Inhibition of nitric oxide (NO) signalling may contribute to the pathogenesis of severe malaria. This thesis examines the impact of Plasmodium infection on three key determinants of nitric oxide synthase (NOS) biochemistry: substrate availability, substrate/inhibitor homeostasis and cofactor availability. Arginine, the NOS substrate, is depleted in human patients with severe Plasmodium falciparum malaria and mice infected with P. berghei ANKA. Using heavy isotope tracer infusions to quantify arginine metabolism in infected mice, we found no evidence of increased catabolism by the enzyme arginase, widely assumed to be responsible for arginine depletion. Genetic knock-out of parasite arginase had no effect on arginine depletion in mice. Instead, our findings link arginine depletion to decreased rates of arginine and citrulline appearance in the plasma of infected mice. Asymmetric dimethylarginine (ADMA) competes with arginine for binding to the NOS catalytic site. We observed elevation of the ADMA/arginine ratio in Gambian children with severe malaria, favouring NOS inhibition. In mice infected with P. berghei ANKA, we found evidence of degradation of dimethylarginine dimethylaminohydrolase 1 (DDAH1), the enzyme primarily responsible for ADMA metabolism. We also observed reduced DDAH activity and accumulation of intracellular ADMA in hepatic tissue of infected mice, suggesting that DDAH dysfunction could contribute to disruption of ADMA/arginine homeostasis. Tetrahydrobiopterin (BH4) is an essential NOS cofactor. In P. berghei ANKA-infected mice, BH4 concentrations were decreased in plasma, erythrocytes and brain tissue, which could inhibit NO synthesis and promote NOS-dependent superoxide production. To reverse deficiencies of NOS substrate and cofactor availability, we infused P. berghei ANKA-infected mice with citrulline, an arginine precursor, and sepiapterin, a BH4 precursor. Restoration of systemic arginine and BH4 availability in infected mice improved whole blood nitrite concentrations, a biomarker of NO synthesis, but did not prevent onset of disease symptoms. These studies have identified biochemical disturbances that may contribute to severe malaria pathogenesis by inhibiting NO synthesis.

Mandatory notification of disease forms the backbone of disease surveillance in New Zealand and overseas. Notification data is used by public health professionals and academics to identify cases requiring public health control, monitor disease incidence and distribution, and in epidemiological research. However, there is emerging evidence that notification rates do not accurately reflect the true extent of notifiable diseases within the community, resulting in the underascertainment of many notifiable cases. While adequate surveillance does not necessarily require that all cases of notifiable disease be captured, the systematic underascertainment of disease can have significant implications for perceived spatial and demographic trends in disease prevalence; potentially threatening the credibility of spatial epidemiological research by under or overestimating the burden of disease in different populations. There is evidence that systematic underascertainment occurs as a result of the differential actions of laboratories and general practitioners. It has also been recognised that that underascertainment can be influenced by a patient's willingness to seek medical attention and participate in laboratory tests. However, few studies have investigated whether these factors systematically influence notification either in New Zealand or overseas. Furthermore, the discipline of health geography has been slow to engage with this topic of public health importance, despite the inherently spatial nature of the processes involved, and the close ties to the geographic literature on health service utilization and healthcare provision. This thesis explores the spatial and temporal variation in notification rates in New Zealand for the period 1997-2005 and the potential relationships between notification rates and different variables. Unlike many underascertainment studies, which have used individual data and capture-recapture methods, data constraints inspired a unique ecological approach to investigating the factors which may be associated with notification in New Zealand. Variables were divided into categories based on Anderson's behavioural model for healthcare utilization and the influence of these variables on notification was determined through multiple regression analyses. The main findings of this research indicate that in New Zealand notification rates have increased during the period 1997-2005 and that there is a north-south gradient in notifications, with substantially lower rates in the North Island than in the South Island. Furthermore, it is also evident that the variables associated with notification vary according to disease, spatial aggregation and spatial scale. Notification rates are significantly associated with a range of predisposing and enabling factors which might influence patient choice to consult for many frequently underascertained diseases. More variation in enteric diseases is explained by the independent variables analysed than the variation in non-enteric diseases. However, further research into these relationships, and underascertainment in general, is required before firm conclusions can be drawn.

