Neglected tropical diseases (NTDs) are a group of conditions that exert disability and poverty on populations that comprise the world’s poorest billion people. These conditions, although caused by different organisms and cause distinct disease, they share geographical distribution within tropical regions, occur during similar ecological conditions and most importantly have similar biological mechanisms that are utilized to facilitate the pathology of these diseases. Proteolytic enzymes like proteases are used in many biological mechanisms such as, migration through tissue and cellular compartments; haemoglobin digestion, evasion of immune system responses and cause necrosis and fibrosis to vital tissues and organs. Genomic, transcriptomic, and proteomic studies on parasites (S. mansoni and F. hepatica), parasitic vector (An. gambiae) salivary glands and snake venom show that these diverse pathogens appear to be utilizing similar molecules to perform similar biological mechanisms. Therefore, it is of interest to ascertain whether a cross-cutting approach in research could facilitate a better understanding of these diseases. Therefore, the initial aim of this work was to investigate molecular parallels of the mechanisms used by these tropical disease pathogens, including parasites, snake venom toxins, and haematophagic parasite vectors, to access their host’s blood stream. Using a bioinformatics-led approach, in combination with immunological and proteomic analyses, this study demonstrated the presence of similar compounds between shared molecular molecules (serine proteases and other proteins) causing pathology in parasites, parasitic vectors and snake venom. This similarity was not only at the bioinformatics level, but presence of cross-reactivity toward parasite proteins was detected using antivenoms and toxin-specific antibodies. In addition, sera collected from patients infected with S.mansoni exhibited an immune response to snake venom. One of this study aims was to investigate possibilities of using homologous proteins in parasitic vectors and snake venom as therapeutic applications. For this purpose, chimeric epitopes of homologous snake venom (Echis ocellatus) and mosquito salivary proteins were designed as primary vaccine that could be boosted by mosquito bites. If succeeded, this method would prevent, or at least reduce, the devastating pathology and death caused by snake venom at a low cost, with limited logistical complications.

Malaria is an ancient vector-borne disease that still has a huge impact on global health. Malaria pathogenesis is developed during the asexual intraerythrocytic stage where P. falciparum modifies the host erythrocytes by exporting repertoires of parasite proteins on to the surface of infected erythrocytes. PfEMP1 is one of these proteins that mediate different functions including the adhesion of IEs to the host receptors such as CD36, ICAM-1 and EPCR. The current study has characterised the adhesion of infected erythrocytes with different PfEMP1 variants to CD36, ICAM-1 and primary endothelial cells. The characterisation was carried using static protein and flow endothelial adhesion assays. First, the characterisation involved an analysis of the binding of recently selected ICAM-1 binding P. falciparum patient isolates on different ICAM-1 variants. The results showed that different isolates have variant-specific binding phenotypes suggesting that there might be variable contact residues on ICAM-1 being used by different parasite PfEMP1 variants. This observation was more emphasised by the adhesion of isogenic isolates that has been confirmed to express ICAM-1 binding domain from IT4 parasites. The second part of the study has characterised the adhesion of IEs with upsC PfEMP1 isolates from HB3 and IT4 isolates on CD36, ICAM-1 and endothelial cells. Three upsC IT4 isolates bound to CD36 and one of these isolates bound to ICAM-1 because it expresses DBLβ-ICAM-1 binding domain. In contrast, HB3 upsC isolates did not show preferential binding to CD36, ICAM-1 and the endothelial cells despite showing cross reactivity with adult hyperimmune sera. Finally, the adhesion of IEs with different length PfEMP1s was analysed. It was concluded that long PfEMP1 adapted to bind efficiently the short, but this might be due to the lack of variety of DBLs for adhesion in the short forms. Therefore, it is suggested that it is the domain constitution rather than size that seems to be important.

