Le paludisme est causé par l'infection et la destruction des érythrocytes par les parasites protozoaires appartenant au genre Plasmodium. Au cours de son développement dans l'érythrocyte,Plasmodium falciparum requiert la biosynthèse massive de membranes dont les principaux constituants lipidiques sont des phospholipides. La phosphatidylcholine (PC) et la phosphatidyléthanolamine (PE) représentent à elles deux environ 80 % des lipides membranaires et l'inhibition de leur biosynthèse est létale pour le parasite. La PC et la PE sont synthétisées par le parasite, principalement via les voies de novo dépendantes de la CDP-choline et de la CDP-éthanolamine (ou voies de Kennedy) en utilisant respectivement la choline et l'éthanolamine comme précurseurs. Ces travaux de thèse se focalisent sur les deux enzymes CTP:phosphocholine etCTP:phosphoéthanolamine cytidylyltransférase (PfCCT et PfECT, respectivement), catalysant les étapes limitantes des voies de Kennedy. Chez Plasmodium, les CCT et ECT possèdent deux domaines cytidylyltransférases (CT) portant l'activité catalytique, séparés par une longue région de liaison. Pour la CCT, cette duplication est retrouvée seulement chez trois organismes, tous faisant partie du phylumdes Apicomplexes : Babesia, Theileria et Plasmodium, alors que la présence de deux domaines CT estune caractéristique retrouvée chez toutes les ECT étudiées à ce jour. La première partie de ce travail de thèse concerne la caractérisation biochimique et l'inhibition la PfCCT Nous avons montré que les deux domaines CT de la PfCCT sont actifs à l'inverse de la PfECT pour laquelle seul le domaine CTN-terminal est catalytiquement actif. A la suite d'un criblage virtuel basé sur la structure de l'enzyme,nous avons identifié un composé princeps capable d'inhiber l'activité de la PfCCT in vitro, la synthèse de PC et la croissance parasitaire. Ce premier composé actif (haut µM) représente une base pour l'optimisation future de nouveaux composés plus efficaces. Dans la deuxième partie de cette thèse,nous avons déterminé le mécanisme catalytique, la spécificité de liaison des ligands et l'organisation structurale de la PfECT grâce à la combinaison d'approches biochimiques et biophysiques. L'ensemble des résultats présentés dans ce manuscrit apportent un éclairage important concernant le fonctionnement de ces deux cibles potentielles et constituent des étapes essentielles à l'élaboration d'une approche thérapeutique. / Malaria is caused by the infection and destruction of red blood cells by protozoan parasitesbelonging to the genus Plasmodium. During its intra-erythrocytic development, Plasmodiumfalciparum requires massive biosynthesis of membranes which are mainly composed of phospholipids.Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) together represent about 80% of thetotal membrane lipids and inhibition of their biosynthesis leads to parasite death. PC and PE aresynthesized by the parasite's machinery mainly through the de novo CDP-choline and CDPethanolamine(Kennedy) pathways using respectively choline and ethanolamine as precursors. Thisstudy focuses on the rate limiting steps of these pathways catalyzed by CTP:phosphocholine andCTP:phosphoethanolamine cytidylytransferases (PfCCT and PfECT, respectively). In Plasmodiumspecies, both CCT and ECT contain two catalytic cores (CT domains) separated by a long linker.Interestingly, for CCT this feature is found only in three organisms, all from the phylum ofApicomplexa: Babesia, Theileria and Plasmodium, whereas the presence of two CT domains is ageneral feature in all ECTs known so far. The first part of this work consists in the biochemicalcharacterization of PfCCT and the investigation of its druggability. We showed that both PfCCT CTdomains are active and display similar kinetic parameters while only the N-terminal CT domain wasactive in PfECT. Subsequent to an in silico structure-based screening of compounds libraries, weidentified a PfCCT inhibitor able to inhibit PC synthesis as well as P. falciparum growth in vitro in thehigh µM range. This compound represents a first step toward the optimization of future more potentcompounds. In the second part of this study, we investigated the catalytic mechanism of PfECT anddeciphered its interactions with its ligands using biochemical, biophysical and structural approaches.Collectively, these results bring new insights into the biochemical and structural properties of thesetwo keys enzymes of the phospholipid metabolism in P. falciparum and pave the way for their futuredevelopment as potential drug target.

