Casos de malária importada, contraídos em região endêmica, mas diagnosticados em um país não-endêmico, são um evento raro mas com desfecho potencialmente fatal. Nosso objetivo foi investigar se a análise de genomas mitocondriais permite inferir a origem geográfica de casos importados de malária vivax diagnosticados nos EUA, comparando os resultados com aqueles obtidos por análise de DNA microssatélite. Foi sequenciado o genoma mitocondrial completo de 63 amostras de P. vivax provenientes de infecções importadas dos EUA, além de 7 amostras do Brasil e 6 do Panamá. A rede de haplótipos com DNA mitocondrial foi construída com 412 sequências e foi possível classificar com precisão a origem geográfica presumida dos isolados da América do Sul, Coréia, Sudeste Asiático e Melanésia, porém os isolados do Sul da Ásia, América Central e África não puderam ser classificados geograficamente. A análise bayesiana realizada com a tipagem de marcadores de microssatélites não apresentou sucesso quanto à classificação geográfica dos isolados de P. vivax. / Cases of imported malaria, infection acquired in an endemic region, but diagnosed in a non-endemic country, are rare but can lead to a fatal outcome. Our objectives were investigate whether the analysis of mitochondrial genomes allows inferring the geographic origin of isolates of P. vivax derived from cases of imported malaria diagnosed in the USA, and compare the performance of mitochondrial genome and DNA microsatellite analysis. We sequenced full mitochondrial genomes from 63 P. vivax isolates collected at the USA from imported infections, and 7 samples from Brazil and 6 Panama. A network of mitochondrial DNA haplotypes was built with 412 genomic sequences and were able to classify accurately isolates from South America, Korea, Southeast Asia and Melanesia according to their presumed geographic origin, but failed to do so with samples from South Asia, Central America and Africa. The Bayesian analysis performed by typing microsatellite markers showed no success on the classification of geographical isolates of P. vivax.

A proteína de ligação em Duffy (PvDBP) dos merozoítos de P. vivax se liga a glicoproteínas de membrana, chamadas de grupo sanguíneo Duffy, conhecidas como DARC. Indivíduos DARC negativos são normalmente resistentes a infecção estágio sanguínea por P. vivax; portanto, PvDBP é um forte antígeno canditato a vacina. Aqui, investigamos respostas de anticorpos contra três proteínas derivadas de PvDBP e MSP119, em 343 indivíduos de uma região Amazônica brasileira. Anticorpos contra Sal III-His, OII-His, Sal III-IV-GST e MSP119-GST foram encontrados em 43,7%, 39,0%, 14,3% e 38,8% dos indivíduos. Os indivíduos FY*BESFY*BES foram os que menos apresentaram anticorpos contra PvDBP, porém encontramos proporções similares de respondedores entre indivíduos com outros genótipos. A análise de sequências revelou muitas variantes da região II de PvDBP em 41 isolados. A mais comum (encontrada em 28,8% dos isolados), difere de Sal I e PNG- O em seis codóns de aminoácidos. Nenhuma variante tipo Sal I, cujo protótipo de vacina está em teste clínico, foi encontrada nos parasitas locais. / The Duffy binding protein (PvDBP) of the P. vivax merozoites, which binds to a erythrocyte membrane glycoprotein known as Duffy blood group antigen for chemokines (DARC). DARC-negative individuals are usually resistant to blood-stage infection with P. vivax; therefore, PvDBP is a major vaccine candidate antigen. Here, we investigated antibody responses to three proteins derived from PvDBP and MSP119, in 343 subjects in the Amazon of Brazil. Antibodies to Sal III-His, OII-His, Sal III-IV-GST and MSP119-GST were found in 43,7 %, 39,0%, 14,3% and 38,8% of the subjects. FY*BESFY*BES individuals were less likely to have antibodies to PvDBP, but we found similar proportions of seropositive subjects with other genotypes. Sequence analysis revealed several region II PvDBP variants in 41 local P. vivax isolates. The most common of them (found in 28,8% isolates) differs from Sal I and PNG-O alleles in six amino acid codons. No Sal I-type variant, from which a PvDBP-based vaccine prototype currently under clinical testing has been derived, was found in local parasites.

