genetics, ecology and epidemiology. in this thesis I use these integrative methods to determine the underlying mechanisms facilitating the spread and persistence of canine rabies virus in Tanzania. Whole genome level characterisation of rabies virus samples was achieved and used in combination with cutting-edge inference techniques to explore spatial patterns of rabies at different spatial scales. Phylogeographic patterns were able to characterise spatial scales of endemic rabies transmission in Tanzania, uncovering strong viral population structure at sub-continental levels with evidence of a more fluid dispersal dynamic at local ( less than 100km2 area) spatial scales . Within-country phylogeographic patterns revealed large regional movements within Tanzania that could be attributed to human-mediated movements and revealed the presence of multiple co-circulating lineages within a single administrative district. Finely resolved incidence data from the Serengeti District complemented with whole genome sequences enabled the exploration of local scales of transmission in more detail. By extending phylogeographic diffusion models to incorporate landscape heterogeneity I was able to uncover evidence supporting landscape predictors of rabies diffusion. While much of the spatial structure was attributable to the effects of isolation by distance, landscape predictors had discernible effects on diffusion. In particular, rivers appeared to act as a barrier to dispersal and road networks facilitated diffusion and I found evidence to support vaccination as an effective control measure for canine rabies in the Serengeti District. Importantly, I also found evidence to support vaccination as resistance to diffusion and therefore an effective control measure for dog rabies. As a complementary approach a space-time-genetic algorithm was used to determine who-infected-whom in the Serengeti District. The model explicitly accounted for the possibility of exogenous sources of infection and how to incorporate genetic data available for only a proportion of samples. Direct transmission events were estimated between 42% of observed cases and highlighted the co-circulation of two major lineages in both time and space. Direct transmission events predominantly occurred over very small distances, less than 1km, but a large proportion of cases had unobserved sources that could represent transmission from dogs in neighbouring regions or larger indirect transmission events. A future development of the model is to delineate between these possibilities to assess the true contribution of exogenous sources to the system dynamic. Ultimately these integrative models are at an early stage of development but highlight the power of genetic data to delineate fine-scale transmission patterns. The results from this thesis suggest that landscape features such as rivers could be exploited as barriers in step-wise vaccination campaigns and highlight the utility of genetic surveillance to monitor control and elimination as rabies management progresses.

It is well established that adenoviruses degrade components of the cellular DNA damage response, such as p53, DNA ligase IV and Mre11, in order to avoid detection from the host cell and thus, promote viral replication. Here we show TAB182, a protein of previously unknown function, is degraded following adenovirus serotype 5 and 12 infection. Similarly to other DNA damage proteins, together with the cellular Cullin 5 (during Ad5 infection) and Cullin 2 (during Ad12 infection). Interestingly, siRNA-mediated knockdown of TAB182 appears to be beneficial for adenovirus infection, as denoted by an increased expression of the adenoviral E1A protein and Cyclin E during adenovirus infection. Together with other studies, we confirm that TAB182 interacts with the large, multi-subunit CNOT complex. This complex has no defined function in mammalian cells, but is known to play a role in gene regulation in yeast. Interestingly, components of the CNOT complex are also degraded during adenovirus infection, whether adenovirus degrades TAB182 as well as CNOT for the same advantage is currently unknown. Cells deficient in TAB182 are hypersensitive to agents that induce DNA replication stress and also exhibit abnormal replication dynamics following release from hydroxyurea-induced fork stalling. In particular, they display increased fork restart and elevated new origin firing following release from hydroxyurea treatment, suggesting that TAB182 prevents fork recovery and suppresses new origin firing following replication stress. Depletion of some components of the CNOT complex is able to rescue the phenotypes observed in TAB182 deficient cells, suggesting that TAB182 and the CNOT complex may act in concert at the replication fork. TAB182 deficient cells display less DNA gaps and breaks but increased levels of 53BP1 bodies in G1 and micronuclei, which are markers of genome instability, following replication stress. Whether TAB182 acts directly at the replication fork, or in conjunction with other proteins known to be involved in replication restart such as helicases, nucleases, or chromatin remodelling complexes, remains to be elucidated.

