This work was undertaken to characterise the interaction of the leukocyterestricted integrin LFA-1 (CD1la/CD18) with its ligands. LFA-1 function is critical for an immune response and, for example, allows leukocyte binding and transmigration across the endothelium, antigen presentation and cytotoxic T lymphocyte-mediated killing. The ligands for LFA-1 are the Intercellular Adhesion Molecules (ICAMs), with ICAM-1, ICAM-2 and ICAM-3 being the best characterised. The binding sites on ICAM-1 and ICAM-3 for LFA-1 were investigated with the use of antibodies and mutated proteins. The following regions were found to have a role in binding LFA-1: the CFG face of ICAM-3 domain 1; domain 2 of ICAM-1; a residue in domain 1 of ICAM-1 that is mutated at high frequency in African populations and is associated with susceptibility to cerebral malaria. Binding of Mg2+ or Mn 2+ to the extracellular region of LFA-1 and intracellular signalling can both stimulate LFA-1 to adhere to ICAM-1, but by different processes. The former mechanism induces a high affinity form of LFA-1, which was shown to be achieved by an inter-domain movement involving the I domain of the LFA-1 a subunit. This is the first physical evidence for a conformational change occurring in an integrin upon activation. The mechanism by which intracellular signalling activates LFA-1 was demonstrated to involve calpaindependent clustering of LFA-1 in the membrane, thus increasing the avidity of LFA-1 for ICAM-1. Leukocyte Adhesion Deficiency-1 and Glanzmann's Thrombasthenia are genetic disorders in which mutations in the integrin genes result in absence of expression or expression of a non-functional integrin. The defects in function of leukocytes from a patient with clinical features of both disorders were studied. The results suggest that the patient has a novel form of integrin dysfunction in which integrins are expressed at normal levels, can be induced to bind their ligands by mechanisms which increase the affinity of interaction, but cannot be stimulated to bind ligand by intracellular signalling pathways.

acase@tulane.edu / Introduction: The Home-based Management of Malaria (HMM) is a cornerstone of malaria control in sub-Saharan Africa (SSA) and is recommended by WHO to provide prompt access to antimalarial treatment for children in under-served areas. Although HMM has been shown to reduce malaria morbidity and mortality with chloroquine, it has not been examined previously in the era of artemisinin-based combination therapies. The objectives of this study were to determine whether HMM reduced: 1] the time from when a mother or guardian realized her child was ill to the time when the child was brought for treatment and 2] malaria morbidity in children less than 5 years of age. Methodology: This cross-sectional retrospective study (2008-2014) was performed in intervention villages (receiving HMM) and control villages (not receiving HMM) to examine the effectiveness of HMM. Key Results: More mothers and guardians were informed about the malaria control activities performed (98% vs. 24%) in intervention than control villages (p < 0.001). Consistent with that result, mothers and guardians in intervention villages sought care for their sick children earlier than mothers in control villages (p < 0.001) and were more likely to obtain treatment from community health workers (CHWs) in their home villages. In contrast, more children were referred for malaria treatment to health posts and health centers from control than intervention villages (p < 0.001). Likewise, more children with complicated malaria were referred for treatment from control villages (p < 0.001), although those conclusions were limited by the small numbers of complicated (severe) malaria cases. Conclusions: These results indicate HMM shortens the time mothers wait before taking their children to receive treatment. Because more children with uncomplicated or complicated malaria are referred for treatment from control than intervention villages, these results indicate that the availability of HMM treatment in the child’s home village reduces morbidity (the risk of severe malarial disease). However, additional studies with larger numbers of subjects will be necessary to determine if HMM reduces mortality. / 1 / Ibrahima SECK