Plasmodium is a genus well known for causing malaria, a life-threatening infection for many people where malaria is endemic. The blood-borne disease is transmitted by the female Anopheles mosquito. Till date, eight parasite species have been reported to cause malaria in humans that include P. falciparum, P. vivax, P. malariae, P. ovale curtisi, P. ovale wallikeri, P. cynomolgi, P. knowlesi and more recently P. simium. Amongst them, the most genetically understood species is P. falciparum, causing most of the deaths in children from malaria. Understanding genome variation at the population level of all malaria species is of utmost importance, including clinical cases with very low parasitemia. To achieve this purpose, we need sufficient amounts of parasite DNA material from the pool of host DNA, which always is overrepresented in clinical infections. We utilized a strategy of selective whole genome amplification (SWGA) technology on P. malariae and P. ovale curtisi (two neglected human infecting malaria parasites that often cause mild yet clinically relevant infections with low parasitemia) to efficiently enrich their genomic DNA for high-quality whole genome sequencing. Previous studies on SWGA applied on P. falciparum and P. vivax showed that SWGA could efficiently enrich the amount of starting DNA material from inadequate amounts of parasites directly from clinical samples without separating the host DNA using specifically designed primer sets. We have successfully designed multiple sets of primers and tested the efficiency of five best primer sets using polymerase chain reaction to enrich the genomes of P. malariae and P. ovale curtisi. The efficiency of primers in enriching the genome was tested on two clinical samples for each of P. malariae and P. ovale curtisi. We were able to enrich the genome of P. malariae with an average of 19-fold (19X) enrichment across both samples. For P. ovale curtisi, we could achieve an enrichment of 3 folds only. Nevertheless, we still obtained a sufficient amount of gDNA to prepare Illumina sequencing libraries and call for SNPs and Indels in a biologically reproducible manner at genome-scale.

MSc (Biochemistry) / Department of Biochemistry / Malaria is caused by protozoa of the genus Plasmodium. Malaria accounts for approximately more than half a million deaths yearly. Of the five species of Plasmodium, P. falciparum accounts for the most deadly form of the disease. P. falciparum survives under various physiological conditions during its life cycle. The parasite employs its molecular chaperones machinery particularly heat shock proteins (Hsps) to protect its protein constituents during physiological stress. Hsps are conserved molecules that constitute a major part of the cell’s molecular chaperone system. P. falciparum Hsps play an important cyto-protective role guaranteeing that the malarial parasite survives under the severe conditions that prevail in the host environment. PFF1010c is a type IV P. falciparum heat shock protein 40. PFF1010c is predicted to be expressed only at the gametocyte stage of the malarial parasite’s life cycle. The aim of the current study was to investigate the expression PFF1010c by parasites and the gametocyte stage as well as characterize the structure-function features of the protein. PFF1010c was successfully expressed in E. coli cells. Despite successful expression of the protein, its purification proved problematic. The attempt to purify PFF1010c was carried out under both native and denaturing conditions. Far Western blot analysis to investigate direct interaction between PFF1010c and PfHsp70-1 was conducted and no interaction was observed. Malarial parasites were harvested at different stages and total protein was isolated. The expression of PFF1010c was confirmed to occur at the gametocyte stage of the parasite’s development using Western blot analysis. This study confirmed that PFF1010c is only expressed at the gametocyte stage of the malarial parasite. Furthermore, PFF1010c was not expressed at the asexual stage. Possible interactors of PFF1010c were predicted by STRING, a bioinformatics based tool. The expression of PfHsp90, PfHop and PfHsp70-1 at the gametocyte stage was investigated and confirmed by Western blot analyses.