The role of the E4 protein in the human papillomavirus (HPV) life cycle is an enigma even though it has varied effects on cell behaviour and organisation in overexpression studies. Full-length E4 proteins are derived from E1^E4 spliced RNA transcripts and E1^E4 proteins from diverse HPV types interact with serine-arginine (SR)-specific protein kinase SRPK1, that regulates diverse cellular functions including RNA splicing. This thesis has sought to address the hypothesis that E1^E4 alters SRPK1 activity and influences SRPK1 functions in the HPV life cycle. This study has uncovered the novel finding that E1^E4 protein of HPV1, but not HPV5, 16 and 18, is a potent inhibitor of SRPK1 activity in vitro and in vivo and inhibition is dependent upon E1^E4 binding to SRPK1. Whilst HPV1 E1^E4 inhibits SRPK1 phosphorylation of cellular (ASF/SF2, SRp20, SC35, 9G8 and SRp75) and viral (HPV E2) SR protein substrates, it has only weak effects on SR protein cellular localisation and on cellular and viral RNA splicing in minigene systems. Addition of the small molecule inhibitor of SRPK, SRPIN340 to organotypic raft cultures of HPV18 genome-containing keratinocytes enhances the morphological features of HPV viral replication suggesting that the HPV may modulate SRPK activity to facilitate the virus life cycle.

EBV lytic replication involves the sequential expression of a large array of antigens that potentially provides a complex antigenic challenge. Despite this number, the primary CD8+ T cell response in infectious mononucleosis (IM) patients appears focused against epitopes found in immediate-early and some early expressed antigens with responses to late antigens typically subdominant. However previous approaches have only focused on a limited number of lytic antigens. To resolve these issues, this study examines the CD8+ T cell repertoire to all EBY lytic antigens in different phases of infection. Polyelonal CD8+ T cell lines were mitogenically expanded from IM patients or expanded in an antigenically unbiased way from post-1M patients and healthy carriers using autologous dendritic cells loaded with lysates of lytically infected cells. Target cells expressing individual lytic antigens along with donor HLA class I molecules were used to challenge polyclonal lines with responses measured by the release of IFNγ. These studies show that the pattern of target antigen choice varies with the phase of infection and is consistent with the idea that CD8+ T cell responses in primary infection are driven by lytically infected B cells. However over time the repertoire of responses may be influenced through antigen cross-presentation.

Dendritic cells are efficient antigen-presenting-cells that can be used in tumour-antigen specific vaccination for malignant disease. Melanoma patients were recently treated with a dendritic cell vaccine expressing gp100 and Melan-A antigens after non-viral (CL22 peptide) transfection. Although clinical and immunological responses were noted, there was no correlation between responses and whole antigen expression levels in the vaccine cells that varied widely. Here, it is established that patient cells expressed detectable levels of Class I restricted epitopes from both antigens, although there was no correlation with whole antigen detection. CL22 transfected dendritic cells could simultaneously present a viral antigen (EBNA1) to CD8 and CD4 T-cells, which had not previously been demonstrated. Using RNA transfection, it was demonstrated that early after transfection cells are whole Melan-A positive yet negative for Class I epitopes and with time Melan-A antigen levels fall whilst Class I epitopes are generated. Loss of whole antigen expression seemed related to lysosomal function and, unlike the viral antigen EBNA1, Class I presentation from Melan-A was lysosome-dependent. For Class II presentation of EBNA1, cellular localisation seems to determine access to the Class II pathway although this depends on the time-scale over which epitope presentation is assessed.

