The nucleocapsid of Hepatitis B Virus is an icosahedral structure composed of 180 subunits of the viral Core Antigen (HBcAg). This protein is 183 amino acids in size and has a highly arginine-rich carboxy-terminal region. It can be expressed at high levels in <i>E.coli</i>, in which it forms nucleocapsid-like core particles which are morphologically indistinguishable from nucleocapsids isolated from infected individuals. The latter property was utilised in this work, the aim of which was to investigate the role of particular amino acids of HBcAg in the determination of the structure of the protein monomer and that of the core particle. Previous work (Stahl <i>et al</i>, 1982) had suggested the importance of the amino-terminal region of the protein in the determination of its structure. This was investigated further in this work by the use of site-directed mutagenesis to create a series of deletion and substitution mutations within this region. These were expressed in <i>E.coli</i> and the importance of amino acid 3 was demonstrated by immunological assay. HBcAg contains four cysteines, all of which are completely conserved among mammalian hepadnaviruses. The role of these cysteines in HBcAg and core particle structure determination and stability was investigated by the use of site-directed mutagenesis to create a series of mutants in which cysteine codons were replaced by serine codons in several combinations, and in the context of both the full-length protein and of a truncated protein lacking the arginine-rich carboxy-terminal region. These proteins were produced in <i>E.coli</i> and formed particles indistinguishable from wild-type particles in structure, even when no cysteines were present. Their behaviour during non-reducing SDS-polyacrylamide gel electrophoresis indicated the presence of different disulphide bond complements in different mutants and allowed a model to be deduced for the arrangement of disulphide bonds within the core particle.

Measuren1ent of disease activity in rheun1atoid arthritis (RA) has been standardised by the developlnent of core sets of variables, constructed by professional experts in rheumatology. However, there is evidence that priorities for treatment of RA differ between health professionals and patients. This thesis describes the exploration of the ilnportant outcOlnes of 'feeling well' and 'n1aintaining or returning to a nonnallife', the developlnent of a patient-generated core set of patients' priority outcon1es, and the relationship between theln. The literature review suggests that the patient perspective is not represented in the existing core sets of outcomes (Chapter 1). The broader literature suggests that contextual factors and individuals' adaptation to chronic illness may affect patients' prioritisation of treatment outcOlnes and their perceptions of well-being (Chapters 2 and 3). A Inixed methods, pragmatic Grounded Theory approach was taken to addressing these issues. Three methods were employed iteratively: in-depth interviews, nominal/focus groups and a postal survey (Chapter 4). The interviews elicited important treatment outcomes and produced thematic frameworks of well-being, and normality in RA, and a model of their interaction (Chapter 5). These informed the nominal/focus groups, which ranked the outcomes, provided an understanding of the factors influencing prioritisation and constructed numerical rating scales (NRS) to measure the din1ensions of well-being (Chapter 6) for use in the survey. The survey produced the patient core set of the eight most important patient outcomes: pain, activities of daily living, joint dan1age, mobility, life enjoyment, independence, fatigue and valued activities. The survey also provided evidence of the accuracy of the n10del derived from the interview data where global well-being was determine by physical, psychological, adaptational and social variables (Chapter 7). The findings have implications for theory, policy, and research methods. First, evidence is given for a greater emphasis on the impact of continued physical impact in theoretical models. Second, criteria for eligibility for anti-TN F therapy is questioned and psychological assessn1ent is advised. Third, the integration of qualitative and quantitative methods is shown to be successful, but has highlighted methodological challenges (Chapter 8). Future work includes testing the proposed patient-generated core set, in comparison to the existing professionally developed core sets.

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Infection by vaccinia virus leads to reorganization of the actin cytoskeleton, changes in cell adhesion, loss of contact inhibition and stimulation of cell motility. Previous work by the group has led to the identification of a viral gene, F11L, which is highly conserved among the orthopoxvirus genomes and is essential for this virus-induced cell mobility. F11L has been shown to interact with the RhoA but does not interact with the two other 'classical’ members of the family Rac1 and Cdc42. Understanding the molecular interaction of F11L and RhoA as well as investigating additional Rho binding partners was the basis of my thesis project with a view to obtain additional insights into the role of Rho GTPase manipulation during the vaccinia life cycle. Deletion of F11L from the virus genome results in reduced virus release and smaller plaque sizes. Characterization of the interaction between F11L and RhoA revealed that it occurs via a region of limited sequence homology to the RhoA effector ROCKI, in the C-terminal part of the molecule. Interestingly, generation of a recombinant mutant virus (F11L-VK) that is unable to bind RhoA leads to an intermediate phenotype between ΔF11L and WR in terms of viral plaque size. Further bioinformatic analysis revealed a region in the N-terminus of F11L with limited sequence homology to hDia2C, a RhoD effector. Biochemical analysis led to the identification of this site as the RhoD binding domain in F11L. Importantly, this study revealed that F11L interacts directly with different Rho GTPases via unique motifs to induce cell motility and facilitate enhanced virus cell-to-cell spread. Furthermore, work done in collaboration with the laboratory of Mariano Esteban in Madrid using a mouse model of infection shows that in contrast to wild-type vaccinia, infection with ΔF11L does not result in animal death. Additionally, the F11L-VK virus shows limited spread in vivo but still induces animal death albeit at a later stage when compared to wild-type vaccinia. These results suggest that F11L has additional functions besides interacting with RhoA, consistent with my observations that it can interact with other Rho GTPases. These studies can provide valuable information about the importance of Rho GTPase manipulation for the life cycle of vaccinia virus but also indicate the potential for the genetic manipulation of F11L in existing poxvirus vectors that may improve their therapeutic potential, safety, immunogenicity and/or oncolytic activity.

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