Meningococcal disease, caused by the bacterium Neisseria meningitidis, is an important cause of morbidity and mortality in young children and adolescents worldwide. There are 12 serogroups with most disease due to meningococci expressing one of five capsular polysaccharide antigens corresponding to serogroups A, B, C, Y and W135. In Europe, the majority of disease-causing strains are of serogroups B and C. No comprehensive vaccine is available against the bacterium due to the difficulty in producing serogroup B vaccines. A number of countries, e.g. UK and the Republic of Ireland have implemented routine meningococcal conjugate C (MCC) vaccine strategies. Due to the high proportion of disease accounted for by serogroup B in Europe and other developed countries, much research is currently being carried out to unearth vaccine candidates that would be protective and give as wide coverage as possible. Such candidates include the antigens PorA, FetA and factor H-binding protein. Potential drawbacks with antigens such as these which are under immune selection are high degrees of variability and lack of cross-immunity. Determination of the distribution, both geographically and temporally, of antigens and their association with clonal complex can aid in the formulation of novel vaccines and assess their potential coverage across Europe. Serological typing schemes involving characterisation of the polysaccharide capsule (serogroup) and outer membrane proteins such as PorA (serosubtype) and PorB (serotype) have been used for a number of years with some success. However, drawbacks associated with these methods include insufficient discrimination, limitations in panels of monoclonal antibodies used in the typing procedures and difficulty in comparison of results among labs. Consequently, in recent years genotypic methods such as multi-locus enzyme electrophoresis (MLEE) and subsequently multi-locus sequence typing (MLST) have been developed. These methods measure the variation in slowly evolving housekeeping genes whereas serological methods measure variation in antigens which are under immune pressure and are therefore more diverse. Combination of phenotypic and genotypic typing methods can offer high levels of discrimination. Molecular studies into meningococcal diversity have offered many important insights into its population biology, which have implications for prevention and control of meningococcal disease. These have included the identification of hyperinvasive lineages and the correlation of genetic type with antigenic type and disease epidemiology. The EU-MenNet programme was established as a pan-European infrastructure for the research and surveillance of European meningococcal disease. Its aim was to coordinate and disseminate the latest molecular isolate characterisation techniques (MLST) and electronic data transfer via the Internet to exploit epidemiological and population genetic studies. Within the EU-MenNet, the European Meningococcal MLST Centre (EMMC) was set up to carry out molecular typing — MLST, PorA and FetA — of European disease isolates from 18 countries over three years 2000, 2001 and 2002. The output of this project will be the largest representative molecular epidemiological study of meningococcal disease in Europe. Assessment of the data produced will give insights into the geographic and temporal distribution and structuring of disease-associated clonal complexes and antigens and their associations. This will give an indication of the meningococcal disease population in Europe and will be invaluable for the current, and ongoing, development and introduction of new meningococcal vaccines.

Cryptococcus neoformans is an encapsulated yeast responsible for severe meningoencephalitis. The importance of epidemiological studies on cryptococcosis has increased since the beginning of the AIDS epidemic. C. neoformans exists in two varieties containing four serotypes, C. neoformans var. neoformans (serotypes A and D) and C. neoformans var. gattii (serotypes B and C). Locally C. neoformans var. neoformans has been associated with pigeon feces during those months having an average temperature of 64.2°F j(17.8°C) and above. Clinical and environmental isolates of C. neoformans obtained from regional hospitals and environmental samplings, respectively, have been grouped into their variety status utilizing canavanine-glycine-bromthymol blue agar. Polyclonal antisera against C. neoformans serotypes A, B, C and D were isolated from challenged rabbits. Serotyping C. neofromans isolates using the polyclonal antisera resulted in 57% (20 of 35) of the serotypes confirmed with a direct immunofluorescent assay utilizing a single monoclonal antibody (E1). Data from the immunofluorescence assay suggest all C. neoformans obtained from regional hospitals (26 of 26) and those isolated from the environment (9 of 9) belong to the A serotype group. These data have provided information leading to the origin of infection for cryptococcosis in our region, which may be beneficial to immunocompromised individuals.