MSc (Chemistry) / Department of Chemistry / See the attached abstract below

La découverte de l'Artémisinine, un 1,2,4-trioxacyclohexane, et le fait que la liaison endoperoxide est essentielle pour son activité antimalarique, a conduit les chimistes à la synthèse de nouvelles molécules contenant le motif endoperoxide pour obtenir de nouveaux leaders. Certains de ces composés peroxidiques sont les 1,2-dioxanes et les 1,2,4,5-tetraoxanes. À cet égard, la première partie de mon travail a été réalisée dans le laboratoire du professeur Giuseppe Campiani à l'université de Sienne et a inclus la synthèse d'analogues 1,2-dioxane du produit naturel Plakortine mais aussi le développement d'une stratégie de synthèse polyvalent du produit naturel 9,10-dihydroplakortine. Une des étapes clés dans notre approche synthétique a été la stéréosélectivité des réactions ainsi que la formation du squelette chiral 1,2-dioxane. En combinant l'époxidation dissymétrique de Sharpless à l'hydroperoxysilylation régiosélective d'alcène catalysée par le cobalt (II) de Mukaiyama-Isayama, la stéréochimie désirée a été obtenue. Dans la seconde partie de mon doctorat qui a pris place dans le laboratoire du professeur Paul O'Neill à l'université de Liverpool, la conception, la synthèse et l'évaluation du potentiel antimalarique de deux nouvelles séries de 1,2,4,5-tetraoxanes ont été réalisées. La première série de tetraoxanes, appelée les Mannoxanes, est une drogue hybride qui possède un noyau tetraoxane et une chaine latérale basique insérée grâce à une réaction de mannich. La seconde série a été préparée par une approche utilisant l'amination réductrice pour inclure la chaine latérale basique sur le noyau tetraoxane. Les deux séries ont montré d'excellentes activités antimalariques (de l'ordre du nanomolaire) contre plasmodium falciparum. / The discovery of artemisinin, a 1,2,4-trioxacyclohexane, and the fact that the endoperoxide bond is essential for its antimalarial activity has led chemists to synthesize new molecules containing the endoperoxide moiety in order to find new antimalarial hits. Some of these peroxidic compounds include the 1,2-dioxanes and the 1,2,4,5-tetraoxanes. In this regard, work on the first part of the PhD (Chapter 2) has been developed in the laboratories of Prof. Giuseppe Campiani at the University of Siena and includes the synthesis of 1,2-dioxane analogues of natural product Plakortin and the development of a versatile synthetic strategy to the natural compound 9,10-dihydroplakortin. One of the key issues in our synthetic approach has been the stereoselectivity of the reactions and the formation of the chiral 1,2-dioxane skeleton. By combining Sharpless asymmetric epoxidation to the Mukaiyama-Isayama Co(II)-catalyzed regioselective hydroperoxysilylation of an alkene, the desired stereochemistry has been obtained. In the second part of the PhD (Chapter 3), which has taken place in the laboratories of Prof. Paul O'Neill at the University of Liverpool, design, synthesis and assessment of the antimalarial potency of two new series of 1,2,4,5-tetraoxanes has been achieved. The first series of novel tetraoxanes are called Mannoxanes and are hybrid drugs that include a tetraoxane and a mannich basic phenol side-chain. The second new series has been synthesized by using a reductive amination approach to include the basic side chain in the tetraoxane molecule scaffold. Both of these series have shown excellent antimalarial activities against Plasmodium falciparum in the low nanomolar range.

Background: Malaria is a major public health issue. Artemisinin-based combination therapies are the WHO recommended treatment for uncomplicated malaria. Plasmodium malariae and Plasmodium ovale infections are considered underestimated and the effectiveness of artemisinin-based combination treatments against them is poorly documented. The aim of this study was to evaluate the efficacy of dihydroartemisinin-piperaquine against low parasite density Plasmodium malariae and ovale infections.  Methods: DNA was extracted from dried blood spots on filter papers with Chelex®-100 or a column-based extraction method. Species detection and determination was conducted by SYBR Green quantitative PCR targeting the cytochrome b gene (cytb-qPCR) followed by restriction fragment length polymorphism analyses. In total, 241 samples from 53 patients enrolled in a clinical trial were analysed. The obtained molecular data were compared with the microscopy data of the study. Results: Only 69 out of 143 microscopy-positive samples were confirmed as positive by cytb-qPCR. Ninety-three samples were identified as parasite negative by both microscopy and PCR. None of the 36 microscopy-defined coinfections were detected in the molecular analysis. The cytb-qPCR success rate was 72.9% (CI95% 61.4-82.6), 75.0% (CI95% 34.9-96.8) and 14.8% (CI95% 6.9-26.2) for parasite densities above 1000 parasites/ μL, between 600-1000 parasites/ μL and below 600 parasites/ μL, respectively. The observed poor qPCR success rate is most likely due to sample degradation under poor storage conditions. Conclusions: This study highlights the impact on the preservation and quality of Plasmodium genomic DNA on dried blood spots, when filter papers are stored for more than 3 years in tropical conditions.