A unique feature of mature Plasmodium falciparum (P. falciparum) parasitized RBC (pRBC) is that they bind to surface molecules of microvasculature endothelium via the parasite-derived surface protein PfEMP1. This ligand is associated with the cytoadherence pathology seen in severe malaria (SM) and recently our group has shown that even when treated with effective anti-malarial drug, pRBC are still able to cytoadhere, therefore, there is a need to find an adjunct treatment (in addition to antimalarial drugs) that can inhibit and reverse the adhesion process. Previous reports have suggested that sulphated glycoconjugates are highly effective at disrupting P. falciparum pRBC rosettes. Here, we investigate that effect by using sulphated polysaccharides and modified heparin for their effect to interrupt pRBC sequestration. We found that not all sulphated compounds or modified heparins were able to interrupt the sequestration process. Consideration of the inhibitory compounds generated some 18rules 19 fore exhibition of inhibitory properties: Sulphate position either at 6-O, or/and 2-O sulphate and N-sulphate is necessary for each compound. In addition, the multivalent effect and drug exhibit low anticoagulant activity also determined an active response to inhibit and de-sequestered P. falciparum pRBC on protein and endothelial cells. Here, we provide evidence that polysaccharides that possess a different level of sulphate, conformational structure and sulphate position act differently. This study also addressed the importance of pH host environment and extracellular matrix (glycocalyx) on the surface of endothelial cells on mediating pRBC binding. It found that pRBC bind significantly higher at pH 7-7.2 to CD36 and ICAM-1. Meanwhile, glycocalyx might interact as an instantaneous binder before pRBC reached ICAM-1 or CD36, unfortunately we cannot prove this due to methods and antibody chosen. The work reported in this thesis opens up new possibilities for therapeutic strategies targeting binding interaction of pRBC to host cells.

Currently employed vector control strategies are experiencing resistance that threaten to limit their effectiveness in controlling malaria transmission. Therefore, development of new genetic vector control strategies may benefit efforts to curb the devastating effects of malaria. Sex-specific gene expression has not been studied in mosquito embryos thus far, but its understanding may lead to creation of new tools for vector control. Here we analysed sex-specific transcription using firstly a targeted gene approach and secondly de novo transcriptional profiling of male and female embryonic transcriptomes. Two genes, doublesex (dsx) and fruitless (fru), belonging to sex determination/differentiation pathway, have sex-specific transcripts described previously in adult An. gambiae, and here, we present evidence that both dsx and fru are also sex-specifically spliced in the embryonic stages. dsx, a final gene in the sex determination pathway and a key regulator of sexual differentiation, is maternally deposited as a female transcript, but establishes a persistent pattern of alternative sex-specific splicing eight hours after egg laying (AEL). fru, a modulator of adult male behaviour, has sex-specific transcription starting at least 16 h AEL. The An. gambiae sex determination pathway was further investigated through isolation of a putative homologue of the D. melanogaster sex determination gene transformer2 (tra2), which was knocked down via RNAi in an attempt to confirm its involvement in Anopheles sexual development. Using a transient reduction in transcript levels, an effect on sex determination phenotype was not observed. In addition, bioinformatics and degenerate PCR approaches identified a large number of candidates with sequence similarity to transformer (tra), though these candidates require additional characterisation. Previous work on sex-specific expression has taken an a priori approach, using microarrays for gene expression analysis. Here, a de novo approach was undertaken and an approximate ~500,000 transcript fragments have been independently sampled using the 454 platform from each transcriptome of male and female embryos and assembled into 17,492 and 16,899 contigs from male and female reads, respectively. In silico subtraction of the two RNA-seq databases revealed a large number of putative male or female specific transcripts, of which 60 were tested using RT-PCR. Among those, two transcripts, displaying male-specific expression, represented two novel genes (YOA and YOB) located on the Y chromosome and sharing partially overlapping exons. YOA is expressed from late larvae through to adulthood. YOB is expressed from 4 hr AEL and continues across all stages through to adulthood. Their amino acid translation has no significant similarity to known proteins, or conserved domains. Furthermore, a functional role could not be assigned since gene silencing approaches did not reveal an obvious phenotype. This study has also emphasised that the current AgamP3.6 gene build is not complete. Deep sequencing of the embryonic transcriptome has produced a large dataset containing 6,436 transcripts that map to previously unannotated genomic regions. These sequences likely represent extensions to untranslated regions, novel exons or unique gene sequences. Our study demonstrates that RNA-seq is a powerful tool to identify sex-specific expression in mosquito embryos and that sex-specific transcription starts very early during embryonic development. The remaining question is whether the data produced within this study can be exploited in the context of a genetic vector control strategy.