Nanotechnology has revolutionised humanity's capability in building microscopic systems by manipulating materials on a molecular and atomic scale. Nan-osystems are becoming increasingly smaller and more complex from the chemical perspective which increases the demand for microscopic characterisation techniques. Among others, transmission electron microscopy (TEM) is an indispensable tool that is increasingly used to study the structures of nanosystems down to the molecular and atomic scale. However, despite the effectivity of this tool, it can only provide 2-dimensional projection (shadow) images of the 3D structure, leaving the 3-dimensional information hidden which can lead to incomplete or erroneous characterization. One very promising inspection method is Electron Tomography (ET), which is rapidly becoming an important tool to explore the 3D nano-world. ET provides (sub-)nanometer resolution in all three dimensions of the sample under investigation. However, the fidelity of the ET tomogram that is achieved by current ET reconstruction procedures remains a major challenge. This thesis addresses the assessment and advancement of electron tomographic methods to enable high-fidelity three-dimensional investigations. A quality assessment investigation was conducted to provide a quality quantitative analysis of the main established ET reconstruction algorithms and to study the influence of the experimental conditions on the quality of the reconstructed ET tomogram. Regular shaped nanoparticles were used as a ground-truth for this study. It is concluded that the fidelity of the post-reconstruction quantitative analysis and segmentation is limited, mainly by the fidelity of the reconstructed ET tomogram. This motivates the development of an improved tomographic reconstruction process. In this thesis, a novel ET method was proposed, named dictionary learning electron tomography (DLET). DLET is based on the recent mathematical theorem of compressed sensing (CS) which employs the sparsity of ET tomograms to enable accurate reconstruction from undersampled (S)TEM tilt series. DLET learns the sparsifying transform (dictionary) in an adaptive way and reconstructs the tomogram simultaneously from highly undersampled tilt series. In this method, the sparsity is applied on overlapping image patches favouring local structures. Furthermore, the dictionary is adapted to the specific tomogram instance, thereby favouring better sparsity and consequently higher quality reconstructions. The reconstruction algorithm is based on an alternating procedure that learns the sparsifying dictionary and employs it to remove artifacts and noise in one step, and then restores the tomogram data in the other step. Simulation and real ET experiments of several morphologies are performed with a variety of setups. Reconstruction results validate its efficiency in both noiseless and noisy cases and show that it yields an improved reconstruction quality with fast convergence. The proposed method enables the recovery of high-fidelity information without the need to worry about what sparsifying transform to select or whether the images used strictly follow the pre-conditions of a certain transform (e.g. strictly piecewise constant for Total Variation minimisation). This can also avoid artifacts that can be introduced by specific sparsifying transforms (e.g. the staircase artifacts the may result when using Total Variation minimisation). Moreover, this thesis shows how reliable elementally sensitive tomography using EELS is possible with the aid of both appropriate use of Dual electron energy loss spectroscopy (DualEELS) and the DLET compressed sensing algorithm to make the best use of the limited data volume and signal to noise inherent in core-loss electron energy loss spectroscopy (EELS) from nanoparticles of an industrially important material. Taken together, the results presented in this thesis demonstrates how high-fidelity ET reconstructions can be achieved using a compressed sensing approach.

The physicochemical nature of the infectious agent in prion diseases creates a significant challenge for decontamination services. It has been shown to be both resistant to standard methods of decontamination, used to inactivate viruses and bacteria, and to associate avidly with surgical stainless steel. Moreover, the pathophysiology of the variant, iatrogenic and sporadic forms of Creutzfeldt-Jakob Disease (CJD) suggests deposition of the infectious agent across a wide range of extraneural, lymphoid tissues, as well as in the skeletal muscle and blood. Coupled with the potential for asymptomatic carriers, there is a significant risk of iatrogenic transmission of CJD through both neurosurgical procedures and standard surgery. This PhD study was undertaken in order to improve methods of instrument decontamination and to evaluate prion detection techniques and their applicability for the assessment of prion inactivation and removal. The project has provided relevant, critical assessment of hospital decontamination procedures, in addition to guidance on how working protocols should be improved to provide a cleaner and safer end product for the patient. Moreover, laboratory studies have been performed to evaluate current methods of prion decontamination in the context of hospital procedures for instrument reprocessing. Challenges faced by sterile service departments, such as soil drying and surface degradation, have been addressed and their impact on the risk of iatrogenic transmission of prions has been investigated. Critically, the use of a fluorescent amyloid fluorophore for the detection of prionassociated amyloid as a marker for disease permitted the investigation of the role of amyloid in infectious disease under denaturing conditions. Correlation of this detection technique with the identification of PrPres by Western blot and infectious disease suggested that, whilst fluorescent detection of prion-associated amyloid was more sensitive than Western blot, PrPres detection was more specific relative to infectivity. Improved fluorophores, with greater sensitivity, have been evaluated which will enhance in situ detection of prions in the future.