No description available.

L’artésunate est désormais le traitement de référence du paludisme grave au plan mondial. Cependant, des cas d’anémie hémolytique différée ont été décrits chez 20% à 25% des voyageurs traités. L’épisode hémolytique survient 2 à 3 semaines après traitement. Environ la moitié des patients vont nécessiter une transfusion sanguine. L’artésunate induit un phénomène original en physiologie humaine : le “pitting” ou épépinage splénique des érythrocytes parasités. Il consiste en l’expulsion du parasite mort de l’érythrocyte hôte lorsque celui-ci traverse une structure microcirculatoire splénique appelée « fente interendothéliale ». Ces érythrocytes pittés retournent sans destruction immédiate dans la circulation générale. Nous avons étudié l’efficacité et la tolérance de l’artésunate intraveineux chez 123 voyageurs atteints de paludisme grave. Cent dix-sept patients ont survécu (95%). Parmi 78 patients suivis plus de 8 jours, 76 (97%) ont eu une anémie au cours du suivi et 21 une hémolyse différée typique (27%). Dans ce sous groupe de patients la chute médiane en hémoglobine a été de 1,3g/dl avec un nadir <7g/dl dans 15% des cas. Un seul patient a été transfusé. Le marquage de la protéine parasitaire Resa, véritable empreinte de l’infection érythrocytaire par Plasmodium falciparum, permet la visualisation des érythrocytes pittés. Chez 21 patients non transfusés le pic de concentration en érythrocytes pittés est survenu durant la première semaine. Chez 9 patients évoluant vers une hémolyse différée le pic de pittés était significativement plus élevé que chez 12 patients présentant d’autres profils évolutifs d’anémie (0,30 vs. 0,07 ; P = 0,0001). Une concentration d’érythrocytes pittés au pic supérieure à 180 millions/l aurait prédit le risque d’hémolyse différée avec une sensibilité de 89% et une spécificité de 83%. Utilisant la technologie ImageStream* l’étude morphologique érythrocytaire chez 4 patients a montré que l’infection plasmodiale suivi de pitting entraine une réduction de surface projetée de 8,9%. Cette altération pourrait contribuer à la réduction de la durée de vie des érythrocytes pittés. La destruction différée des érythrocytes infectés et épargnés par le pitting durant le traitement par artésunate est un mécanisme original d’anémie hémolytique. Ce travail a permis de structurer l’espace nosologique de l’anémie post-thérapeutique au cours du paludisme, de clarifier la physiopathologie de l’hémolyse différée et d’identifier certains de ses mécanismes. Malgré l’incidence élevée de l’hémolyse différée, l’anémie résultante n’est préoccupante que dans 15% des cas et ne remet pas en cause le bénéfice de l’artésunate par rapport à la quinine dans le traitement du paludisme grave. La concentration précoce des érythrocytes pittés pourrait être un marqueur prédictif intéressant de la survenue d’une hémolyse différée post-artésunate. / Worldwide, artesunate is now the recommended treatment for severe malaria. However cases of delayed hemolytic anemia have been described in 20% to 25% travelers treated with artesunate. The episode usually occurs 2 to 3 weeks after the end of the treatment. About half on the inpatients need blood transfusion. Artesunate induces an original phenomenon called splenic “pitting” of parasitized erythrocyte. The dead parasite is expelled from the host erythrocyte when it comes through a microcirculatory structure called inter-endothelial slit. These pitted red blood cells go back to the general blood circulation without destruction. We have studied efficacy and tolerance of intravenous artesunate in 123 patients with imported severe malaria, of whom 117 have survived (95%). Among 78 patients followed more than 8 days, 76 (97%) suffered from anemia during follow-up and 21 had a typical delayed hemolysis pattern (27%). In this sub group the median loss of hemoglobin was 1.3g/dl with a nadir below 7g/dl in 15% of them. Only one patient was transfused. The labelling of Resa protein, a plasmodium protein included in the bilayer membrane of the red blood cell, allowed the visualization of pitted cells. In 21 patients non transfused, the pitted cells peak occured during the first week post treatment. In 9 