La déformation de matériaux élastique dont l'une au moins des dimensions est petite apparaît dans un grand nombre de structures naturelles ou artificielles pour lesquelles une courbure spontanée est présente. Dans ces travaux de thèse, nous couplons plusieurs approches théoriques à des expériences macroscopiques sur des rubans élastiques afin de comprendre la dynamique d'enroulement de biomembranes ouvertes d'un trou. La motivation est issue d'observations récentes d'enroulements obtenues au cours de la sortie de parasites de la Malaria de globules rouges infectés, et de l'explosion de vésicules polymère. Dans une première partie, nous étudions théoriquement la stabilité d'un pore et la propagation de l'enroulement sur une biomembrane sphérique ouverte. Nous modélisons de façon géométrique l'enroulement toroïdal de la membrane par une spirale d'Archimède de révolution et décentrée. Avec cette hypothèse, nous montrons que la stabilité du pore vis-à-vis de l'enroulement dépend fortement de la tension de ligne et du cisaillement et nous discutons ces résultats dans le cadre de l'enroulement de membranes MIRBCs. De plus, en prenant en compte les différentes sources de dissipation, nous obtenons un très bon accord entre les données expérimentales obtenues pour les MIRBCs et la dynamique d'enroulement obtenue par le calcul. Notre approche montre en particulier que la dissipation dans la membrane due à la redistribution de la matière durant l'enroulement domine sur la dissipation visqueuse dans le milieu.Cependant, la complexité de la géométrie sphérique, ainsi que le nombre limité d'observations microscopiques à l'échelle de la membrane sont une entrave au développement de modèles plus détaillés qui permettraient de décrire complètement le couplage entre écoulement et déformation. Nous avons donc étudié dans une seconde partie la déformation d'enroulement dans le cas de rubans élastiques ayant une courbure spontanée dans différents milieux visqueux et pour différentes conditions élastiques. A grands nombres de Reynolds, en raison de la localisation de la courbure pour les rubans au cours de la propagation du front d'enroulement le long du matériau, nous montrons que l'enroulement atteint rapidement une vitesse de propagation constante. Dans ce régime, le ruban s'enroule sur lui-même de façon compacte, sur un cylindre dont la taille est prévue à partir de la solution de l'onde stationnaire pour l'Elastica. A faible nombre de Reynolds, cependant, se rapprochant des conditions d'enroulement d'une membrane microscopique, nous mettons en évidence l'influence des forces de lubrification sur la nature non-compacte de l'enroulement. La taille globale de la spirale de ruban augmente dans le temps conduisant à une diminution de la puissance élastique libérée et donc à une diminution de la vitesse. Nous discutons dans quelle mesure ces résultats peuvent faire avancer la modélisation de l'enroulement dans les MIRBCs et les vésicules polymère. / Curling deformation of thin elastic surfaces appears in numerous natural and man-made structures where a spontaneous curvature is present. In this thesis, we couple theoretical approaches and macroscopic experiments on elastic ribbons to understand the dynamics of curling of opened bio-membranes, motivated by the need to better understand recent microscopic observations during egress of Malaria infected red blood cells (MIRBC) and bursting of artificial polymersomes.In a first part, we study theoretically pore stability and curling propagation of an initially opened spherical bio-membrane. We model geometrically curling deformation as the revolution of a decentered Archimedean spiral, leading to a prescribed toroidal wrapping of the membrane. In this configuration, we show how the stability of a pore to curling depends strongly on both line-tension and shear elasticity and we discuss these results in relation to the curling of MIRBCs membranes. Moreover, taking into account viscous dissipations, the consequent dynamics we calculate agrees quantitatively well with experimental data obtained during opening of MIRBCs. Our approach shows in particular how the membrane dissipation resulting from the surface redistribution dominates curling dynamics over outer viscous dissipation.However, the complexity of the spherical geometry and the lack of detailed images in microscopic observations hamper the development of more accurate models where the coupling between flow and deformation is fully understood. Subsequently, we study in a second part the curling deformation of macroscopic naturally curved elastic ribbons in different viscous media and elastic conditions. At high Reynolds numbers, due to the tendency of ribbons to localize bending deformations when a curling front travels down the material, we show that curling reaches rapidly a constant propagating velocity. In this regime, the ribbon wraps itself into a compact roll whose size is predicted through the solitary wave solution of the associated Elastica. At low Reynolds numbers, however, closer to the hydrodynamic conditions of curling in microscopic membranes, we show that the strong lubrication forces induce a non-compact curling. The overall size of the spiraling ribbon increases in time leading to a temporal decrease of the released elastic power and therefore a consequent decrease in velocity. We discuss how such discovery sheds a new light on the modeling of curling in MIRBCs and polymersomes.