Today, malaria remains the biggest killer of the third world, killing over a million people every year, despite intensive research efforts. Carbamoyl phosphate synthetase II (CPSII) is the first and rate-limiting enzyme in pyrimidine biosynthesis of Plasmodium falciparum, the causative agent of malaria. PfCPSII is a unique target for DNAzyme therapy due to the presence of two unique insertion sequences of 700bp and 1800bp that exist within the mature mRNA transcript. Previous studies have demonstrated that exogenous delivery of nucleic acids such as ribozymes and DNAzymes targeting PfCPSII insertion II effectively inhibited the growth of P. falciparum cultures at sub-micromolar levels. The objective of this study was to investigate the insertion sequences within CPSII from rodent malaria species P. berghei, P. chabaudi and P. yoelii in order to further validate the insertions as DNAzyme targets in vivo. In addition, the insertions were isolated from another human malaria parasite, P. vivax. All Plasmodium CPSII genes investigated encoded two highly hydrophilic insertion sequences of similar size and nature, in the precise position seen in PfCPSII. Although these insertions are poorly conserved, border and internal regions of high homology are present. Thirty-one new DNAzymes were designed to target the P. berghei CPSII insertion II region, seventeen of which demonstrated the ability to cleave the target RNA. Of these, four showed significant cleavage activity, with the DNAzyme MD14 cleaving greater than half the target RNA within five minutes. These DNAzymes were then further characterised for kinetic behaviour. Again, MD14 displayed favourable kinetics of cleavage and was chosen as a suitable candidate in an in vivo rodent malaria trial. Analysis of parasitaemia from the MD14 treated mice indicated the administration of MD14 effected a highly statistically significant reduction of parasitaemia, although this reduction was low (6.3%). More efficient DNAzyme delivery methods were investigated in order to improve DNAzyme efficacy and included the novel use of porphyrin conjugated DNAzymes. The porphyrin-conjugated DNAzymes improved uptake into parasitised red blood cells and significantly reduced parasite growth in vitro at nanomolar levels.

Les moustiques anophèles sont les vecteurs du parasite Plasmodium l’agent du paludisme. Le parasite subit des pertes massives pendant son cycle de développement chez l’anophèle, ce qui suggère que les moustiques sont capables de développer une réaction immunitaire efficace contre le parasite. La connaissance de l’immunité et de la résistance des moustiques au genre Plasmodium provient principalement de systèmes de laboratoire qui utilisent des espèces de parasites de rongeurs ou d’oiseaux comme modèles du paludisme humain. Les observations présentées dans cette thèse suggèrent que certains gènes comme Tep1 et LRIM1 sont des médiateurs de réponses antiparsitiques contre différents Plasmodiums dans différents vecteurs. Cependant, le degré d'efficacité avec laquelle un moustique est capable de réduire le nombre de parasites peut être variable surtout entre combinaison de souche de moustique et de souche de parasite différentes, selon que la paire soit hautement compatible ou non.

A better understanding of the role of antigen-presenting cells (APCs) in host resistance to malaria is essential to unravel the complex interactions between the host and the parasite. This would improve the design of malaria vaccines. Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has been utilized as a vector to deliver vaccine candidate antigens. We assessed the immunogenicity of a recombinant BCG-expressing (BCG-CS) circumsporozoite protein (CSp) as a malaria vaccine candidate. Immunization of BALB/c mice with BCG-CS augmented the numbers of dendritic cells (DCs) in draining lymph nodes and in the spleen. The activation markers MHC-class-II, CD40, CD80, and CD86 on DCs were significantly upregulated by BCG-CS as compared to wild-type BCG (wt-BCG). In vitro stimulation of bone marrow-derived DCs and macrophages with BCG-CS induced IL-12 and TNF-α production. BCG-CS induced higher phagocytic activity in macrophages as compared to wt-BCG. Finally, BCG-CS induced CSp-specific antibodies and IFN-γ-producing memory cells. Taken together, we found that BCG-CS is highly efficient in activating innate immune responses and could effectively prime the adaptive immune system. Heterologous prime–boost approaches using vectors are optimal strategies to improve a broad and prolonged immunogenicity of malaria vaccines. We have demonstrated in BALB/c mice that priming with a replication-defective human adenovirus serotype 35 (Ad35) vector encoding CSp (Ad35-CS), followed by boosting with BCG-CS, maintained antibody responses and significantly increased levels of long-lived plasma cells (LLPC) and IFN-g-producing cells in response to CSp peptides. The increased number of IFN-g-producing cells induced by the combination of Ad35-CS/BCG-CS and the sustained type 1 antibody profile, together with high levels of LLPCs, may be essential for the development of long-term protective immunity against liver-stage parasites. Fulani and Dogon, two sympatric ethnic groups living in northeastern Mali, are characterized by a marked difference in the susceptibility to P. falciparum malaria. We investigated whether APCs obtained from Fulani and Dogon children exhibited differences in terms of activation status and toll-like receptor (TLR) responses during malaria infection. We observed decreased activation of APCs and markedly suppressed TLR responses in Dogon children as compared to Fulani. These findings suggest that APCs and TLR signaling may be of importance for the protective immunity against malaria observed in the Fulani. In conclusion, this thesis provides new insights that could facilitate a rational design of novel vaccines against malaria. Furthermore, the results elicit some immunological bases of the APC activation underlying the differences in host susceptibility to malaria infections. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: In Press. Paper 2: Manuscript.

