Candida albicans is a major fungal pathogen causing life threatening systemic infections in immunocompromised humans. While in the host C. albicans is exposed to a range of stresses during phagocytosis by host innate immune cells, including reactive oxygen species (ROS), cationic fluxes, and fluctuations in pH. The ability of C. albicans to adapt to such stresses is essential for survival and pathogenesis. Despite this, however, there is still much to be learnt regarding the stress responsive mechanisms mounted by this major pathogen. Hence, the overarching goal of this project was to provide novel insight into the cellular processes necessary to enable stress adaptation and virulence of C. albicans. To facilitate this, quantitative fitness analysis (QFA) of two C. albicans deletion libraries was performed using inducers of superoxide, cationic, and alkaline pH stresses. GO term analysis of sensitive genes highlighted distinct and overlapping biological processes, molecular functions, and cellular components enriched during adaptation to each stress. Notably, the importance of ion binding for resistance to cationic and superoxide stress was revealed, whereas cell wall biogenesis was enriched for alkaline pH stress. QFA also identified several regulatory genes not previously implicated in stress responses, including the Pho4 transcription factor. Cells lacking PHO4 were acutely sensitive to all three stresses tested and thus the role of Pho4 in mediating stress resistance was investigated further. Additional phenotypic testing revealed pho4Δ cells display impaired resistance to several organic and metal cations, and defects in morphogenic switching. Similar to Pho4 function in S. cerevisiae, deleting PHO4 in C. albicans completely abolished acquisition and accumulation of phosphate stored as polyphosphate (polyP) in the vacuole. Consistent with stress resistance and nutrient acquisition being important virulence determinants in C. albicans, cells lacking PHO4 were acutely sensitive to macrophage-mediated killing, and displayed attenuated virulence in Caenorhabditis elegans and murine models of infection. Further analysis of the role and regulation of Pho4 in stress adaptation in C. albicans revealed that in addition to the essential role of Pho4 in phosphate acquisition and storage, which enables survival in phosphate limiting and alkaline pH conditions, Pho4 function is also important for metal ion homeostasis which is essential for cationic and superoxide stress resistance. vi As C. albicans only causes systemic infections in immunocompromised hosts, the final objective of this study was to explore whether the immune status of the host dictated the importance of key stress regulators in promoting the virulence of this fungal pathogen. Although the Hog1 stress activated protein kinase and the Pho4 transcription factor were demonstrated to be essential for C. albicans virulence in the model mini host C. elegans, both were dispensable for virulence upon infection of immunocompromised worms. These findings infer that robust stress responses of C. albicans may only be required for virulence when immune responses are evoked in an immunocompetent host. Taken together, the data presented in this thesis highlight that metabolic adaptation is essential for the survival of C. albicans to host-imposed stresses, and that the immune status of the host may govern the importance of stress protective mechanisms in mediating the virulence of this major fungal pathogen.

No description available.