Le parasite Plasmodium falciparum, responsable des formes graves du paludisme chez l'homme, est transmis par Anopheles gambiae, son principal vecteur en Afrique sub-saharienne. Les nouvelles stratégies de lutte contre la maladie visent à limiter ou à interrompre le développement du parasite chez le moustique vecteur, et il est donc nécessaire d'améliorer notre compréhension des interactions entre le vecteur, son environnement et le parasite. L'objectif de ce projet de thèse a été de caractériser la flore microbienne du vecteur An. gambiae en conditions naturelles de transmission, d'étudier le rôle des principales espèces bactériennes colonisant l'estomac du moustique sur le développement de P. falciparum et de mesurer l'influence des bactéries sur la réponse immunitaire des moustiques femelles et leur capacité à transmettre le parasite. Pour mener à bien ce projet, nous avons collecté des populations de moustiques sauvages au Cameroun pour la caractérisation de la flore microbienne, nous avons ensuite exposé des moustiques de la colonie de laboratoire Ngousso à des cultures bactériennes puis infecté ces moustiques avec des isolats naturels de P. falciparum. Notre étude a montré que les souches bactériennes naturelles de l'intestin du moustique Serratia, Pseudomonas et Escherichia réduisaient la prévalence et l'intensité de l'infection et que le degré d'inhibition variait selon les taxons bactériens et les porteurs de gamétocytes. L'analyse des flores bactériennes des différents épithéliums de l'insecte par pyroséquençage a révélé des similarités entre la flore intestinale et celles retrouvées dans les ovaires et les glandes salivaires pour un même moustique. Les analyses d'expression suggèrent que la régulation de l'expression des gènes l'immunité par les bactéries intestinales pourrait participer à la modulation de la réponse antiplasmodiale. Les mécanismes impliqués dans les interactions bactéries-Plasmodium-vecteur sont complexes et multifactorielle et la modélisation de l'ensemble des interactions qui permettent à P. falciparum d'accomplir son cycle chez le moustique vecteur sera nécessaire pour envisager de nouvelles méthodes de lutte efficaces et durables. / Plasmodium falciparum, the parasite responsible for the severe form of malaria, is transmitted by Anopheles gambiae, its major vector in sub-Saharan Africa. Novel strategies for malaria control envision interrupting the sporogonic development in An. gambiae, then it is important to better understand vector*environment*parasite interactions that underlie parasite transmission. The aim of this project was to characterize the microbial flora of An. gambiae in natural conditions, to study the role of the main bacterial strains on sporogonic development using natural isolates of parasites and to measure the influence of bacterial exposure on the mosquito immunity and its successive ability to transmit P. falciparum. To carry out this project, we used wild mosquito populations from Cameroon to characterize the mosquito microbial flora, next we challenged female mosquitoes of the Ngousso colony to bacterial strains and then infected the mosquitoes with natural isolates of P. falciparum. Our study showed that Serratia, Pseudomonas and Escherichia isolated from the mosquito midgut reduced infection prevalence and intensity and that the effect of the bacterial exposure on parasite infection levels varied between bacterial strains and gametocyte carriers. The analysis of the 454 sequencing of the different mosquito epithelia revealed intriguing similarities between bacterial communities in the midgut, ovaries and salivary glands of a single mosquito. Expression analyses suggested that immune gene regulation by midgut bacteria could help the mosquitoes to mount an effective antiplasmodial response. Mechanisms involved bacteria-Plasmodium-vector interactions are complex and rely on multiple factors. Deeper investigations on these interactions that allow P. falciparum to complete its cycle in the mosquito vector will be necessary for modeling parasite transmission in the field and for developing new methods for effective malaria control.

Magister Public Health - MPH / Background: Cerebral malaria (CM) kills up to 25% of its patients and about one third of its survivors develop neurological impairments (NIs). With advancements in diagnostic and management techniques for CM, more children are likely to survive. The increase in the number of CM survivors may increase the prevalence of children with NIs. In Malawi, rehabilitation for children with NIs is mostly institution-based with erratic community-outreach services, resulting in poor long-term outcomes. To date, community-based rehabilitation (CBR), a comprehensive rehabilitation approach that also addresses socio-economic impact of NIs and may supplement institution-based rehabilitation services, has not been well explored and documented. Presented here are experiences and perceptions on CBR programmes for NIs following CM in Blantyre, Malawi.