patients with typical delayed hemolysis pattern, the pitted cells peak was higher than in the 12 patients with other kind of anemia patterns (0.30 vs. 0.07 ; P = 0.0001). A pitted cells concentration above 180 millions/l would have predicted the risk of late hemolysis with 89% sensitivity and 83% specificity. Red blood cell morphology was studied using ImageStream* technology in 4 patients. It has shown that infection and pitting process induces a membrane projected area loss of 8.9%. This loss could explain the reduced life span of the pitted red blood cells. Differed destruction of the erythrocytes first infected and then spared by pitting process during the treatment with artesunate is a new pattern of hemolytic anemia during malaria. This work has provided a nosological framework of post therapy hemolysis during malaria, a clarified pathophysiology of delayed hemolysis and has identified potential explanatory mechanisms. Notwithstanding the high incidence of hemolysis, the resulting anemia is severe in 15% of the patients and does not jeopardize the advantage of artesunate compared to quinine in the treatment of severe malaria. Early pitted cells concentration could be a surrogate marker to determine the risk of delayed hemolysis and anemia after artesunate treatment.

Orientador: Fabio Trindade Maranhão Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T14:00:00Z (GMT). No. of bitstreams: 1 Kayano_AnaCarolinaAndradeVitor_M.pdf: 4619978 bytes, checksum: 3bf657caae96f9c4faf7fcdf7e519956 (MD5) Previous issue date: 2011 / Resumo: Para superar o problema do aumento de resistência às drogas, os medicamentos tradicionais são fontes importantes na investigação de potenciais novos antimaláricos. Caesalpinia pluviosa, mais conhecida como 'sibipiruna', é originária do Brasil e estudos mostraram que este gênero apresenta várias propriedades farmacológicas, incluindo a atividade antimalárica. O extrato bruto obtido da casca foi submetido ao fracionamento com diferentes solventes resultando em sete frações. Para avaliar a citotoxicidade do extrato e frações em células MCF-7 foi realizado o ensaio de MTT. Essas amostras foram testadas in vitro contra P. falciparum cloroquino sensível (3D7) e resistente (S20) e in vivo em camundongos infectados por P. chabaudi. A interação da fração etanólica 100% de C. pluviosa com o artesunato foi avaliado e análises de espectrometria de massas foram realizados. As frações etanólica 100% e metanólica 50% apresentaram atividade antimalárica significativa em concentrações não tóxicas, e o ensaio de interação medicamentosa do artesunato com a fração etanólica 100% foi sinérgico. Essa fração foi capaz de inibir significativamente a parasitemia dos animais de forma dose dependente após 4 dias de tratamento (0-3 dia pós infecção). Além disso, análise de espectrometria de massas revelou a presença do íon m/z 303.0450, sugerindo a presença de quercetina. No entanto, em uma segunda análise com o padrão de quercetina mostrou íons diferentes como m/z 137 e 153. Nossos resultados mostram que a fração etanólica 100% de C. pluviosa apresentou atividade antimalárica in vitro em concentrações não tóxicas e esse efeito foi potencializado com a presença de artesunato. Além disso, essa atividade antimalárica foi também sustentada após o tratamento in vivo de camundongos infectados. Finalmente, as análises de espectrometria de massas sugerem que um novo composto, provavelmente um isômero da quercetina, possa estar relacionado à atividade antimalárica da fração etanólica 100% / Abstract: To overcome the problem of increasing drug resistance traditional medicines are an important source for investigation of potential new antimalarials. Caesalpinia pluviosa, commonly named 'sibipiruna', is originated from Brazil and studies showed that this genus present multiple therapeutic properties, including antimalarial activity. Crude extract obtained from stem bark was purified with different solvents, resulting in seven fractions. MTT assay was performed to evaluate cytotoxicity in MCF-7 cells. The crude extract and its fractions were tested in vitro against chloroquine-sensitive (3D7) and -resistant (S20) strains of Plasmodium falciparum and in vivo