Malaria transmission blocking vaccines (TBV) have been prioritized as an intervention to facilitate malaria elimination, but tools are required to support roll-out and evaluation. This thesis presents research that aids development of pre-fertilization TBV candidate 10C (amino acids 159-428 of Pfs48/45). Gametocytes must be detected to identify the infectious reservoir and support mosquito infectivity studies. I proposed filter papers as a novel, cost effective, practical approach for collection and detection of mRNA in low density gametocytes. Comparing 3 filter papers, 2 RNA extraction methods and 2 molecular detection techniques, I concluded Whatman 903 and Whatman 3MM filter papers, combined with guanidine based nucleic acid extraction and detection using QT-NASBA, were operationally most appealing and most sensitive. To identify natural recognition to 10C and 230CMB (a vaccine candidate including amino acids 444-730 of Pfs230), cross sectional surveys were performed sampling school children (n=510) in 3 countries. I demonstrated naturally exposed individuals had antibodies against 10C and 230CMB which displayed age dependent acquisition (p<0.03). Supportive datasets demonstrated 10C and 230CMB antibodies are significantly associated with >90% transmission reducing activity (TRA) (p<0.003). To assess the TRA of 10C-immunized rats against genetically diverse parasites, I sampled venous blood from naturally infected participants in Burkina Faso (n=53), and performed direct membrane feeding assay combined with serum replacement using European control serum spiked with IgG from 10C vaccinated rats. I demonstrated 10C vaccine induced IgG significantly reduced transmission in 4/5 participants who were infectious and infected >2 mosquitoes. This resulted in 80.9-100% reduction in oocyst prevalence (p<0.042), and 85.2-100% reduction in oocyst density (p<0.023). My research identified an attractive combination of tools for detecting low density gametocytes to facilitate sampling in remote field settings. I advanced progress of 6 10C vaccine candidate by indicating antibodies are acquired following natural malaria exposure and are associated with functional TRA.