Este trabalho observacional teve como objetivo investigar os fatores associados com o desenvolvimento da malária ao longo da gestação e as implicações da infecção por Plasmodium no momento do parto e da malária clínica ao longo da gestação para o crescimento fetal e para o nível de hemoglobina materna no parto, em uma região de alta endemicidade para a doença, onde há cocirculação de P. falciparum e P. vivax, na Amazônia Brasileira. Ao todo 1.180 gestantes participaram das análises de fatores associados ao desenvolvimento de malária gestacional e 1.140 - as gestantes que tiveram filhos nascidos vivos não gêmeos - participaram das análises da associação entre ocorrência de malária gestacional e os desfechos gestacionais abordados neste estudo. A prevalência de malária clínica antenatal, diagnosticada por microscopia de gota espessa, foi de 8,0%, sendo 74,6% dos casos notificados como P. vivax; a prevalência de infecção por Plasmodium no momento do parto, diagnosticada por qPCR, foi de 7,5% - ao todo 12,5% das gestantes que participaram deste estudo tiveram malária clínica antenatal ou infecção por Plasmodium no parto. A grande maioria (89,9%) das infecções por Plasmodium no parto foram assintomáticas. Morar em área rural foi o principal fator associado à malária gestacional na população deste estudo. No que se refere ao impacto da malária para os desfechos de crescimento fetal e nível de hemoglobina no parto, observou-se que malária antenatal provocou redução média de 0,36 (intervalo de confiança [IC] 95%, - 0,57 - -0,14 p = <0,01) no escore z de peso ao nascer, de 0,31 (IC 95%, - 0,54 - -0,08, p = 0,01) no escore z de comprimento ao nascer, e de 0,34 g/100 mL (IC 95%, -0,62 - -0,05 p = 0,02) na concentração de hemoglobina no momento do parto. Foi observado que um único caso de malária vivax antenatal foi suficiente para afetar negativamente o crescimento fetal e a concentração de hemoglobina materna. Quando foram analisados somente os casos de malária vivax repetidas, o efeito negativo foi ainda mais pronunciado. Entre as 637 amostras de cordão umbilical que tiveram diagnóstico molecular feito, 4 (0,6%) foram positivas para P. vivax e 2 (0,3%) para P. falciparum, totalizando uma prevalência de 0,9% de malária congênita na população de estudo. Apenas um dos casos de malária congênita apresentou sintomas. Foram observadas falhas de notificação no banco de dados do sistema de Informação Epidemiológica (SIVEP Malária), e há indicação de falhas no tratamento de malária na gestação no Brasil. Os resultados deste estudo confirmam que a malária vivax na gestação pode ter implicações graves para a saúde materno-fetal. No âmbito local os resultados deste estudo evidenciam a malária na gestação como problema de saúde pública nas regiões endêmicas do Brasil. / The main objectives of this observational study were to investigate the associations between malaria in pregnancy (MiP) and sociodemographic and biological variables of mothers and estimate its impact on birth weight and length and maternal hemoglobin in 1,180 women from Juruá Valley, the main malaria hotspot in Brazil. Antenatal malaria episodes, 74.6% of them due to Plasmodium vivax, were microscopically diagnosed in 8.0% of the women and were associated with an average reduction in birth weight z-scores of 0.36 (95% confidence interval [CI] -0.57 - -0.14 p = <0.01) and in birth length z-scores of 0.31 (CI 95%, - 0.54 - -0.08, p = 0.01), compared with malaria-free pregnancies. Affected mothers had a mean decrease in hemoglobin concentration at delivery of 0.34 g/100 mL (CI 95%, -0.62 - -0.05 p = 0.02). Although repeated antenatal vivax infections were associated with poorer birth outcomes, even a single vivax malaria episode was associated with a significant reduction in birth weight and length and maternal hemoglobin. Overall, 7.5% women had the parasites DNA found in peripheral blood at delivery. Most (83.1%) of these 89 perinatal infections were due to P. vivax and only 7.9% of them progressed to symptomatic disease after delivery. P. vivax and P. falciparum DNA was found in 0.6% and 0.3% of 637 cord blood samples examined, respectively, but only one newborn developed clinical neonatal malaria. Living in rural areas was the main factor associated with gestational malaria in the study population. Moreover, we found further evidence that health-care providers often fail to comply with the countrys malaria treatment guidelines for pregnant women in Brazil. We retrospectively found that Primaquine was prescribed for more than half of the antenatal P. vivax infections diagnosed during the study, not only in the first trimester (when the pregnancy status might be unknown) but also in infections correctly reported as MiP. Our results further challenge the notion that vivax malaria is relatively benign during pregnancy and call for better strategies for its prevention.