Background: This research was conducted in 2010, in Akobo County, Jonglei State, South Sudan, a region with a long history of inter-ethnic conflict. Consideration of children in situations of armed conflict tends to focus on their protection and frequently portrays children as passive victims. Previous research and evaluations of child participatory programming, however, provide powerful testimonies as to the capacities and desire of children to be more involved. The aim of this research was to explore children’s health needs from a child perspective and to determine existing and potential opportunities and challenges for children to participate in health decision making. Methodology: This research uses qualitative and quantitative methods for different but well defined purposes within the same overall research project. Qualitative methods including interviews, focus group discussions, non-participant observations and workshops were used to explore knowledge and perspectives related to children’s health needs, children’s risk exposure and available means of protection as well as children’s roles, skills and capabilities to engage in decision making. Subsequently, a cross sectional mental health survey was conducted to investigate exposure to traumatic events, Post-traumatic Stress Disorder (PTSD), anxiety and depression using the Harvard Trauma Questionnaire (HTQ) and the Hopkins Symptom Checklist (HSCL-25). Positive outcomes were identified using the Post-traumatic Growth Inventory (PTGI). Multivariate linear regression analysis was used to define associations between variables. Results: One hundred and forty-four children aged 7-18, 88 adult community members and 20 staff of service providers participated in the qualitative study. Psychological distress was identified as the main perceived health threat and as a potential challenge to children’s participation. The qualitative findings further illustrate children’s suffering, but also the resilience and adaptability of children affected by armed conflict and their capacity and motivation to contribute and take action to improve their everyday life. Adult community members showed a high level of trust and belief in children’s strength, ability and willingness to address issues, take risks and make decisions. At the same time, adults voiced great concerns about losing authority and control over their children if children were given more rights. Interviews with service providers showed that half of them had consulted with children at some point during program implementation. A higher degree of children’s participation, where children have the initial idea and decide how the project is to be carried out, with adults available but not taking charge, was found to be an issue of concern to child mandated agencies alone. Three hundred and fifty-three children aged 12-18 participated in the cross-sectional mental health survey. The survey findings showed a high prevalence of experienced traumas and adverse mental health outcomes: 64.5% of the children met symptom criteria for PTSD, 72.2% of the children met symptom criteria for anxiety and 65.4% of the children met symptom criteria for depression. Linear regression analysis showed statistically significant relationships between orphan hood (p<0.01), ‘material deprivation’ (p<0.001), ‘witnessed general violence’ (p<0.01), ‘witnessed death, abduction and injury of loved ones’ (p<0.01) and adverse mental health outcomes. The PTGI demonstrated a high prevalence of positive change (PTG) as a result of the most traumatic event in children in all five categories: ‘spiritual change’ (73%), ‘relating to others’ (67%), ‘personal strength’ (60%), ‘appreciation of life’ (54%) and ‘new possibilities’ (52%). Regression analysis showed a significant positive relationship between PTG and post-traumatic stress (p<0.001) suggesting that growth and symptom severity may be independent of each other, that is, both growth and psychological distress can co-exist. Conclusion: The direct impact of armed violence has significantly contributed to extremely high levels of trauma exposure while the long term consequences of conflict such as poverty, the destruction of social networks and family relationships have deprived children of their support system. Our findings indicate that PTG and posttraumatic stress can go hand in hand. According to the theory that PTG results from the struggle with highly challenging life circumstances these findings indicate that the trauma categories associated with post-traumatic growth have threatened children’s pre-trauma view in a significant way, thus fostering PTG in the individuals attempt to assimilate the traumatic event into a new, modified world view. This study further demonstrated that children’s participation can provide an important opportunity in conflict settings to address mental health and to re-build or maintain positive relationships among children and between adults and children.