in P. chabaudi-infected mice. In vitro interaction with artesunate and C. pluviosa fraction was assessed and mass spectrometry analyses were conducted. At non-toxic concentrations the 100% ethanolic and 50% methanolic fractions presented significant antimalarial activity against both 3D7 and S20 strains, and drug interaction assays with artesunate showed a synergistic effect with 100% ethanolic fraction. This fraction was able to inhibit mice parasitemia significantly and in a dose dependent manner after 4 days treatment (0-3 post-infection). Moreover, mass spectrometry analyses revealed the presence of an ion corresponding to m/z 303.0450, suggesting the presence of quercetin. However, a second set of analyses, with the standard quercetin, showed distinct ions of m/z 137 and 153. Our findings show that the 100% ethanolic fraction of C. pluviosa exhibited antimalarial activity in vitro at non-toxic concentrations and this effect was potentiated with the presence of artesunate. Moreover, this antimalarial activity was also sustained in vivo after treatment of infected mice. Finally, mass spectrometry analyses suggest a new compound, most likely an isomer of quercetin, related with antimalarial activity of the 100% ethanolic fraction / Mestrado / Imunologia / Mestre em Genética e Biologia Molecular

Orientador: Fabio Trindade Maranhão Costa / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-29T19:18:42Z (GMT). No. of bitstreams: 1 Bastos_MarceleFontenelle_D.pdf: 23599395 bytes, checksum: 51577c364bf25d45e672a187e5d0bc4a (MD5) Previous issue date: 2015 / Resumo: Acredita-se que o sequestro de eritrócitos infectados por Plasmodium falciparum (Pf-EIs) na microvasculatura de órgão vitais, contribua para a patogênese de síndromes graves da malária, como a malária cerebral (MC), síndrome da angústia respiratória grave, anemia grave e malária na gravidez. Apesar do tratamento com drogas antimaláricas eficazes, mortalidade significativa ainda é observada em casos graves da doença. Assim, tem sido sugerido o uso de terapias adjuvantes. Nesse sentido, polissacarídeos sulfatados, como a heparina, têm demonstrado capacidade em inibir a citoaderência de P. falciparum a vários receptores do hospedeiro, inibir a invasão de merozoítos e romper rosetas. A heparina foi utilizada no passado como tratamento para malária grave, no entanto o seu uso foi interrompido devido à ocorrência de efeitos colaterais graves, tais como hemorragia. Além disso, muitos desses polissacarídeos sulfatados são derivados de mamíferos, o que aumenta o risco de contaminação por agentes patogênicos, como príons. Apesar de muitos compostos terem sido testados como terapia adjuvante para diferentes aspectos patogênicos da malária grave, nenhum destes demonstrou evidência inequívoca de melhora dos pacientes nos testes clínicos. Sendo assim, nesse estudo, investigamos a ação de dois polissacarídeos sulfatados extraídos de invertebrados na citoadesão e desenvolvimento de P. falciparum. Já foi demonstrado que esses compostos; o condroitim sulfato fucosilado (FucCS), extraído do pepino-do-mar Ludwigothurea grisea, e o análogo da heparina (heparam sulfato), extraído do molusco bivalve Nodipecten nodosus; possuem ação anticoagulante e antitrombótica, porém em menor escala do que a heparina comercial. Além disso, apresentam efeito anti-inflamatório e antimetastático. Aqui, nós mostramos que o FucCS e o heparam sulfato (HS) de molusco foram eficazes em inibir a citoadesão de P. falciparum em condições estáticas e de fluxo a células endoteliais de pulmão humano (HLECs). Eles também foram capazes de inibir o desenvolvimento parasitário por interferir na invasão de merozoítos. Além de romper rosetas eficientemente. Ainda, o FucCS inibiu a adesão de Pf-EIs a criocortes de placenta. Finalmente, a remoção das cadeias de fucose sulfatadas presentes na estrutura do FucCS praticamente aboliu o efeito inibitório do composto, evidenciando a importância dessas cadeias para sua atividade. Sendo assim, sugerimos o FucCS e o HS de molusco como candidatos promissores a terapia adjuvante no tratamento da malária grave e na prevenção ao agravamento da doença, além de abrir perspectivas para continuação e aprofundamento