Northern Pakistan, Khyber Pakhtunkhwa (KP) is low malaria endemic area characterised by seasonal transmission with predominantly vivax malaria. Migration of high number of Afghan Refugees in 1978 into KP led to concerns for an increase in malaria, as the malaria incidence in this group was reportedly high compared to the local Pakistani population. Considerable progress has been made in controlling malaria through operational research in the camps where the Afghan refugees reside. However, this process requires effective, repeatable active surveillance tools for monitoring malaria control as availability of accurate data is the major challenge at present. The aim of this PhD project was to generate current information on malaria infection rates through parasite prevalence and malaria exposure using antimalarial antibody responses. The project also investigated the risk factors of malaria and heterogeneity in the geographic distribution of malaria in the camps by using GIS data with serological responses and parasite prevalence data. As an ancillary objective the project aimed to determine the prevalence of G6PO deficiency in the study population. A cross-sectional survey was conducted in five Afghan refugee camps of KP between June and September in 2010. Blood samples were obtained on filter paper from 2526 individuals and tested by rapid diagnostic test, paraSite species specific PCR and ElISA for antibody responses to Plasmodium vivax and Plasmodium falciparum. A questionnaire was administered to collect household and individual based information to determine the potential risk factors of malaria. Heterogeneity in malaria was observed between the studied camps based on seroprevalence, which ranged from 17%-45% for P. vivax and 3% to 11% for P. falciparum. Variation in P. vivax infection prevalence was also detected between the camps, which ranged from 0.4-9% (ROT) and 5-15% (peR). Variation in the distribution of malaria was also found within the camp using spatial/GIS data with clear foci of infection identified in 4 of 5 camps. The results showed that as expected parasite based prevalence measures (ROT and peR) are significantly lower than serological measure of exposure. P. falciparum infection prevalence (ROT and PCR) and seroprevalence was found to be extremely low with P. vivax infections predominant. Age seroprevalence changes were more pronounced for P. vivax than P. falciparum and seroconversion rate was strongly associated with parasite rate. Increasing age .and poorly built houses were associated with increasing risk, while staying in the same camp for the last 6 months and using measures to reducing vector biting such as repellents repellent, coils or insecticide spraying were associated with reduce risk of falciparum malaria. The risk of vivax malaria was observed to increase with increasing age, sharing house with cattle and having fever within 24 hours or two weeks and a reduction in the risk was seen in the individuals who reported use of Insecticide treated Bed Nets (ITN) night prior to surveyor used self protection measures from vector. The 563C-T polymorphism of G6PD gene was observed in only 2 unrelated individuals out of 505 individuals tested (O.4%). In conclusion, both parasitological and serological measures were able to detect spatial variation in infection and exposure to malaria at the micro epidemiological level within the camp. This data will help to provide beneficial and up-to-date information to manage control activities in the study area.

The majority of reports of imported malaria in Europe come from the UK, France, Germany and Italy. Most of those affected are of African origin visiting friends and relatives (VFRs). There is little understanding of the factors that enhance and constrain effective malaria prevention practices in this group. Two studies were undertaken to investigate this. An epidemiological analysis of 13813 reports made in the UK between 2001 and 2008 showed that 58% lived in London, where 83% of infections were caused by Plasmodiumfalciparum. The rate per 100000 offalciparum infections in the capital in 2008 in those categorised as of "Black African" ethnicity was 131.0 (CI: 120.0-142.1), compared to 0.3 (CI: 0.2-0.5) amongst the "White British". To investigate factors influencing the behaviours of VFRs, semi-structured interviews were undertaken with African VFRs resident in the UK visiting Nigeria and Ghana (n = 20), malaria patients (n= 6), practice nurses (n= 10), GPs (n = 10), community pharmacists (n= 7) and hospital consultants (n= 3). Factors influencing the use of mosquito avoidance methods included knowledge about the local environment, a perceived inevitability of contracting malaria, and a desire to use methods common amongst host families. Chemoprophylaxis use was influenced by perceptions of susceptibility, by previous experiences of malaria, perceptions of the seriousness of malaria, and peer pressure. Relevant structural factors included the cost of chemoprophylaxis and difficulties in accessing health advice. This research showed that in managing malaria, VFRs choose pragmatically between two parallel social and environmental contexts and the structural constraints associated with each. A novel conceptual framework shows the complex manner in which these interact, and may be appropriate in different countries and also in future research investigating other travel-related diseases in migrants. Practical recommendations for research priorities within the UK imported malaria context are made.