A malária, doença causada pelo protozoário do gênero Plasmodium, está entre as doenças que mais causam mortes os países subdesenvolvidosn. O hospedeiro é infectado por meio da picada do mosquito do gênero Anopheles, que introduz o parasita durante a hematofagia. As formas mais graves são causadas pelo Plasmodium vivax e o Plasmodium falciparum. As regiões mais afetadas por estas formas são África Subsaariana, Ásia, América Central e Sul. Desde o começo do século XXI, a Organização Mundial de Saúde (OMS) busca erradicar a doença, porém o P.falciparum se mostrou resistente aos fármacos antimaláricos existentes, dificultando a eficácia do tratamento. Isto, entre outros fatores, como mortalidade e alto índice de infecção, tornam necessárias novas pesquisas para a descoberta de novos fármacos mais seguros e eficazes contra a malária. Estudos têm mostrado como um alvo promissor para a criação de novos antimaláricos, a cisteína protease falcipaína, a qual se apresenta em três isoformas no parasita, sendo elas, falcipaína 1, 2 e 3. A falcipaína 2 está ligada com a hidrólise da hemoglobina, e seus inibidores vem sendo estudados como alternativas na busca de agentes antimaláricos. Derivados de semicarbazona, tais como o nitrofural e o hidroximetilnitrofural demonstraram atividade inibitória de cisteíno proteases parasitárias. Utilizando estratégias modernas de planejamento de fármacos e por meio da integração entre técnicas computacionais e experimentais, realizou-se o planejamento, síntese e avaliação biológica de compostos derivados dos ditiocarbazatos e tiossemicarbazonas, bioisosteros de semicarbazona, como inibidores da cisteíno protease falcipaína 2, no intuito de obter novos antimaláricos. Aplicaram-se técnicas de modelagem molecular em três séries de compostos (A, B e C), sendo a A e B derivados dos ditiocarbazatos e a C das tiossemicarbazonas. Estes estudos sugerem, três compostos da série A, quatro na série B e três na C com maior potencial para inibição da falcipaína 2. Isso devido aos resultados teóricos indicarem condições favoráveis ao ataque nucleofílico da cisteína 42 catalítica da falcipaína 2 às tiocarbonilass presentes nos compostos planejados. Estes derivados foram sintetizados, analisados por espectroscopia de ressonância magnética de 1H e 13C, espectroscopia de IV, ponto de fusão e pureza caracterizando sua formação. Após a obtenção, os compostos foram enviados para ensaios biológicos frente ao parasita P. falciparum. Os compostos testados não apresentaram inibição, porém é sabido que muitos inibidores enzimáticos não são ativos contra o parasita mesmo tendo alta potência contra a enzima, isto devido às barreiras a serem ultrapassadas até chegar ao alvo bioquímico, deste modo faz-se necessário ensaios contra a enzima para validar nossa hipótese. / Malaria, a disease caused by the protozoan of the genus Plasmodium, is among the most deadly diseases in poor countries. The host is infected through the bite of the mosquito of the genus ,i>Anopheles, which introduces the parasite during hematophagy. The most severe forms are caused by Plasmodium vivax and Plasmodium falciparum. The regions most affected by these forms are Sub-Saharan Africa, Asia, Central and South America. Since the beginning of the 21st century, the World Health Organization (WHO) has sought to eradicate the disease, but P. falciparum has been resistant to antimalarial drugs treatment. Among other factors, such as mortality and high infection rates, new research is needed to find new, safer and more effective drugs against malaria. Studies have shown as a promising target for the creation of new antimalarial drugs, the cysteine protease falcipain, which is present in three isoforms in the parasite: falcipain 1, 2 and 3. Falcipain 2 is linked to the hydrolysis of hemoglobin, and its inhibitors have been studied as alternatives in the search for antimalarial agents. Derivatives of semicarbazone such as nitrofural and hydroxymethylnitrofural demonstrated inhibitory activity of parasitic cysteine proteases. Using modern strategies for drug design and the integration of computational and experimental techniques, the design, synthesis and biological evaluation of compounds derived from dithiocarbazates and thiossemicarbazones, semicarbazone biosynthesis as inhibitors of cysteine protease falcipain 2 were carried out in order to new antimalarials. Molecular modeling studies were performed in three series of compounds (A, B and C), with A and B being derived from dithiocarbazates and C from thiossemicarbazones. These studies suggest three compounds in the A series, four in the B series, and three in the C group with the greatest potential for inhibition of falcipain 2. This is due to the theoretical results indicating favorable conditions for the nucleophilic attack of the catalytic cysteine of falcipain 2 on thionyls present in the compounds planned. These derivatives were synthesized, analyzed by 1H and 13C magnetic resonance spectroscopy, IR spectroscopy and melting point, characterizing their formation. After being obtained, the compounds were sent for biological assays against the P. falciparum parasite. The compounds tested did not show inhibition, but it is known that many enzyme inhibitors are not active against the parasite even though they have high potency against the enzyme, this is due to the barriers to be overcome until reaching the biochemical target, thus enzyme to validate our hypothesis.