Introduction: The process of sequestration involving specific cytoadhesion between parasite ligands expressed on the surface of the parasitised red blood cells (pRBC) and host vascular endothelium contributes to pathogenesis of severe falciparum malaria. A major polymorphic surface antigen implicated in cytoadhesion is the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 is encoded by the var multigene family that is sub-divided into three main groups: A, B and C, according to sequence similarities in coding and non-coding sequences. PfEMP1 has variant adhesive phenotypes, some of which interact with ABO blood groups to form rosettes and some involved in apparent formation of platelet-mediated clumps of infected erythrocytes that are thought to contribute to disease severity. With heavy HIV burden concentrating in areas with high malaria rates, co-infections are common. Both HIV and malaria interact with the host immune system, resulting in a complex activation of immune cells and subsequent dysregulated production of cytokines and antibodies. However, there is limited information on the molecular mechanisms of interaction between the two infections. Methods: Real time PCR was used to: 1) compare abundance of the three main var groups and measure the level of cytokine production and receptor expression utilising the resources of a clinicopathological study of 20 Malawian fatal paediatric malaria patients divided into three diagnostic groups: circulating and sequestered parasites (CM1); circulating and sequestered parasites plus perivascular pathology (CM2) and parasitaemic control (PC) groups; and 2) determine the effect of host ABO blood group on expression of var/PfEMP1 subtypes mediating platelet-mediating clumping in 65 Malawian paediatric patients with uncomplicated malaria (UM). Results: While there were no significant associations between ABO blood antigen groups with the clumping phenotype in UM patients, an abundance of var upsA and C transcripts were expressed in CM2 and the PC (p ≤ 0.001) groups. However, a very different expression pattern was observed in the CM1 group, which were mostly HIV positive (80%), with var gene group upsB being more abundant than in the other two diagnostic groups ( p ≤ 0.001). This result was supported by different cytokine/receptor upregulation between HIV positive and HIV negative children, with significant upregulation of TNF in HIV negative children (p ≤ 0.05). Conclusions: This data suggests that perivascular pathogenesis in naturally infected children is associated with differential var gene expression in the body. HIV disruption of cytokine release affects receptor regulation and influences parasite antigen expression.

There are insufficient data on the epidemiology and clinical features of HIV in the Middle East. WHO statistics show the Kingdom of Saudi Arabia (KSA) to be one of the least affected countries globally. However, the Saudi National Program for HIV Control reported a 34.6% increase in cases in 2008 from the previous year. Jeddah region has the highest proportion of HIV cases in KSA (40%). Infection risk data are not always complete and coinfection rates have not been studied. The first part of these studies included a retrospective longitudinal case review of all patients attending the Jeddah clinic to obtain a clearer view of clinical and epidemiological features of that population There are few publications about resistance to antiretroviral therapy (ART) in the Arabian Peninsula, including KSA, and most are heavily biased towards assessment of patients with sequential treatment failure. Wider access to resistance testing has only become available recently and baseline local resistance patterns are largely unknown. The aim of the second part of the study was to determine patterns of ART resistance in a systematic fashion in treatment-naïve HIV positive Saudi patients and to document the presence and frequency of novel resistance HIV markers in Jeddah. Study Part 1. Clinical features and epidemiology of HIV and coinfection with TB and/or viral hepatitis in a large clinic in Jeddah, Kingdom of Saudi Arabia Aims: To describe demographic and clinical features of HIV infection in clinics and hospitals in Jeddah and to document prevalence and risks for coinfections with tuberculosis (TB) and/or hepatitis Methods: Retrospective study including all HIV positive Saudi adults attending the main treatment centre in Jeddah in the 11 years (2000-2010). Data were systematically collected from case files and summarised. Statistical comparisons included univariate analyses with a p value <5% considered significant. Results: 1383 HIV positive adults were reviewed, median (range) age 40 (18-86) years, of whom 1026 (74.2%) were male. Risk factors included heterosexual transmission in 709 (51.3%), men having sex with men (MSM) in 264 (26%), blood products in 148 (10.7%), injecting drug use (IDU) in 97 (7%) and not identified in 165 (11%). The predominant clinical presentation was with respiratory symptoms 611 (44%), followed by gastrointestinal manifestations