Insects pose large problems for health and agriculture; several major diseases are transmitted by insect vectors, and huge losses in food production occur due to insect pests. Current strategies for insect control are inadequate, with several disadvantages. Various novel approaches are being developed, aimed at either suppressing wild populations, or replacing them with insects refractory to disease-causing pathogens. Some possible strategies utilise Wolbachia, a genus of maternally inherited intracellular bacteria. Wolbachia can manipulate host reproduction through a phenomenon known as cytoplasmic incompatibility (CI), which leads to spread of Wolbachia through populations. CI could potentially be used as a gene drive system for spread of pathogen-resistance genes, or for population suppression via the release of incompatible males. Furthermore, some strains of Wolbachia have been found to inhibit virus transmission by mosquitoes; there have been successful field trials of population replacement exploiting this inhibition. At present, little is known about the mechanisms of CI, or viral inhibition by Wolbachia. Discussed in Chapter 2, candidate genes for CI involvement were identified by sequencing the genome of the non-CI strain wAu and comparing it to that of the CI strain wMel. One candidate was used to transform Drosophila melanogaster, but it did not induce a CI phenotype. Chapter 3 discusses further identification and investigation of candidate genes for CI involvement by comparing RNA-seq data from wAu and the CI strain wRi, and from D. simulans either uninfected or infected with one of these two strains. Similarly, candidate genes for viral inhibition were identified and investigated by comparing RNA-seq data from Aedes albopictus mosquitoes that were uninfected, naturally infected with wAlb, or transinfected with wMel, which inhibits dengue in this species. Other potential insect control strategies involve the release of modified insects with a genetic system that leads to population suppression. Discussed in Chapter 4, attempts were made using comparative genomics to identify the sex-determining locus in the mosquito Ae. aegypti, which could be utilised in genetic control systems. Some possible regions were identified, but the locus was discovered by another group before the work was completed. Chapter 4 also discusses the identification using RNA-seq of germline-specifically expressed or spliced genes in Ae. aegypti and the agricultural pest Ceratitis capitata; these could be used to control gene expression in various genetic insect control systems. Overall this thesis demonstrates the use of high-throughput sequencing analyses to identify and investigate candidate genes of importance to insect control. It will hopefully serve as a platform for further research in this important field.

Malaria control is heavily reliant on the use of insecticides for interventions such as Indoor Residual Spraying (IRS) and Long Lasting Insecticide Nets (LLINs). Widespread resistance to insecticides in the malaria vector Anopheles gambiae threatens these interventions. To ensure the continued effectiveness of malaria control strategies, novel insecticides and insecticide synergists must be formulated, and in order to do so, new insecticide targets must be investigated. Digestion of huge volumes of vertebrate blood in the midgut of An. gambiae mosquitoes results in the production of large concentrations of free haem: a potentially cytotoxic molecule. Amongst the adaptations haematophagous arthropods have for limiting haem mediated toxicity is haem degradation by the haem oxygenase system. Haem oxygenase, with its obligate redox partner, cytochrome P450 reductase, catalyses the hydrolysis of haem into biliverdin, producing carbon monoxide and ferrous iron. To investigate the biochemical properties of An. gambiae haem oxygenase (AgHO), the enzyme was cloned and expressed in E. coli. The purified AgHO was observed to catalyse the degradation of haem to biliverdin, releasing CO and Fe2+, in the presence of insect and human CPR reducing systems. AgHO bound haem stoichiometrically, with a KD closer to that of H. sapiens and C. diphtheriae than that of D. melanogaster. Application of the HO inhibitors Sn-protoporphyrin and Zn-protoporphyin to mosquitoes as a bloodmeal supplement revealed a dose-dependent reduction in egg laying capability of An. gambiae. It is unclear whether this is due to a detrimental effect on mosquito health because of haem mediated cytotoxicity, or if this reduction in reproductive fitness is due to a key role for AgHO in the reproductive system. A range of truncated CPRs from insects (An. gambiae, A.mellifera, C. quinquefasciatus, D. melanogaster, R. prolixus) and H. sapiens were also cloned and expressed. The six enzymes were biochemically characterised side-by-side using cytochrome c reduction assays in order to determine whether there are any differences in activity between those CPRs in blood-sucking insects and others. There were no clear trends in terms of enzyme kinetics which associated with a phenotype of haematophagy. The insect enzymes studied had higher Vmax values than H. sapiens with respect to both NADPH (1.6-7.8 fold higher) and cytochrome c (1.5-5.8 fold higher). Also compared were the enzymes' sensitivity to nucleotide analogue inhibitors and DPIC. HsCPR more sensitive to the nucleotide analogues than insect CPRs (by 2-30 fold). DPIC however was more inhibitory to insect CPRs by up to 20 times, giving it potential as an insecticide synergist. Again, there were no clear trends aligning inhibition profiles with a blood-feeding habit. In conclusion, AgHO has been found to be a true haem oxygenase, with biochemical characteristics in keeping with well characterised HOs such as H. sapiens HO-1 and C. diptheriae HmuO. AgHO inhibition appears to play a key role in oviposition and mosquito fecundity, and therefore may be a potential target for insecticides and insecticide synergists. Taken together, these results suggest that knowledge of these enzyme targets may aid the future development of novel insecticides.