Since the start of Roll Back Malaria (RBM) in 1998 funding for malaria control has increased dramatically, resulting in the current peak of $2.5billion spent on global malaria control annually. Vector control has been a major source of expenditure, with the focus in sub-Saharan Africa being free Long-Lasting Insecticidal Net (LLIN) distribution and Indoor Residual Spraying (IRS). Use of pyrethroid insecticides in agriculture and rapid scaling up of pyrethroid LLINs and IRS for malaria vector control has led to the development and spread of pyrethroid resistance in Anopheles gambiae malaria vectors. In community use, the level of insecticide resistance at which malaria control is compromised remains uncertain, but experimental hut trials in Benin, an area of high frequency pyrethroid resistance, showed that holed pyrethroid Insecticide Treated Nets (ITNs) failed to protect sleepers from being bitten and no longer had a mass killing effect on malaria vectors. If LLINs and IRS are to remain effective it is essential that new public health insecticides are developed to address the growing problem of resistance. All insecticides that are currently recommended by the World Health Organization Pesticide Evaluation Scheme (WHOPES) for LLIN or IRS belong to just four classes of chemistry that act on nerve and muscle targets; namely pyrethroid, organophosphate (OP), carbamate, and organochlorine (DDT). The Global Plan for Insecticide Resistance Management (GPIRM) states that in areas of pyrethroid resistance or high LLIN coverage, alternative insecticide classes should be used for IRS in a rotation. Rotation of insecticides is very difficult to implement due to a lack of new public health insecticides. The Stockholm Convention on Persistent Organic Pollutants (POPs) came into effect in 2004, yet the use of DDT (classified as a POP) for malaria control has been allowed to continue under exemption since then due to a perceived absence of equally effective and efficient alternatives. Alternative classes of insecticide for IRS such as pirimiphos-methyl (OP) and bendiocarb (carbamate) have a relatively short residual duration of action (2-6 months according to WHOPES). In areas of year-round transmission, multiple spray cycles are required resulting in significantly higher costs for malaria control programs and user fatigue. For continued cost-effectiveness of IRS programs it is important to develop new longer-lasting formulations of currently available insecticides, while also developing insecticides with new modes of action. Pyrethroids are the only insecticides that are currently recommended by WHOPES for LLIN. Therefore, it is essential to develop and evaluate new insecticides for LLIN before effectiveness of pyrethroid LLIN is compromised. 6 This thesis consisted of a sequence of tests to evaluate the efficacy of several new formulations of WHOPES recommended insecticides and novel insecticides both in the laboratory and against wild mosquitoes entering experimental huts. Specifically these studies have shown that:  Addition of eave baffles in experimental huts succeeded in reducing the potential for mosquito escape and is preferable to the assumption of doubling veranda catch to allow for unrecorded escapes (research paper 2).  A Capsule Suspension (CS) formulation of pirmiphos-methyl used for IRS showed a significant improvement in terms of longevity on mud, concrete and plywood when compared with the previously recommended Emulsifiable Concentrate (EC) formulation in laboratory and experimental hut bioassays (research paper 3).  A new formulation of deltamethrin with polymeric binder (SC-PE) for IRS showed only a slight improvement over the existing Water Dispersible Granules (WG) formulation in bioassays, but both formulations equalled DDT in experimental huts and should provide annual mosquito control. Deltamethrin SC-PE or WG should only be considered for use by malaria control programs where there is low pyrethroid LLIN coverage (research paper 4).  In experimental hut trials, chlorfenapyr (pyrrole) IRS was equivalent to alphacypermethrin against pyrethroid susceptible An. arabiensis but superior against pyrethroid-resistant Cx. quinquefasciatus. The unique non-neurological mode of action shows no cross-resistance to existing resistance mechanisms and should be successful for control of pyrethroid resistant mosquitoes (research paper 5).  In experimental hut trials, chlorfenapyr ITNs produced relatively high mortality rates of pyrethroid susceptible An. arabiensis but due to low irritability there was only a small reduction in blood-feeding (research paper 8). Mortality rates were similar to those produced by deltamethrin ITN.  Unlike neurotoxic insecticides, such as pyrethroids and carbamates, chlorfenapyr owes its toxicity to the disruption of molecular pathways which enable cellular respiration to occur. Conventional 3 minute contact bioassay based on WHOPES guidelines is suitable for pyrethroids but does not predict field performance of 7 chlorfenapyr, which is metabolic in nature and sensitive to temperature and the phase of the insect’s circadian activity rhythm (research paper 9).  Combining chlorfenapyr with a more excito-repellent pyrethroid on mosquito nets produced higher levels of blood-feeding inhibition than chlorfenapyr alone, in tunnel tests with both pyrethroid susceptible and resistant strains of Cx. quinquefasciatus (research paper 10).  Restricting insecticide to particular surfaces of the nets (top only or sides only) indicated that An. arabiensis contacts both the top and sides of a mosquito net during host-seeking behaviour. These results support the rationale behind the ‘2-in-1’ mosquito net, in which the top of the net is treated with a non-pyrethroid insecticide and the sides with pyrethroid (research paper 11).

It is now generally accepted that if nothing is done and insecticide resistance in malaria vectors especially to pyrethroids eventually led to widespread failure of current vector control strategies, the progress achieved so far in reducing the burden of malaria could be reversed. Interventions and operational tactics capable of controlling insecticide resistant malaria vector populations and delaying the evolution of resistance need to be urgently identified and properly investigated. One important insecticide resistance management strategy is to expose vector populations to a combination of unrelated insecticides. In this study I investigated the potential of this combination concept to control and manage the spread of indoor resting insecticide resistant African malaria vectors. A series of field evaluations were performed in experimental huts in selected malaria endemic sites to investigate; 1.the impact of combining non-pyrethroid IRS or wall linings with pyrethroid LLINs against malaria vector populations with different levels of insecticide resistance and 2.The efficacy of LLINs treated with a pyrethroid and an alternative compound against pyrethroid resistant mosquitoes. The capacity of the combined intervention approach to delay the spread of insecticide resistance genes was investigated via genotyping studies. I demonstrate that the use of combined interventions and mixture net with unrelated insecticides is an effective way to improve the control of pyrethroid resistance malaria vectors. However, the performance of these combinations will undoubtedly depend on the levels and type of resistance encountered. Where resistance to both insecticides exists, improved control is unlikely. While the use of single interventions would likely exacerbate resistance the combinations would be less beneficial for preventing selection of insecticide resistance when resistance genes are already well established. The impact of these findings on malaria vector control and resistance management is discussed.