Thesis advisor: Marc A.T. Muskavitch / Malaria is a life-threatening infectious disease caused by inoculation of the apicomplexan Plasmodium parasite into vertebrate hosts. Transmission of the parasite is mediated by the Anopheles mosquito, which has the capacity to efficiently transmit the parasite from host to host, as the disease vector. There are many factors that make anopheline mosquitoes competent vectors for disease transmission. The hematophagous (blood-feeding) behavior of the female mosquito is one of most fundamental factors in physical transmission of parasites, because the ingestion of blood from an infected host allows parasite entry into the mosquito and the completion of parasite sexual reproduction. In addition to this blood-feeding behavior, there are a host of biological (i.e., parasite replication) and behavioral factors (i.e., mosquito chemosensation, host preference) that contribute to the high vectorial capacity of these vector species. There are over four hundred Anopheles species worldwide, approximately forty of which are considered epidemiologically critical human malaria vectors. Anopheles gambiae, the primary vector in malaria-endemic sub-Saharan Africa, is responsible for the largest number of malaria cases in the world and is therefore one of the most important vectors to study and target with control measures. Currently, vector-targeted control strategies remain our most effective tools for reduction of malaria transmission and incidence. Although control efforts based on the deployment of insecticides have proven successful in the past and are still widely used, the threat and continuing increases of insecticide resistance motivate the discovery of novel insecticides. In this thesis, I provide evidence that G protein-coupled receptors (GPCRs) may serve as “druggable” targets for the development of new insecticides, through the modulation of developmental and sensory processes. In Chapter II, “A critical role for the Drosophila dopamine 1-like receptor Dop1R2 at the onset of metamorphosis,” I provide evidence supporting an essential role for this receptor in Drosophila melanogaster metamorphosis via transgenic RNA interference and pharmacological methods. In An. gambiae, we find that the receptor encoded by the mosquito ortholog GPRDOP2 can be inhibited in vitro using pharmacological antagonists, and that in vivo inhibition with such antagonists produces pre-adult lethality. These findings support the inference that this An. gambiae dopamine receptor may serve as a novel target for the development of vector-targeted larvicides. In Chapter III, “RNAi trigger delivery into Anopheles gambiae pupae,” I describe the development of a method for injection directly into the hemolymph of double strand RNA (dsRNA) during the pupal stage, and I demonstrate that knockdown of the translational product of the SRPN2 gene occurs efficiently, based on reductions in the levels of SRPN2 protein and formation of melanized pseudo-tumors, in SRPN2 knockdown mosquitoes. This method was developed for rapid knockdown of target genes, using a dye-labeled injection technique that allows for easy visualization of injection quality. This technique is further utilized in Chapter IV, “Uncovering the Role of an Anopheles gambiae G Protein-Coupled Receptor, GPRGR2, in the Detection of Noxious Compounds,” where the role for GPRGR2 in the detection of multiple noxious compounds is elucidated. We find that pupal stage knockdown of this receptor decreases the ability of adult Anopheles gambiae to identify multiple noxious compounds. While these findings provide a strong link between GPRGR2 and a very interesting mosquito behavior, they may also provide opportunities to develop better field-based strategies (i.e., insecticides baited traps) for vector control. The goal of this thesis is to understand the functional roles of selected mosquito GPCRs that may serve as targets for the development of new vector-targeted control strategies. Exploiting these GPCRs genetically and pharmacologically may provide insights into novel vector control targets that can be manipulated so as to decrease the vectorial capacity of An. gambiae and other malaria vectors in the field, and thereby decrease the burden of human malaria. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

ABSTRACT: Background: Malaria is endemic throughout Ghana and continues to be a major public health concern especially among pregnant women and children under the age of five. The Ministry of Health (MoH) estimates that over the past ten years, there have been 2-3 million cases of malaria each year, representing 40 percent of outpatient cases, while severe malaria accounts for 33-36 percent of in-patients. Malaria also accounts for 25 percent of the deaths in children under the age of five (GHS, 2001). Correct identification of risk factors could focus interventions at reducing malaria mortality in children. Demographic Surveillance System (DSS) sites have been established and they generate high quality population based longitudinal health and demographic data. The DSS conduct Verbal Autopsies to determine probable causes of death. Objective: This study examines factors affecting childhood