in 312 (22%), while 29% (408) were asymptomatic. Past or present TB coinfection (clinical and/or radiology) was found in 208 (15%); 59 (5.3%) had hepatitis B serology positive (HBsAg positive) and 82 (7.4%) had hepatitis C coinfection (antibody positive). TB was associated with IDU (RR 1.67 (CI 1.13-2.41) p< 0.01) and having been in prison (RR 1.83 (1.18-2.85) p< 0.01) and these two risk factors were closely linked themselves. HBV coinfection was not linked with IDU (RR 1.89 (0.93-3.85) p=0.08) but was linked to being in prison (2.38 (1.25-4.54) p <0.01), while HCV was strongly linked with IDU (RR 4.22 (2.71-6.57) p<0.01) and MSM but not with imprisonment (RR 1.94 (1.04-3.63) p=0.07) Conclusion: HIV/AIDS and related coinfections are medical problems in Saudi Arabia with many social challenges. The Saudi National Program for HIV Control actively addresses prevention of HIV and provision of high quality care for those affected. More detailed studies are needed on clinical patterns in outpatient and inpatient settings and on locally appropriate prevention programmes in high risk groups. Study Part 2. HIV resistance in ART naïve patients in a large treatment centre in Jeddah, Kingdom of Saudi Arabia Aims: To document the prevalence and types of ART resistance in ART naïve HIV patients in Jeddah, and to compare the efficacy of Next Generation Sequencing (NGS) with standard (Sanger) genotypic methods. Methods: Plasma samples were collected from all ART-naïve patients sequentially attending the HIV clinic at King Saud Hospital, the main HIV treatment centre in Jeddah, between November 2012 and February 2013. Plasma was saved and HIV protease (Prot) and reverse transcriptase (RT) regions were sequenced according to routine in-house diagnostic protocols (Sanger) at Liverpool Specialist Virology Centre. The Stanford database was used for interpretation of resistance profiles. Neighbour-joining phylogenetic analysis was performed on samples with adequate sequence data for both Prot and RT. NGS sequencing was later performed at the Public Health England reference laboratory in Colindale. Results: Blood samples were collected from 109/116 (94%) eligible patients approached to join the study. 71 (65%) were male and 52 (48%) had been diagnosed with HIV within the last 6 months. HIV RNA was successfully amplified from 96, and sequence data obtained from 93 Prot amplicons and 87 RT amplicons by Sanger and 105 by NGS. Mutations at putative resistance sites for NNRTI NRTI and PI were detected by Sanger in 9/87 (10.3%), 1/87 (1.1%) and 6/93 (6.5%) and 24/105 (22.9%), 6/105 (5.7%) and 34/105 (32.4%) respectively by NGS. Those with significant potential to confer resistance to NNRTI were found in 9/87 (10.3%), with resistance to NRTI in 1/87 (1.1%) and PI in 6/93 (6.5%). 4.5% of the samples showed resistance to efavirenz and nevirapine. The most common HIV-1 subtype was C (38%); although a cluster of CRF2_A (7%) was also prominent. Conclusion: Clinically significant resistance is emerging (16 %) in this population. A variety of other markers included some clustering suggesting local transmission of primary resistance. The results are probably generalizable in KSA and we recommend the introduction of routine resistance testing for all HIV positive patients in the region before starting ART. Overall: These findings provide evidence for introduction of several changes to enhance the National HIV/AIDS programme in the Kingdom of Saudi Arabia.

Pneumococcal disease is preceded by nasopharyngeal colonization, which is also the source of transmission. Current pneumococcal conjugate vaccines protect against invasive disease and reduce carriage in children, but are less effective against mucosal disease and have limited serotype coverage. There is an urgent need for new vaccines and colonization has been suggested as an alternative endpoint in vaccine licensure. Experimental human pneumococcal carriage, although potentially risky, offers a way to examine colonization in the context of vaccination. Experimental carriage also allows the investigation of the impact of a pathogen on the immunological complexity and normal microbiota of humans, both of which cannot be done using animal models. We developed a safe and reproducible method of experimental human pneumococcal carriage, described bacteriological and immune factors associated with carriage, and examined the density and duration of experimental carriage. The data presented in this thesis show that experimental human pneumococcal carriage was safe and reproducible. There were important bacteriological differences between pneumococcal strains that affected carriage. Asymptomatic upper respiratory tract viral infection increased both the risk of pneumococcal colonization and the levels of mucosal Factor H, leading to increased colonization density. This model will be useful in further studies of pneumococcal pathogenicity and host protection against carriage and disease. The model may also be used to select vaccine candidates by protective efficacy in blocking experimental carriage.