Transcriptional, cytological and culture-based analyses of Mycobacterium tuberculosis (Mtb) in sputum have revealed multiple traits indicating the presence of a persister-like or dormant mycobacterial population. Between patients, variable proportions of bacilli in sputum appear to be slow or non-growing, contain lipid bodies (LBs) and depend on exogenous Resuscitation promoting factors for growth. More recently by using Auramine O/Nile-red staining the presence of non-acid-fast (NAF) Mtb-like bacilli containing abundant LBs has been noted. Based on these findings, Mtb in sputum may present in multiple populations and express distinctive transmission adapted phenotypes. Identifying these phenotypes and replicating them in in vitro settings may lead to important new understanding of Mtb in vivo. To study the suspected NAF Mtb cells in sputum, immunofluorescence (IF), peptide nucleic acid (PNA) probe and fluorescence Kinyoun methods were developed and studied. The IF and PNA methods detected only minor components of sputum and variable proportions of in vitro grown cells. Various conditions such as freeze thawing, growth phase and biofilm cultures were shown to alter Auramine NAF proportions. In contrast the fluorescence Kinyoun method labelled the majority of Mtb cells in the preparations studied and provides a promising method for future studies when combined with a suitable LB stain. The capacity of biofilm cultures to replicate the Mtb bacillary populations in sputum was studied. Three phases of biofilm cultures (pellicle, planktonic and attached layers) were studied for gene expression, cytological, growth, antibiotic tolerance and [superscript 3]H-uracil labelling properties comparable to the Mtb phenotypes seen in sputum. The three layers replicated to differing degrees the sputum phenotypes including LB and NAF content, and modest Rpf-dependancy. Attached and planktonic cells gave well correlated transcriptional patterns. Overall, it appears plausible that biofilm grown cells in patient’s lungs could contribute to the populations seen in sputum.

In this thesis, the author set out to investigate the impact of Highly Pathogenic Avian Influenza H5NI (!-IPA! H5Nl) on the livelihoods and food security of a subset of poor women in the household poultry sector in Egypt. Egypt has experienced one of the worst outbreaks of HP AI (H5N l) outside Asia and is now one of six countries where the virus is endemic among the poultry population. The discovery of the Swine Flu virus (LPA! HINI) in Egypt in 2009 and its present co-existence with HPAl (H5NI) has alarmed the international community. In light of the above the findings presented in this thesis become particularly relevant. On a global level, there is a realization that efficient HPAI (H5Nl) control cannot be based on epidemiological data alone. Such control depends on a thorough understanding and appreciation of the interconnectedness of epidemiological, social, and economic factors that contribute to HPAI (H5NI) vulnerability. Therefore, this thesis explored the interrelationship between the three major influences on HP AI (H5N I) endemicity in Egypt: poverty and livelihoods, risk perceptions and food security. A mixed method approach underpinned the analysis. To date, the control of HPAl (H5N I) in Egypt has been challenging. Part of the problem has been a lack of understanding of underlying conditions and motives that influence preventive behaviours at the household level. Indeed, the analysis of risk demonstrated that perceptions of human infection were low and despite recognising the benefits of many biosecurity behaviours, the overall adoption of such behaviour was poor. By disaggregating local indicators of wealth and poverty the study was able to ' reach beyond' traditional classifications of poultry keepers and explore differences between groups. The 'package' approach described in this thesis enabled a more intricate understanding of how marriage/widowhood, education levels and access to resources influenced a wide range of issues such as vulnerability to HP AI (H5N I), risk perceptions, preventive behaviours, food security and coping strategies. In doing so, the thesis was able to demonstrate the need for more targeted approach to knowledge transfer and awareness at the community level.

