Histidine-rich glycoprotein (HRG) is a plasma protein that regulates key cardiovascular processes such as coagulation, angiogenesis and immune response. The protein consists of six distinct functional domains: two N-terminal domains (N1 and N2), two proline-rich regions (PRR1 and PRR2), a central histidine-rich region (HRR) and a C-terminal domain. The HRR binds Zn²⁺, which alters the affinity of HRG towards various ligands including the anticoagulant, heparin. A key aim of this study was to structurally characterise HRG. The 1.93 Å crystal structure of the HRG N2 domain presented here represents the first crystallographic snapshot of the molecule. The N2 domain is cystatin-like and N-glycosylated at Asn184. An S-glutathionyl adduct was observed at Cys185, providing in vivo evidence that release of an anti-angiogenic HRR/PRR fragment is controlled in part by a redox mechanism, representing a novel further role for GSH in regulation of angiogenesis. Since Zn²⁺ regulates some of the functions of HRG, the dynamics of Zn²⁺ in plasma were investigated using a combination of ITC, ELISA and thrombin assay systems. Zn²⁺ is normally associated with albumin in circulation, but its ability to bind Zn²⁺ is allosterically inhibited upon fatty acids binding to albumin. Elevated plasma fatty acid levels are associated with some disease states. It is proposed that this may alter the proportion of Zn²⁺ bound to HRG, which could in turn activate thrombin to promote coagulation. These studies provide evidence to suggest that Zn²⁺-dependent activation of HRG (following fatty acid binding to albumin) may play a role in the development of haemostatic complications in susceptible individuals. Finally, the Zn²⁺ binding ability of albumin was probed in order to locate unidentified sites using recombinant albumin mutants. H9A, H67A, E252A, D256A and H288A mutants all exhibited diminished Zn²⁺ binding ability, indicating that these residues are involved directly or indirectly in Zn²⁺ binding.

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In regions where endemic measles virus circulation has been interrupted, laboratory confirmation of measles is like puzzle solving. The complexity of these puzzles depends on the available pieces of clinical, epidemiological and laboratory information. The main goal of this dissertation is to evaluate diagnostic laboratory tools to aid in suspected case confirmation in these settings. First, protocols to elute measles IgM and IgG antibodies from blood spots dried onto filter paper were compared to propose one that will permit the recovery of the maximum volume of eluted sample in the minimum time, effort and cost. An easy-to-implement protocol is proposed for the rapid extraction of serum for measles/rubella serology in outbreak situations for use in the World Health Organization Global Measles and Rubella Laboratory Network. Second, due to inherent limitations of measles specific IgM enzyme immunoassays and molecular methods used for measles confirmation, not all suspected cases can be resolved. For example, IgM and RNA may not be detected in vaccinated cases with waning immunity (secondary vaccine failures) and presenting with modified measles. The observation is made that serological parameters of elevated titers of high avidity neutralizing antibodies correlate with measles secondary vaccines failures and may be useful biomarkers for confirming secondary vaccine failures that cannot be confirmed otherwise. Third, a highly accurate measles IgG avidity enzyme immunoassay for vaccine failure classification is described. Detection of low avidity antibodies using this highly sensitive and specific avidity assay can complement existing measles diagnostic tools in confirming suspected cases when routine IgM testing may be inconclusive. Therefore, these diagnostic approaches can provide additional laboratory information to resolve suspected cases irrespective of vaccination status. Together, data presented in this dissertation may assist in enhancing measles control and surveillance in elimination settings. / En les regions on s'ha interromput la circulació del virus del xarampió endèmic, la confirmació del xarampió a nivell de laboratori és com resoldre trencaclosques. La complexitat d'aquests trencaclosques depèn de les peces disponibles d'informació clínica, epidemiològica i de laboratori. L'objectiu principal d'aquesta tesi doctoral és avaluar eines de diagnòstic de laboratori per a ajudar en la confirmació de casos sospitosos en regions on el xarampió està eliminat. En primer lloc, es van comparar protocols per a eluir les IgM i IgG anti-xarampionoses de mostres de taques de sang seca sobre paper de filtre i proposar un protocol que permetés la recuperació del volum màxim de mostra eluïda en el mínim temps, esforç i cost. Es proposa un protocol de fàcil implementació dins de la xarxa de laboratoris de rubèola i xarampió de l'Organització Mundial de la Salut per a ser usat en situacions de brot. En segon lloc, les IgM anti-xarampionoses i l’ARN del virus del xarampió poden no ser detectats en casos vacunats amb disminució de la immunitat (fallada vacunal secundària) i amb simptomatologia de xarampió modificada. L'observació de que paràmetres serològics de títols elevats d'anticossos anti-xarampionosos neutralizants i d'alta avidesa correlacionen amb fallades vacunals secundàries permet proposar aquests paràmetres com a biomarcadors útils per a confirmar aquestes fallades vacunals quan d'altra manera no podrien confirmar-se. En tercer lloc, es descriu un assaig immumoenzimàtic per a determinar l'avidesa de les IgG contra el virus del xarampió per a la classificació de fallades vacunals. Aquest assaig és altament sensible, específic i precís. La detecció d'anticossos de baixa avidesa mitjançant aquest assaig pot a més complementar les eines actuals de diagnòstic de xarampió en la confirmació de casos sospitosos quan les proves rutinàries de IgM no són concloents. Per tant, aquestes estratègies diagnòstiques poden proporcionar informació de laboratori addicionals per a resoldre casos sospitosos de xarampió independentment del seu estat de vacunació. Les dades presentades en aquesta tesi doctoral poden ajudar a millorar el control del xarampió i vigilància epidemiològica allà on el xarampió ja està eliminat.