do estudo desses compostos no tratamento da malária / Abstract: It is believed that sequestration of Plasmodium falciparum-infected erythrocytes (Pf-iEs) in the microvasculature of vital organs, contributes to the pathogenesis of severe malaria syndromes such as, cerebral malaria (CM), severe respiratory distress, severe anemia and malaria in pregnancy (MiP). Despite treatment with effective antimalarial drugs, high mortality is still observed in severe cases of the disease. Thus, the use of adjuvant therapies has been suggested. Accordingly, sulfated polysaccharides, such as heparin, have been shown to prevent P. falciparum cytoadherence to several host receptors, inhibit merozoite invasion and disrupt rosettes. Heparin was used in the past as treatment for severe malaria, however its use was abandoned due to the occurrence of serious side effects such as bleeding. Moreover, many of these compounds are derived from mammals, which increase the risk of contamination by pathogens, such as prions. Although many compounds have been tested as adjunct therapy to different pathophysiological features of severe malaria, none of them showed clear evidence of patients¿ improvement in clinical trials. Therefore, in this study, we investigated the action of two sulfated compounds extracted from invertebrates in P. falciparum cytoadhesion and development. It has been shown that these compounds; chondroitin sulfate fucosylated (FucCS), extracted from the sea cucumber Ludwigothurea grisea, and the heparin analogue (heparan sulfate), extracted from the bivalve mollusk Nodipecten nodosus; have anticoagulant and antithrombotic action, but on a smaller scale than the commercial heparin. They also have anti-inflammatory and antimetastatic effect. Here, we show that FucCS and mollusk heparan sulfate (HS) were effective in inhibiting P. falciparum cytoadhesion, under static and flow conditions to human lung endothelial cells (HLECs). They were also able to block parasite development by interfering with merozoite invasion, and to disrupt rosettes efficiently. In addition, FucCS inhibited Pf-iEs adhesion to placenta cryosections. Finally, removal of sulfated fucose branches on the FucCS molecule virtually abolished its inhibitory effect, indicting a central role played by these structures. Then, we suggest FucCS and mollusk HS as promising candidates for adjunct therapy in the treatment of severe malaria and in preventing disease worsening. Also, we open new avenues to understand the mechanisms of action of these compounds in malaria treatment / Doutorado / Imunologia / Doutora em Genética e Biologia Molecular

O parasita Plasmodium falciparum causa a forma mais grave da malária humana.Para evadir a resposta imune do hospedeiro, as formas assexuadas do parasita podem usar variação antigênica ou podem se diferenciar em formas sexuais como estratégia para sobreviver e garantir a sua transmissão para o mosquito.A base molecular desses processos ainda é pouco compreendida. Por manipulação genética, nos identificamos a participação de um fator de transcrição da família ApiAP2 ( PF3D7_1143100), no controle da transcrição de genes variantes e no desenvolvimento em formas sexuais na fase intraeritrocítica. Demonstramos ainda que um outro membro desta família, PF3D7_1466400, não é essencial no ciclo assexual de P. falciparum, já que seu silenciamento não afeto o normal desenvolvimento do parasita. / The parasite Plasmodium falciparum causes the most severe form of human malaria. To evade the host immune response, asexual parasite forms can employ antigenic variation or differentiation to gametocytes as a means to survive and secure their transmission to the mosquito. The molecular basis behind these processes is still poorly understood. By genetic manipulation, we indentified the participation of a ApiAP2 transcription factor, PF3D7_1143100, in the control of variant gene transcription as well as in the switching from asexual to sexual development in the intraerythrocytic stage. We also demonstrate that the ApiAP2 transcription factor PF3D7_1466400 is not essential in the asexual stage of P. falciparum, since its knockdown did not affect the normal development of the parasite.