En 2016, 216 millions de cas et 445000 décès liés au paludisme ont été reportés par l'Organisation Mondiale de la Santé (OMS), 90 % de ces cas ont eu lieu sur le continent africain, très majoritairement causés par Plasmodium falciparum. Chez les femmes enceintes le paludisme peut avoir de lourdes conséquences en termes de morbidité et de mortalité aussi bien pour la mère que pour l'enfant à venir. Outre l'utilisation de moustiquaires imprégnées, l'OMS recommande l'utilisation d'un traitement préventif intermittent par sulfadoxine-pyriméthamine (SP) pour les femmes enceintes des zones endémiques d'Afrique sub-saharienne. La résistance du parasite à la SP est un réel problème en l'absence d'alternative thérapeutique et le niveau de résistance est déjà très élevé dans certaines régions d'Afrique. Cette résistance est causée par différents polymorphismes nucléotidiques simples (SNPs) sur les gènes codant pour DHPS et DHFR. Ces mutations modifient la structure 3D de ces enzymes, diminuant leur liaison à la SP. Ce travail s'est attaché à faire un état des lieux récent de la prévalence des mutations des gènes pfdhfr et pfdhps à travers 7 sites d'Afrique Centrale. Un octuple mutant combinant trois mutations sur le gène pfdhfr et cinq mutations sur le gène pfdhps (CirnI + vagKgs) a été découvert à Yaoundé en 2015 ; nous l'avons retrouvé au Nigeria et au Cameroun mais pour la première fois à un haut niveau de prévalence à Maroua (52,2%). Sur plusieurs sites d'Afrique Centrale, nous décrivons l'augmentation de la prévalence de la mutation K540E, typique des parasites résistants de l'Afrique de l'Est et sur laquelle se base l'implémentation de la SP par l'OMS. D'autre part, à partir d'un modèle d'homologie de pfDHPS, nous avons utilisé des techniques de dynamique moléculaire pour mieux comprendre les modifications de la structure 3D liées à différents haplotypes d'intérêts retrouvés en Afrique Centrale. Cette partie de l'étude propose une autre approche pour évaluer et visualiser l'effet structurel des mutations, élément supplémentaire à la compréhension de leur impact sur la résistance à la SP. / Malaria was responsible for 445 000 death and 216 million new cases worldwide in 2016, 90% of these cases occurred in Africa. Pregnant women are particularly susceptible to malaria, resulting in medical consequences on both mother and child. In the endemic countries of sub Saharan Africa, in addition to the use of insecticidal nets, the World Health Organization (WHO) recommends a preventive treatment against Plasmodium falciparum malaria in pregnancy using Sulfadoxine Pyrimethamine (SP). Resistance of the parasite to SP is a significant problem in the absence of alternative treatment and the level of resistance is very high in some regions of Africa. This resistance is mediated by several single nucleotide polymorphisms (SNPs) in the genes coding for DHPS and DHFR, changing the 3D structure of the two target proteins and decreasing drug binding. This study offers an updated view of the epidemiology of these mutations throughout Central Africa. We identified for the first time the octuple mutant combining 3 mutations on pfdhfr gene and 5 mutations on pfdhps gene (CirnI + vagKgs), discovered in 2015 in Yaoundé, as the major haplotype in Maroua, northern Cameroon (52,2%). On several Central-African sites, we describe the increase of the prevalence of the typically East-African K540E mutation on which is based the implementation of SP by the WHO. Knowledge of the prevalence of resistance markers is crucial for the adaptation of SP recommendation in Central Africa. This study also explores the changes occurring in the 3D structure of the most common mutated pfDHPS haplotypes identified in our epidemiology study, using molecular dynamics on a homology model of the protein. It offers a new vision of the effect of the mutations on the structure, a further step to understanding the impact of mutations in resistance.

No description available.

Yes / The prospect of eradicating malaria continues to be challenging in the face of increasing parasite resistance to antimalarial drugs so that novel antimalarials active against asexual, sexual, and liver-stage malaria parasites are urgently needed. In addition, new antimalarials need to be affordable and available to those most in need and, bearing in mind climate change, should ideally be sustainable. The West African climbing shrub Cryptolepis sanguinolenta is used traditionally for the treatment of malaria; its principal alkaloid, cryptolepine (1), has been shown to have antimalarial properties, and the synthetic analogue 2,7-dibromocryptolepine (2) is of interest as a lead toward new antimalarial agents. Cryptolepine (1) was isolated using a two-step Soxhlet extraction of C. sanguinolenta roots, followed by crystallization (yield 0.8% calculated as a base with respect to the dried roots). Semi-synthetic 7-bromo- (3), 7, 9-dibromo- (4), 7-iodo- (5), and 7, 9-dibromocryptolepine (6) were obtained in excellent yields by reaction of 1 with N-bromo- or N-iodosuccinimide in trifluoroacetic acid as a solvent. All compounds were active against Plasmodia in vitro, but 6 showed the most selective profile with respect to Hep G2 cells: P. falciparum (chloroquine-resistant strain K1), IC50 = 0.25 µM, SI = 113; late stage, gametocytes, IC50 = 2.2 µM, SI = 13; liver stage, P. berghei sporozoites IC50 = 6.13 µM, SI = 4.6. Compounds 3–6 were also active against the emerging zoonotic species P. knowlesi with 5 being the most potent (IC50 = 0.11 µM). In addition, 3–6 potently inhibited T. brucei in vitro at nM concentrations and good selectivity with 6 again being the most selective (IC50 = 59 nM, SI = 478). These compounds were also cytotoxic to wild-type ovarian cancer cells as well as adriamycin-resistant and, except for 5, cisplatin-resistant ovarian cancer cells. In an acute oral toxicity test in mice, 3–6 did not exhibit toxic effects at doses of up to 100 mg/kg/dose × 3 consecutive days. This study demonstrates that C. sanguinolenta may be utilized as a sustainable source of novel compounds that may lead to the development of novel agents for the treatment of malaria, African trypanosomiasis, and cancer.