malaria mortality in Northern Ghana, using longitudinal data collected by the Navrongo DSS during the period 1995- 2000. It deals especially with the role of socioeconomic factors (mother’s education, family wealth index based on the possessions and housing characteristics and residence, and possession of bed net) and the demographic characteristics (child’s sex and age, and mother’s age). Design: Secondary data analysis of longitudinal data collected by the Navrongo Health Research Centre. Multinomial logistic regression was used to compare the relative risk in three groups of children i.e. those who died of Malaria and those who died of other causes to those who survived as base. Results: Overall, for the deaths due to malaria, older children (1-5years) had a higher risk (RRR 1.4, 95%CI 1.25-1.57 P <0.0001) of dying compared to the infants. Equally, children born of older mothers (maternal age at birth of child >30 years) had a higher risk (RRR 1.28, 95%CI 1.15-1.42 P <0.0001). However, maternal education and residence had a protective effect, with children born of mothers who had some education (RRR 0.79, 95%CI 0.67-0.93 P=0.004) and residing in urban area (RRR 0.61, 95%CI 0.46-0.82 P=0.001) having a lower risk. Similarly, those children whose families are in the highest wealth index had a lower risk (RRR 0.76, 95%CI 0.63-0.91 P=0.003). Interestingly, the same factors were associated with deaths occurring due to other causes, but with varying degree of association. Whereas sex of child was not associated with malaria deaths, being female offered a lower risk of dying from other causes (RRR 0.9, 95%CI 0.84-0.98 P=0.017). It was observed that children in the older age group (1-5 years) were at higher risk of dying (RRR 1.14, 95%CI 1.05-1.25 P=0.002) just as those born of older mothers (RRR 1.16, 95%CI 1.07-1.26 P <0.0001). Even in this group, maternal education (RRR 0.87, 95%CI 0.76-0.98 P=0.023), a higher wealth index (RR 0.87, 95%CI 0.77-0.99 P=0.032 and RRR 0.63 95%CI 0.54-0.73 P <0.0001 for the two highest categories of wealth indices respectively), and area of residence (RRR 0.67, 95%CI 0.55-0.83 P <0.0001) offered a reduction in the risk of dying. Conclusion: The study identified the risk factors (age and sex of the child and mother’s age, maternal education, wealth and residence of the family) associated with malaria mortality and other causes of death in childhood in northern Ghana and this should help formulate cost effective interventions such as health education.

A thesis submitted to the Faculty of Science University of the Witwatersrand Johannesburg in fulfillment for the requirements of the degree of Master of Science. 14 May 2015. / Peptidomimetic compounds have been shown to exhibit both anti-HIV and anti-malarial activity. A multicomponent reaction was used to create a library of peptidomimetic compounds with an α-hydroxy-β-amino acid unit. The Passerini reaction between an aldehyde, carboxylic acid and isocyanide was used to prepare compounds containing both ester and amide functionalities. These compounds were then subjected to a deprotection-acyl migration strategy giving rise to the target compounds. This approach, known as the Passerini Amine Deprotection Acyl Migration (PADAM) sequence was successfully used to create a library of novel peptidomimetic compounds. From this library, 22 compounds were tested for activity against HIV and malaria. The Passerini reaction gives rise to a product containing a new stereogenic centre, and as the starting aldehyde used (N-Boc-phenylalaninal) has a stereogenic centre, the products were isolated as a mixture of diastereomers. Our research was also focused on finding ways of influencing the stereoselectivity of the reaction and the separation of the resulting diastereomers. The diastereomeric ratio of the Passerini products was found to be approximately 2:1 for all the reactions performed. This ratio could be modified slightly when using certain carboxylic acids and isocyanides that were either very bulky or had a stereogenic centre. Attempts to enzymatically resolve the diastereomeric products were not successful after trials using a library of 25 lipase enzymes. However, use of preparative HPLC enabled the successful separation of most of the diastereomeric mixtures, affording compounds with high purity. X-ray crystallography enabled us to identify the major diastereomers as having the R,S configuration, whilst the minor diastereomers had the S,S configuration at the two stereogenic centres. A possible explanation for the observed stereoselectivity is based on the Felkin-Anh chelation control model. It suggests that mono-protected amino aldehydes follow a chelation controlled mechanism in nucleophilic addition reactions. Chelation occurs, albeit in the form of hydrogen bonding, between the NH and carbonyl oxygen. The library of compounds was tested for activity against both HIV-1 and malaria. Only three compounds showed moderate activity against the malaria parasite, inhibiting parasitic growth by 37-42% at 5 μM respectively. Significantly, all of the active compounds contained an adamantyl moiety. Unfortunately no anti-HIV activity was seen for any of the compounds tested in the HIV-assay.