Plasmodium falciparum e Toxoplasma gondii são protozoários parasitas pertencentes ao filo Apicomplexa. Apirases são enzimas metabolizadoras de nucleotídeos extracelulares. Nesta tese mostramos pela primeira vez a presença de um membro desta família de enzimas em P. falciparum, o qual foi capaz de degradar ATP extracelular. Análises por RT-qPCR revelaram a expressão da apirase durante todo o ciclo intraeritrocítico. A adição de inibidores desta classe de enzimas foi capaz de prejudicar o desenvolvimento dos parasitas e a invasão de novas hemácias pelos merozoitos, sugerindo assim um papel da apirase nestes processos. A via de sinalização por Ca2+ é universal e vital para todas as células. Para melhor entender a fisiologia celular de P. falciparum construímos uma nova linhagem de parasitas transgênicos, PfGCaMP3, que nos tornam capazes de monitorar a dinâmica de Ca2+ sem o uso de protocolos invasivos de marcação. De modo semelhante utilizamos uma nova linhagem de T. gondii expressando de forma estável o indicador de Ca2+ GCaMP3 para estudar o papel deste íon na saída da célula. T. gondii possui o Ca2+ necessário para promover este processo, entretanto Ca2+ extracelular age como um fator intensificador neste passo essencial do ciclo lítico. / Plasmodium falciparum and Toxoplasma gondii are protozoan parasites that belong to phylum Apicomplexa. Apirases are metabolizing enzymes of extracellular nucleotides. In this work we show for the first time the presence of an apyrase in P. falciparum, which was able to degrade extracellular ATP. RTqPCR analysis revealed the expression of apyrase throughout the intraerythrocytic cycle. Addition of apyrase inhibitors was able to impair the development of the parasites and the invasion of new erythrocytes by merozoites, thus suggesting a role of apyrase in these processes. Calcium signaling is universal and vital to all cells. To better understand the cellular physiology of P. falciparum we construct a new strain of transgenic parasites, PfGCaMP3, which enable us to monitor the Ca2+ dynamics without using invasive protocols. Similarly we use a new strain of T. gondii that stably express the Ca2+ indicator GCaMP3 to study the role Ca2+ in parasite egress. T. gondii has the Ca2+ required to promote this process, however extracellular Ca2+ acts as an enhancer factor in this crucial step of the lytic cycle.

Nesta dissertação, foi caracterizada a via de biossíntese de tiamina (Vitamina B1) nas três formas intraeritrocitárias de P. falciparum. Foram realizadas marcações metabólicas, utilizando diferentes precursores radioativos envolvidos na biossíntese de tiamina, já descritos para outros organismos. A utilização do precursor [1-14C] acetato de sódio demonstrou que a via de biossíntese de tiamina encontra-se ativa em todos os estágios intraeritrocitários de P. falciparum. Investigamos os precursores que poderiam estar envolvidos na biossíntese do intermediário tiazol, e nossos dados sugerem que a cistéina é a doadora do enxofre presente na molécula de tiamina; que o aminoácido tirosina pode ser o precursor da biossíntese de tiamina, e nicotinamida não é utilizada como precursor em P. falciparum. Também se avaliou o efeito da fosmidomicina e 3ClDHP e foi demonstrado que ambos propiciaram uma inibição no crescimento dos parasitas. Estes dados sugerem que a via de biossíntese de tiamina, pode ser explorada como alvo para drogas antimaláricas, devido ausência em humanos. / In the present work we have demonstrated the biosynthesis of thiamin (vitamin B1) in the intraerytrocytic stages of P. falciparum. We have demonstrated active biosynthesis of thiamine in the three parasite stages metabolically labeled with [1-14C] sodium acetate. We also investigated which precursors could be involved in the biosynthesis of the thiazole intermediate, by metabolic labelling with different precursors. Our data suggest that the sulphur present in the thiamine molecule is formed from cysteine white that tyrosine can be the precursor of thiamine biosynthesis. Nicotinamide is not utilized as a precursor in P.falciparum. We also investigated the effect of fosmidomycin (an inhibitor of the DOXP reductoisomerase in the MEP pathway) and 3CIDHP (an analogue of bacimethrin) in vitro cultures and both showed an inhibitory effect on parasite growth. These data suggest that the biosynthesis of thiamine can be an attractive target for the development of antimalarial drugs since this pathway is absent in humans.

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.

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.