The liver fluke Fasciola hepatica seriously undermines food production and is now recognised as a neglected tropical disease with up to 17 million humans infected. This thesis reports the development of in vitro maintenance methods for both juvenile and adult life stages of F. hepatica so that functional assays can be developed using RNA interference (RNAi). Juvenile fluke were maintained for 201 days (more than two fold longer than reported previously) in chicken serum in RPMI 1640. These worms grew considerably (- 240x their size at excystment) and exhibited significant development of reproductive and digestive tissues. The best balance of growth and survival in NEJs was seen with 50% chicken serum in RPM I and is therefore recommended as in vitro maintenance medium for juvenile F. hepatica. Two novel Fasciola specific tegumental genes (designated Teg1 and Teg5) were characterized and their potential as new control targets examined using RNAi. In juveniles, Teg1 and Teg5 exhibit temporal expression and immunostaining revealed the expression of both proteins on the tegumental surface where they are likely to be involved in the host-parasite interaction. Teg5 was also localised to the nervous system of juvenile flu~e. Teg1 and Teg5 genes were knocked down in juvenile F. hepatica in both RPMI 1640 and in 20% chicken serum in RPMI (80-90% knockdown was achieved) over a variety of timescales. However, no significant RNAi phenotypes were recorded. The first successful RNAi of adult genes (Teg1, Teg5, and cathepsin L) is also reported following the development of an injection-based RNAi-trigger system. The work in this thesis has significantly advanced the development of new tools to support functional genomics efforts in liver fluke.

No description available.

The fungal vacuole is an acidic membrane bound compartment, containing a range of hydrolytic enzymes. Its functions include recycling of cellular proteins through degradation, storage of cellular metabolites, and homeostasis of the cytoplasmic environment. In the model eukaryote Saccharomyces cerevisiae, vacuolar function is non-essential for vegetative growth, since mutant strains deficient for vacuole function are viable. However, vacuolar function appears to be more important for survival under conditions of nutritional, osmotic, and temperature stress. Furthermore, mutants deficient in vacuolar hydrolase activity are unable to sporulate. This suggests that the vacuole plays an important role during processes of adaptation and differentiation. The vacuole has been well characterised in the model fungi S. cerevisiae, and Aspergillus nidulans, but to date little is known about the functions of the vacuole in the human fungal pathogen Candida albicans. It has been postulated that the vacuole is likely to play a central role in the adaptation of C. albicans to host environments during the process of infection. Moreover, the vacuole has previously been observed to undergo rapid expansion during the emergence of a germ-tube from a yeast cell, to occupy the majority of the parent yeast cell. This process of the yeast-hyphal switch has been implicated in virulence. The class-C vps mutants of S. cerevisiae are defective in vacuole biogenesis and lack a vacuolar compartment. In this study C. albicans homologues of the S. cerevisiae class-C VPS genes, have been identified. Consistent with a role in vacuole enlargement during the yeast-hyphae switch, transcription of a VPS18 like ORF (CaVPS18) was found to be increased during the process of germ-tube formation. Disruption of a C. albicans VPS11 homologue (CaVPSll), resulted in a number of phenotypes similar to that of the class-C vps mutants of S. cerevisiae. Furthermore, a cavpsll null strain was delayed in the emergence of germ-tubes upon serum induction of filamentous growth, and had a reduced apical extension rate compared to its parental strain. These results support a model whereby vacuole enlargement is necessary to support the rapid emergence and extension of the germ-tube from the parent yeast cell. Further analysis of vacuolar function in C. albicans should elucidate some of the processes underlying the yeast-hypha switch.