ContexteSi l’efficacité de la stratégie de prise en charge du paludisme au niveau communautaire n’est pas remise en cause à Madagascar, la mise en place de la nouvelle politique nationale incluant le remplacement de la chloroquine par la combinaison artésunate plus amodiaquine et l’introduction des tests de diagnostic rapide (TDR) dans la prise en charge des fièvres chez les enfants de moins de cinq ans suscite plusieurs inquiétudes. ObjectifsLe principal objectif de notre travail a été d’évaluer la stratégie de prise en charge des fièvres chez les enfants de moins de 5 ans au niveau communautaire. Pour évaluer la stratégie de prise en charge,trois objectifs spécifiques ont été abordés, à savoir, (i) l’étude de l’efficacité thérapeutique de la combinaison fixe « artesunate-amodiaquine » dans la prise en charge des fièvres chez les enfants de moins de 5 ans au niveau communautaire, (ii) l’évaluation des performances des tests de diagnostic rapide utilisés au niveau communautaire et (iii) la description de la situation épidémiologie du paludisme au niveau communautaire et l’évaluation de l’impact des stratégies de lutte actuellement mises en œuvre.MéthodologiesDeux études longitudinales ont été mises en place. La première étude (objectifs 1 & 2) a été conduite de février 2008 à février 2010 (24 mois) chez les enfants de moins de 5 ans en zone de transmission stable (district de Manakara) et en zone de transmission instable (district de Moramanga). La deuxième (objectif 3) s’est déroulée entre février 2009 et mars 2011 en zone de transmission stable (district de Manakara). Résultats & ConclusionObjectif 1 : les données obtenues lors de notre étude, nous permettent d’affirmer l’excellente efficacité thérapeutique de la combinaison fixe « artesunate-amodiaquine ». Nous avons observé que le taux de guérison clinique global était de 98,4% au bout de 28 jours et 97,9% au bout de 42 jours. La compliance au traitement a été estimée à 83,4%. Aucun effet indésirable grave n'a été observé. : Objectif 2 : Cette étude a permis de confirmer que les performances diagnostiques des agents de santé communautaire utilisant les TDR en termes de sensibilité, spécificité, VPP et VPN étaient supérieures à 85%, que la concordance entre les résultats de la microscopie et des TDRs, estimée par la valeur Kappa était excellente (83%) et que les TDR étaient stable même conservés au niveau communautaire. L’introduction des TDR au niveau communautaire semble être une stratégie efficace pour améliorer la prise en charge des malades fébriles, pour réduire la surconsommation d’antipaludiques (et donc le coût des traitements inutilement utilisés) et pour réduire la pression de sélection exercée par cette surconsommation. Objectif 3 : Nous avons pu démontrer que les mesures de prévention et de traitement prises au niveau communautaire étaient efficaces avec la mesure objective de la réduction de la prévalence du paludisme. Nous avons également mis en évidence une variation importante de la prévalence du paludisme inter-villages dans une même commune de quelques kilomètres carrés, suggérant des interventions ciblées en fonction des risques liés à la situation géographique, agricole, et climatique. Ce travail a permis de proposer une première approche méthodologique qu’il serait souhaitable d’étendre pour collecter les données de prévalence et d’incidence dans les autres communes, et préciser ainsi les besoins ciblés en TDR et ACTs à Madagascar. / Context:One should think back of the effectiveness of malaria case management strategy at the community level Otherwise, questions will remain unanswered on the setting up of the new national Policy including the replacement of the chloroquine with combination artesunate and amodiaque and the introduction of rapid diagnostic test (RDT) for fever case management among children under five.Objective:The principal goal of our essay is to assess fever case management strategy among children under five years old at the community level. To proceed to the assessment, three specific objectives were tackled: (i) Therapeutic effectiveness of the fixed combination (artesunate-amodiaque) in fever case management among children under five at the community level. ii)the assessment of RDT performances used at the community level and iii) description of malaria epidemiological situation at the community level and the assessment of the impact of the fight against malaria strategies implementedMethodology: Two long depth studies were applied. The first study (Objective 1 and 2) was conducted in February 2008 to February 2010 (24 months) among children under five years old in stable transmission area (Manakara district) and in changeable transmission area (Moramanga district). The second study (objective 3) was held in February 2009 and March 2011 in stable transmission area (Manakara).Outputs and Conclusion:Objective 1: Data collected during our study enabled us to confirm the excellent therapeutically effectiveness of the fixed combination “artesunate-amodiaque”. We have noticed that the global clinical recovery rate is 98.4% after 28 days and 97.9% after 42 days of treatment. Compliance with the treatment was estimated at 83.4%. Not any adverse effect was noticed.Objective 2: This study enabled us to confirm the diagnostic performances of community health workers using RDT in terms of sensitiveness, specification. VPP and VPN were more 85% than the reliability between microscopic results and the RDTs estimated by the kappa value was excellent (83%) and RDTs were stable even kept at the community level. The introduction of RDT at the community level seems to be an effective strategy to improve sick people case management, to reduce overconsumption of products anti-malaria (and so reduce the cost of treatments used uselessly) and to reduce the selection pressure from this overconsumption.Objective 3: We could prove that prevention and treatment measures at the community level were effective with the objective measure of reducing malaria rate. We could bring to evidence an important variation of malaria rate between villages of one same commune from few kilometers of distance, suggesting targeted interventions depending on the risks linked to the geographic, agricultural and climate situations. This work enabled us to suggest a first methodological approach that is better to extend to collect prevalence data and in adverse circumstances in other communes and to mention the RDT targeted needs and ACTs in Madagascar.