Scrub typhus is a potentially fatal infection that threatens one billion persons in the Asia-Pacific region and is caused by the obligate intracellular bacterium, Orientia tsutsugamushi. How this organism facilitates its intracellular survival and pathogenesis is poorly understood. Intracellular bacterial pathogens utilize the Type 1 (T1SS) or Type 4 secretion system (T4SS) to translocate ankyrin repeat-containing proteins (Anks) into the host cell to modulate host cell processes. The O. tsutsugamushi genome encodes one of the largest known bacterial Ank libraries as well as Type 1 and Type 4 secretion systems (T1SS and T4SS), which are expressed during infection. In silico analyses of the Anks’ C-termini revealed that they possess characteristics of T1SS secretion signals. Escherichia coli expressing a functional T1SS was able to secrete chimeric hemolysin proteins bearing the C-termini of 19 of 20 O. tsutsugamushi Anks. In addition to infecting endothelial cells, O. tsutsugamushi infects professional phagocytes. To better understand why these innate immune cells are unable to eliminate O. tsutsugamushi, we addressed the activity of host NF-κB proinflammatory transcription factor. Screening of O. tsutsugamushi infected cells at an MOI of 1 revealed inhibition of NF-κB nuclear accumulation as early as 8 hours in HeLa and bone-marrow derived macrophage cells. When stimulating infected cells with TNF-α, IκBα degradation still occurs, however NF-κB dependent gene transcription remains downregulated. Immunofluorescence microscopic analysis of TNF-α treated cells ectopically expressing all O. tsutsugamushi Anks revealed that two nuclear trafficking Anks, Ank1 and Ank6, result in a significant decrease in NF-κB nuclear accumulation. Additionally, these Anks also significantly inhibited NF-κB dependent gene transcription. Co-immunoprecipitation experiments revealed that both Anks interact with importin-β1, exportin-1, and the p65 NF-κB subunit. Treating cells with importazole significantly reduces the nuclear accumulation of Ank1 and Ank6. Finally, treating infected cells or cells ectopically expressing Ank1 or Ank6 with leptomycin B resulted in restoration of NF-κB nuclear accumulation. With these data, we propose that O. tsutsugamushi secretes Ank1 and Ank6 to initially interact with importin-β1, which permits their nuclear entry where they then interact with NF-κB and subsequently exportin-1 to prevent NF-κB nuclear accumulation.

Background Indigenous peoples with traditional lifestyles worldwide are considered particularly vulnerable to climate change effects. Large climate change impacts on the spread of infectious vector-borne diseases are expected as a health outcome. The most rapid climate changes are occurring in the Arctic regions, and as a part of this region northernmost Sweden might experience early effects. In this thesis, climate change effects on the lives of Sami reindeer herders are described and 30 years of weather changes are quantified. Epidemiology of the climate sensitive human infection tularemia is assessed, baseline serologic prevalence of tularemia is investigated and the disease burden is quantified across inhabitants in the region. Methods Perceptions and experiences of climate change effects among the indigenous Sami reindeer herders of northern Sweden were investigated through qualitative analyses of fourteen interviews. The results were then combined with instrumental weather data from ten meteorological stations in a mixed-methods design to further illustrate climate change effects in this region. In two following studies, tularemia ecology and epidemiology were investigated. A total of 4,792 reported cases of tularemia between 1984 and 2012 were analysed and correlated to ecological regions and presence of inland water using geographical mapping. The status of tularemia in the Swedish Arctic region was further investigated through risk factor analyses of a 2012 regional outbreak and a cross-sectional serological survey to estimate the burden of disease including unreported cases. Results The reindeer herders described how the winters of northern Sweden have changed since the 1970s – warmer winters with shorter snow season and cold periods, and earlier spring. The adverse effects on the reindeer herders through the obstruction of their work, the stress induced and the threat to their lifestyle was demonstrated, forcing the reindeer herders towards the limit of resilience. Weather data supported the observations of winter changes; some stations displayed a more than two full months shorter snow cover season and winter temperatures increased significantly, most pronounced in the lowest temperatures. During the same time period a near tenfold increase in national incidence of tularemia was observed in Sweden (from 0.26 to 2.47/100,000 p<0.001) with a clear overrepresentation of cases in the north versus the south (4.52 vs. 0.56/100,000 p<0.001). The incidence was positively correlated with the presence of inland water (p<0.001) and higher than expected in the alpine and boreal ecologic regions (p<0.001). In the outbreak investigation a dose-response relationship to water was identified; distance from residence to water – less than 100 m, mOR 2.86 (95% CI 1.79–4.57) and 100 to 500 m, mOR 1.63 (95% CI 1.08–2.46). The prevalence of tularemia antibodies in the two northernmost counties was 2.9% corresponding to a 16 times higher number of cases than reported indicating that the reported numbers represent only a minute fraction of the true tularemia. Conclusions The extensive winter changes pose a threat to reindeer herding in this region. Tularemia is increasing in Sweden, it has a strong correlation to water and northern ecoregions, and unreported tularemia cases are quite common.