<p>The study aims to assess the characteristics of donor funding for national tuberculosis, malaria and HIV programmes to Zambia over an 8 year period in order to inform it&rsquo / s more effective and efficient utilization.</p>

There are many aspects of the immunology of P. falciparum infection that are not understood. Genetic approaches are of great value for dissecting the complexity of immune responses to malaria in natura by providing new insights into molecular interactions between the parasite and the host. The work presented in this thesis had two major aims: to investigate the role of IFN-γ signalling in susceptibility to malaria; and to understand the biological basis of the low susceptibility to malaria shown by the Fula people of West Africa. We conducted genetic association studies to investigate the role of four candidate loci: IFNG, IFNGR1, IFNGR2 and IRF1. We observed significant associations between common genetic variation at the IRF1 locus and the ability to control P. falciparum infection. Our studies did not provide evidence for a major role of this gene in determining susceptibility to severe malaria. Using allele-specific expression assays we obtained preliminary results suggesting the existence of a regulatory element(s) in the 5’ upstream region of the IRF1 locus. IRF1 polymorphisms regulating gene expression could affect the production of inflammatory cytokines and the control of infection, but not the immune-based pathogenesis of severe disease. To understand the biological basis of the resistance to malaria shown by the Fula, we analysed HLA class II polymorphism and confirmed previous data showing that the Fula from Burkina Faso are genetically distinct from sympatric Mossi and Rimaibé. We then compared the expression profiles of PBMCs and CD4+CD25+ cells isolated from healthy adults of Fula and Mossi ethnicity. In the Fula we observed higher expression of several genes related to Th1 and Th2 function and reduced expression of important genes related to immune tolerance: FOXP3, CTLA4, TGFB and TGFBRs. These results suggest a functional deficit of T regulatory cells in the Fula and identify key genes as good candidates for future genetic association studies.

Structural biology lays the molecular foundation for the modern field of life sciences. In this thesis, X-ray crystallography is the primary resource for atomic detail structural information and is the major technology employed in our research. Three examples show how structural biology addresses the basic processes of life. Firstly, two crystal structures of R21, corresponding to two biological states, reveal a new activation mechanism of SAR-endolysin, which not only complements the previous model, but is also more generally applicable to the endolysin family. The structural information was further corroborated by NMR data in solution. The second example is the crystal structure of mycobacteriophage lysin B, which identified the function of the protein, and tackles the unique problem of how mycobacteriophage circumvent the mycolic acid-rich outer membrane of mycobacterium. The last example is the homology modeling of the Plasmodium ribosomal L4 protein. The action mode for the drug in Plasmodium was proposed based on that, which accounts for the anti-malaria effect of azithromycin.

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<p>The study aims to assess the characteristics of donor funding for national tuberculosis, malaria and HIV programmes to Zambia over an 8 year period in order to inform it&rsquo / s more effective and efficient utilization.</p>