No description available.

No description available.

No description available.

Malaria continues to cause immense public health pressure to poor and developing countries in the tropical world hindering economic development. Malaria control using long-lasting insecticide-treated bed nets (LLINS) and indoor residual spraying (IRS) have contributed significantly to the reduction of malaria transmission. However, this reduction in transmission is not uniform. Particular foci ‘hotspots’ that do not respond as anticipated to control tools have become common in low-moderate transmission settings like The Gambia. While several factors contribute to heterogeneity in malaria transmission, the roles that malaria vectors with differing behaviour, ecology and susceptibility to insecticides play in these hotspots is poorly understood. This thesis aimed to investigate whether vector-related factors might contribute to malaria transmission heterogeneity in The Gambia. First, I demonstrated how the three major malaria vectors; Anopheles gambiae s.s., Anopheles arabiensis and An. coluzzii varied in relative abundance between villages of varying transmission intensities. Susceptibility to deltamethrin and DDT varied significantly among species and locales with An. gambiae s.s. from the east of the country by far the most resistant. There was a correlation between An. gambiae s.s. phenotypic resistance to DDT and malaria transmission in 2013, driven by the east of the country which experiences higher malaria transmission. The major driver of insecticide resistance to DDT and deltamethrin was the target site mutation, Vgsc-1014F although another target site mutation Vgsc-1575Y and a metabolic gene variant Gste2-119T were also implicated, albeit to a far lesser extent. The frequency of Vgsc-1014F in An. gambiae s.s. remained low in a second sampling year in western populations but approached fixation in in the east. Generally, whether resistant or susceptible, malaria vectors in the study villages primarily fed on humans, except An. funestus, sampled for the first time in the centre of the country, which fed more on cows. As a preliminary study, I investigated whether resistant populations naturally exposed to insecticides in the wild would still be resistant when older, since laboratory studies typically suggest a decline with age. Results suggested that selection by insecticides may result in resistance remaining in older mosquito cohorts, and marker-based prediction of resistance remained effective. Finally, I used microsatellites to study population genetic structure of An. gambiae s.s. to investigate the origin and likelihood of spread of the strong insecticide resistance phenotypes in the east of The Gambia. Results suggested that although distance was the primary isolating factor among population samples, different ecologies representing coastal marine and drier inland ecosystems may also play a part in partitioning populations from the east and west of the country, likely maintained by large scale rice farming in the central area, dominated by An. coluzzii. Strong differentiation of eastern populations from Senegalese samples did not support a hypothesis of gradual spread of resistance, and Vgsc-L1014F differentiation far exceeded the neutral signal implicating local selection. Resistance to pyrethroids and DDT observed in The Gambia either represented one or more de novo mutations or rare migrants from Senegal subjected to varying local selection pressures. The results presented in this thesis highlight how local variation in characteristics of vector populations is important in driving malaria heterogeneity. To tailor control interventions to tackle high malaria transmission zones, it will be useful for National Malaria Control Programmes to investigate the type of malaria vectors present, their behaviours and insecticide resistance profiles.