O Antígeno 1 de Membrana Apical (AMA-1) é um dos mais promissores candidatos a vacina contra a fase sanguínea da malária. No presente estudo, geramos uma nova proteína recombinante baseada no ectodomínio de AMA-1 de P. vivax (PvAMA-1) a partir do gene sintético com códon otimizado para expressão secretada na levedura Pichia pastoris. Por ELISA, a proteína PvAMA-1 foi reconhecida por 62,5% dos soros de pacientes da Amazônia brasileira infectados por P. vivax. A imunogenicidade de PvAMA-1 foi avaliada em camundongos BALB/c, utilizando protocolos homólogos e heterólogos de indução e reforço com DNA plasmidial e/ou proteína recombinante, emulsificada em Adjuvante de Freund. Os resultados mostraram que a eficiência da imunização é dependente da presença da proteína emulsificada em adjuvante forte. As imunizações subseqüentes foram realizadas com a proteína recombinante emulsificada em diferentes adjuvantes: sais de alumínio (Alum), Monophosphoryl Lipid A (MPLA) de Bordetella pertussis, flagelina FliC de Salmonella Typhimurium, saponina Quil A e Adjuvante Incompleto de Freund (AIF). Os resultados demonstraram que as imunizações na presença de Quil A e AIF foram capazes de induzir altos títulos de anticorpos IgG e uma resposta de isotipos de IgG mais balanceada, caracterizando um padrão do tipo Th1/Th2. Títulos de anticorpos mais baixos, mas também expressivos, foram obtidos na presença de Alum, MPLA, Alum + MPLA e Alum + FliC. A análise da resposta proliferativa, por citometria de fluxo utilizando marcação com CFSE, mostrou que células esplênicas CD3+CD4+, assim como CD3+CD8+, de animais imunizados com PvAMA-1 na presença dos adjuvantes Alum, Alum + MPLA, Quil A e AIF foram capazes de proliferar especificamente in vitro. Por imunofluorescência, soros policlonais de camundongos imunizados com o antígeno na presença de MPLA e Quil A foram capazes de reconhecer a proteína nativa expressa em isolados de P. Vivax da Ásia. Visando futuros testes pré clínicos em primatas não humanos, a proteína PvAMA-1 foi produzida em boas práticas de laboratório (BPL) por uma companhia especializada e o potencial imunogênico da proteína foi confirmado em imunizações posteriores. Em conjunto, nossos resultados demonstram que PvAMA-1 é um antígeno promissor para ser explorado em protocolos de imunização contra malária vivax, individualmente, ou em combinação com outros antígenos. / The Apical Membrane Antigen 1 (AMA-1) is one of the most promissing vaccine candidates against the erythrocytic stage of malaria. In the present study we generated a new recombinant protein based on AMA-1 ectodomain of P. vivax (PvAMA-1) using a synthetic codon optimized gene for yeast secreted expression. By ELISA, the protein PvAMA-1 was recognized by 62,5% of the sera from Brazilian Amazonia patients infected by P. vivax. The immunogenicity of PvAMA-1 was evaluated in BALB/c mice using homologous and heterologous protocols of prime and boost with plasmidial DNA and/or recombinant protein emulsified in Freund adjuvant. The results showed that the efficiency of immunization is dependent on the presence of a strong adjuvant. The following immunizations were conducted using the protein emulsified in different adjuvants: aluminium salts (Alum), Monophosphoryl Lipid A (MPLA) of Bordetella pertussis, flagelin FliC of Salmonella Typhimurium, saponin Quil A and Incomplete Freund\'s Adjuvant (IFA). The results showed that immunizations in the presence of Quil A e IFA were able to induce high IgG titers and a more balanced IgG isotypes response, featuring a standard type Th1/Th2. Lower antibody titers, but also significant, were obtained in the presence of Alum, MPLA, Alum + MPLA and Alum + FliC. The proliferative cellular analysis, by flow cytometry using CFSE staining, showed that splenic cells CD3+CD4+, as well as CD3+CD8+ from immunized mice that received PvAMA-1 with Alum, Alum + MPLA, Quil A and IFA were specifically able to proliferate in vitro. By immunofluorescence, the polyclonal sera from mice immunized with the antigen in the presence of MPLA and Quil A were able to recognize native protein expressed in Asian P. vivax isolates. Aiming future pre-clinical assays in non-human primates, the protein PvAMA-1 was produced in good laboratory practices (GLP) conditions by a specialized company and its immunogenic efficiency was confirmed in later immunizations. Altogether, our results showed that PvAMA-1 is a promissor antigen to be explored in immunization protocols against vivax malaria, individually, or combined with other antigens.