The first line of defence against viral infection is the interferon (IFN) response, which must be overcome by a virus for successful replication. Pattern recognition receptors detect virus which triggers induction of IFNβ. Secreted IFNβ stimulates the JAK/STAT signal transduction pathway and the upregulation of IFN stimulated genes (ISGs) culminating with expression of hundreds of antiviral proteins. Bunyamwera virus (BUNV) is the prototype virus for the genus Orthobunyavirus and the family Bunyaviridae. BUNV is a trisegmented single stranded negative sense RNA virus whose genome comprises the Large (L), Medium (M) and Small (S) RNA segments. The L segment encodes the RNA polymerase, the M segment the two glycoproteins Gn and Gc and a non-structural protein NSm, and the S segment the nucleoprotein and a non-structural protein NSs in overlapping reading frames. The NSs protein interferes with RNA polymerase II mediated transcription thereby inhibiting cellular mRNA production, including IFN mRNA, and hence it is the primary IFN antagonist. A recombinant virus, rBUNdelNSs, that is unable to express the NSs protein, does not inhibit cellular transcription and is thus a strong IFN inducer. The aim of this thesis was to understand how IFN inhibits BUNV replication. Cells stimulated into the antiviral state by IFN treatment were protected against BUNV infection but addition of IFN 6 hours (or later) post infection had little effect on the replication cycle. However, addition of IFN immediately following infection conferred restriction on BUNV replication by initially increasing viral protein synthesis and then by blocking translation of positive sense viral RNA. To identify ISGs with anti-BUNV activity, I screened a panel of 26 cell lines that inducibly express individual ISGs. To aid screening, recombinant BUNV that expressed green fluorescent protein (GFP) were employed, including an NSs deletion virus with GFP fused to the Gc, rBUNGceGFPdelNSs, that I created and characterised. By a combination of virus yield assays, Western blotting and fluorescence techniques, three cell lines that inducibly express PKR, viperin or MTAP44 were shown to restrict BUNV replication. More detailed studies revealed PKR to restrict BUNV RNA and protein synthesis, but when PKR was knocked-down in IFN competent A549 cells viral replication was not blocked in cells pre-treated with IFN. Viperin inhibited viral protein synthesis and virally-induced host cell protein synthesis shut-off. Additionally, viral RNA synthesis was restricted by viperin and this was dependent on the CX₃CX₂C motif 1 of viperin. Taken together, these data show that the restriction of BUNV replication mediated by IFN is an accumulated effect of several different ISGs acting on different stages of the viral life cycle.

Title: ‘Extending Scottish Exception Reporting Systems Spatially and Temporally’ Abstract: Exception reporting systems allow medical conditions and micro-organisms to be automatically monitored for unusually high levels. Typically, statistical models are used to predict expected levels. Where observed levels exceed the predicted ones by some pre-determined amount, an ‘exception’ is reported to give warning. We focus on developing suitable models for use in the predictive component of such systems. Two particular systems are extended spatially to monitor counts at the regional health board level. The first of these uses call data from the 24-hour medical helpline NHS24, to monitor particular medical syndromes. The second uses counts of positive lab identifications of micro-organisms collected by Health Protection Scotland (HPS). Regional incidences tend to have very small counts, and for these, we use negative binomial Generalized Linear Models (GLMs). However, GLMs assume that observations are independent, which is rarely, if ever, the case in the systems we consider. Two approaches are investigated for dealing with serial correlation and capturing local trend, both of which improve the models. We also investigate links between the health boards and investigate if these links can be used to further improve the models. A new system is produced for monitoring daily all-cause mortality in Scotland, using data collated by the General Register Office. Fitting models to this data is challenging because of the sharp peaks present in the annual seasonality; to address this, we use Generalized Additive Modelling. There is also a marked delay in the reporting of deaths, which must be dealt with if the system is to detect unusually high levels of mortality in a timely fashion. We present a straight forward ‘correction’ to do this. Combining these elements, a mortality surveillance system is produced, which has been used by HPS to monitor mortality during the swine flu pandemic (2009).