No description available.

Tsetse flies (Diptera: Glossinidae) are the vectors of African trypanosomes which cause human sleeping sickness and nagana to animals. Despite their importance as vectors, tsetse flies are largely refractory to trypanosome infections after the first two bloodmeals. Fly immunity is thought to play a crucial role in determining the outcome of initial parasite establishment in the fly midgut. As a result of defending against trypanosomes and haem released during blood digestion, redox genes probably play a significant regulatory role in midgut physiology of tsetse flies. The primary objective of this PhD thesis was to test the hypothesis that superoxides are a core protective device for tsetse flies against invading trypanosomes. To first examine whether inducible nitric oxide synthase (NOS), a putative pro-oxidant enzyme, acts as one of the barriers to trypanosome establishment in the tsetse midgut, temporary depletion of NOS from the midgut using RNA interference (RNAi) was performed. This caused a significant increase (p=0.011) in midgut infection prevalence (2-fold) compared to when NOS was reduced post-infection. It had the most noticeable effect when knockdown occurred before the trypanosome infection event. Further evidence for this was given by the inhibition of NOS by oral administration of the NOS inhibitor L-NAME which also caused a significant increase in midgut infections (p=0.048). Further, indirect evidence of the anti-trypanosomal role of NOS is seen in the fact that NOS activity increased almost 2-fold in trypanosome-infected flies compared to uninfected flies. By contrast, in tsetse NOS levels did not change in response to bacterial infections with Gram-negative Escherichia coli or Gram-positive Staphylococcus aureus suggesting this is quite a specific response to trypanosome infection. An indirect approach was taken to measure reactive oxygen species (ROS) levels in tsetse flies. ROS levels were correlated with trypanosome prevalence in infected flies under various physiological conditions known to impact susceptibility: fly age, starvation, fly sex, bloodmeal fractions, immune-stimulation with bacteria, knockdown of immune genes (tsetseEP protein) and puparial conditions. The indirect evidence gathered suggested that changes in ROS levels cannot explain changes in susceptibility seen in tsetse under this range of physiological conditions. The only result suggesting a role for ROS in trypanosome infection was the observation of a significant (p=0.029) increase in hydrogen peroxide levels in flies infected with live trypanosomes compared to uninfected flies. Consequently, a direct approach was attempted to look for a role of ROS in trypanosome establishment in tsetse midgut. Gene knockdown via RNAi of genes involved in ROS generation (dual oxidase – Duox) and removal (oxidation resistance 1 - OXR1) was attempted. Knockdown of transcript levels of Duox resulted in increased midgut infection prevalence suggesting its involvement in controlling parasite establishment. However, despite repeated attempts, transcript levels of OXR1 could not be knocked down. A bioinformatic approach including phylogenetic analyses of NOS, Duox and OXR1 was taken for comparative analysis with other dipterans. In addition, manual extension of the sequences of these genes was carried out and the extended protein sequences of these genes are now comparable in length to Drosophila orthologs, incorporating necessary domains and residues essential for their functionality. Available resources such as a 454 cDNA tsetse midgut transcriptome enabled the identification of 70 genes which were differentially expressed in tsetse fed trypanosomes. Trypanosome infection was found to have a substantial effect on metabolic and cellular processes in the fly, in addition to the emerging common classes such as serine proteases, adhesion proteins, reactive intermediates and immune-related genes. Finally, with the recent release of the Glossina morsitans morsitans genome, a selection of antioxidant genes was manually annotated to include transcript and protein sequence predictions. Consequently, a comparative analysis with the model system, D. melanogaster was carried out. The essential components of the antioxidant system appear to be conserved, with a few exceptions. In addition, lack of differential expansion of antioxidant gene families among Glossina and other dipterans including Drosophila reflects their close relation. Furthermore, we are now able to validate antioxidant genes identified in the past and find novel genes such as a second thioredoxin reductase in tsetse. Overall, this research involves both functional and bioinformatic approaches to offer new insights into the roles of oxidants in response to trypanosomes in the tsetse fly.