In Western Europe the incidence of tick-borne encephalitis (TBE) has increased over the last 30 years, coupled with changes in distribution. Modifications in the TBE enzootic cycle, through a combination of changes in temperature, vertebrate abundance and habitat suitability may have increased the risk of TBE in recent years. In Switzerland, analysis using satellite-derived climate data demonstrated that the environment of areas with TBE since the 1980s and areas that recently became endemic for TBE have become more similar between 2001 and 2009. This was coupled with an increase in April, May and June temperature, which could have affected the tick population and/or human exposure to ticks. Deer and boar abundance also changed in some cantons. In Germany, spatio-temporal modelling demonstrated the importance of temperature, vertebrate abundance and unemployment in the incidence and distribution of TBE between 2001 and 2009. Changes in TBE reporting, April, May and June temperature, vertebrate abundance and pesticide use may have contributed to increases in TBE in 1992 and 2001. Human exposure patterns, however, appear to be as important as the enzootic cycle in shaping the incidence of TBE, not only in determining the overall trend but also in interacting with the weekly, seasonal and yearly patterns of tick hazard to give the observed incidence. In Switzerland, in weeks with warm, sunny weather, human exposure to ticks is promoted and short-term increases in tick bites are seen. Human outdoor activity also shifts the seasonal pattern of tick bites, when compared with tick questing. There was no apparent increase in time spent in outdoor activities between the 1990s and 2000s in Italy, Germany and Austria, but survey data demonstrated that walking and hiking were already popular activities across Europe by the 1990s. The popularity of mushroom and berry foraging as a source of income in Latvia, Lithuania and Poland, coupled with the expense of vaccination, provide an inverse link between economic wellbeing and TBE risk. Correspondingly, in 2009, the economic recession was associated with an increase with TBE in these three countries.

Axonal domains are required for proper neuron function. These domains are unstable and degenerate concurrent with the inflammation in multiple sclerosis (MS) and the inflammatory disease models experimental autoimmune encephalomyelitis (EAE) and lipopolysaccharide (LPS) induced inflammation. Previous studies from our laboratory have shown that the axon initial segment (AIS) is maintained independently of the presence of myelin, but that AIS disruption is seen in MS as well as EAE and LPS-mediated inflammation. AIS loss can be interrupted in the early stage of EAE using the anti-inflammatory drug Didox. However, the potential for Didox directed repair of the AIS in later stages of disease has not been investigated. Here, we utilize two models of CNS inflammation to assess the possibility of reversing AIS pathology. Based on our findings, we present the first evidence that AIS degeneration, an axonal pathology observed in MS and in chronic inflammation, is reversible.

This study using a quantitative, descriptive design with a questionnaire investigated cultural, religious and social factors that might impact on ARV treatment in HIV/AIDS-infected women in Botswana. The study found that the majority never missed any doses, a few missed doses once or twice, and a small minority missed more than three times. The respondents’ perception of cultural influence on treatment of HIV/AIDS in women revealed that the majority (70%) believe culture has an influence on the treatment. Social factors also impacted on ARV adherence. A few of the respondents indicated that side effects and the number of pills prevented ARV medication adherence. The main reason for non-adherence, however, was forgetfulness. There had been an improvement in the majority of the respondents’ health status and quality of life. Maximizing adherence is essential. Providers and patients both have responsibilities in this regard. / Health Studies / M.A.(Health Studies)

Toll-like receptors (TLRs) are a major group of pattern recognition receptors expressed on the surface of immune cells that recognize molecular patterns associated with all classes of pathogenic microorganisms. TLR4 recognizes the lipopolysaccharide component of Gram-negative bacterial cell walls and is the only TLR known to induce signaling through both the MyD88 and TRIF pathways. Basigin, a ubiquitous cell adhesion molecule, is a member of the immunoglobulin superfamily that has the ability to influence cell signaling mediated by the MyD88 and TRIF pathways, the same signaling pathways induced by the TLR4 receptor protein. Analysis of the Basigin protein sequence indicates the presence of a hydrophilic glutamate residue within the hydrophobic transmembrane domain, but no consensus binding sites for MyD88 or TRIF. The purpose of this study was to determine if Basigin uses TLR4 for signal transduction. It is hypothesized that Basigin interacts with TLR4 and that the glutamate residue plays a role in the interaction. Enzyme-linked immunosorbent binding assays were performed using endogenous TLR4 and recombinant Basigin proteins. These analyses demonstrated that binding of Basigin to TLR4 was significantly greater than that of the control protein and that the glutamate residue in the Basigin transmembrane domain does play a role in the interaction between Basigin and TLR4 as well as many hydrophobic residues in the Basigin transmembrane domain. The data suggest that Basigin interacts with TLR4 to influence signaling cascades using MyD88 and TRIF.