Ce travail avait pour objectif, de caractériser le poids des infections au cours de la grossesse et d’étudier la construction de l’immunité anti-PfEMP1 chez le jeune enfant. La première partie, aborde la conséquence des infections et l’efficacité des traitements chez la mère. Cette étude réalisée sur la cohorte de femmes d’une étude prospective au Bénin, a démontré l’impact des infections à bas bruit sur le taux d’hémoglobine maternel et le faible poids de l’enfant. Le TPI-SP, a montré les limites quant à sa capacité à débarrasser les femmes enceintes infectées au moment du traitement de leur parasite. Nos résultats soutiennent davantage la nécessité de trouver des moyens alternatifs de prévention qui offrent une meilleure couverture de la grossesse. La deuxième partie aborde la construction de l’immunité anti-PfEMP1 dans la première année de vie chez l’enfant. Un résultat majeur de cette étude est la démonstration que l’acquisition des anticorps contre les PfEMP1 associés aux complications du paludisme est dépendante des infections patentes de l’enfant. La troisième partie aborde les phénotypes des parasites responsables de diverses formes cliniques du paludisme chez l’enfant Africain âgé de 0 à 5 ans. Cette étude a permis de mettre en évidence un marqueur de mauvais pronostic du paludisme cérébral. Sur un deuxième volet, nous avions montré que les isolats adhérant faiblement à ICAM-1, transcrivent fortement les gènes codant pour les PfEMP1 contenant des motifs DC8. Ces résultats soulèvent la question du rôle de EPCR dans la physiopathologie du neuropaludisme. Le travail développé dans cette thèse a permis de décrire pour la première fois la construction de l’immunité anti-PfEMP1 dans la première année de vie, de mettre à jour les connaissances sur la physiopathologie du paludisme cérébral chez le jeune enfant et de dégager des pistes à explorer prioritairement dans la perspective du développement d'un vaccin contre les formes graves du paludisme. / This work aimed to characterize the burden of P. falciparum infections during pregnancy and to study the construction of the anti-PfEMP1 immunity in the early life. The first part discusses the consequence of infection and the effectiveness of treatments in the mother. This study on the cohort of women from a prospective study in Benin, demonstrated the impact of infections with low parasitemia on the maternal hemoglobin and low weight of the child. IPT-SP, has shown the limits of its ability to rid infected pregnant women in the processing of their parasite. Our results further support the need to find alternative means of prevention that provide better coverage of pregnancy. The second part treated the construction of the anti-PfEMP1 immunity in the first year of life in children. A major finding of this study is the demonstration that the acquisition of antibodies against the PfEMP1 associated with complications of malaria depends on patentes infections in children. The third part studies parasites phenotypes responsible of various clinical forms of malaria in African children aged 0-5 years. This study allowed finding a marker of poor prognosis of cerebral malaria. On a second component, we showed that isolates bind to ICAM-1, highly transcribe the genes encoding PfEMP1 containing DC8. These results raise the question of the role of EPCR in the pathophysiology of cerebral malaria. The work developed in this thesis has allowed describing for the first time the construction of the anti-PfEMP1 immunity in the first year of life, to update knowledge on the pathophysiology of cerebral malaria in young children and identify Options to be primarily from the perspective of developing a vaccine against severe forms of malaria.

Please read the abstract in the section 00front of this document / Dissertation (MSc (Biochemistry))--University of Pretoria, 2005. / Biochemistry / unrestricted

Malaria is the cause of over one million deaths per year, primarily in African children. The parasite responsible for the most virulent form of malaria, is Plasmodium falciparum. Protein structure plays a pivotal role in elucidating mechanisms of parasite functioning and resistance to anti-malarial drugs. Protein structure furthermore aids the determination of protein function, which can together with the structure be used to identify novel drug targets in the parasite. However, various structural features in P. falciparum proteins complicate the experimental determination of protein three dimensional structures. Furthermore, the presence of parasite-specific inserts results in reduced similarity of these proteins to orthologous proteins with experimentally determined structures. The lack of solved structures in the malaria parasite, together with limited similarities to proteins in the Protein Data Bank, necessitate genome-scale structural annotation of P. falciparum proteins. Additionally, the annotation of a range of structural features facilitates the identification of suitable targets for structural studies. An integrated structural annotation system was constructed and applied to all the predicted proteins in P. falciparum, Plasmodium vivax and Plasmodium yoelii. Similarity searches against the PDB, Pfam, Superfamily, PROSITE and PRINTS were included. In addition, the following predictions were made for the P. falciparum proteins: secondary structure, transmembrane helices, protein disorder, low complexity, coiled-coils and small molecule interactions. P. falciparum protein-protein interactions and proteins exported to the RBC were annotated from literature. Finally, a selection of proteins were threaded through a library of SCOP folds. All the results are stored in a relational PostgreSQL database and can be viewed through a web interface (http://deepthought.bi.up.ac.za:8080/Annotation). In order to select groups of proteins which fulfill certain criteria with regard to structural and functional features, a query tool was constructed. Using this tool, criteria regarding the presence or absence of all the predicted features can be specified. Analysis of the results obtained revealed that P. falciparum protein-interacting proteins contain a higher percentage of predicted disordered residues than non-interacting proteins. Proteins interacting with 10 or more proteins have a disordered content concentrated in the range of 60-100%, while the disorder distribution for proteins having only one interacting partner, was more evenly spread. Comparisons of structural and sequence features between the three species, revealed that P. falciparum proteins tend to be longer and vary more in length than the other two species. P. falciparum proteins also contained more predicted low complexity and disorder content than proteins from P. yoelii and P. vivax. P. falciparumprotein targets for experimental structure determination, comparative modeling and in silico docking studies were putatively identified based on structural features. For experimental structure determination, 178 targets were identi_ed. These targets contain limited contents of predicted transmembrane helix, disorder, coiled-coils, low complexity and signal peptide, as these features may complicate steps in the experimental structure determination procedure. In addition, the targets display low similarity to proteins in the PDB. Comparisons of the targets to proteins with crystal structures, revealed that the structures and predicted targets had similar sequence properties and predicted structural features. A group of 373 proteins which displayed high levels of similarity to proteins in the PDB, were identified as targets for comparitive modeling studies. Finally, 197 targets for in silico docking were identified based on predicted small molecule interactions and the availability of a 3D structure. / Dissertation (MSc)--University of Pretoria, 2008. / Biochemistry / unrestricted

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.

Le paludisme a déjà sévi dans 80% des pays chinois. Après plusieurs décennies d’efforts, la prévalence du paludisme a considérablement diminué et la Chine est sur le point de l’éliminer dans tout le pays. Les informations sur les vecteurs du paludisme ne sont toujours pas bien documentées, ce qui pourrait entraver l'élaboration de stratégies de surveillance appropriées et la certification par l'OMS. Le principal risque pour l'élimination du paludisme est la réintroduction de la maladie à partir de cas de paludisme importés. De plus en plus de cas de paludisme importés sont causés par des citoyens chinois revenant récemment de pays touchés par le paludisme. Les informations sur leurs personnages, leurs conducteurs et leur voie d’introduction dans différentes zones fourniront aux décideurs politiques des données factuelles où et quand effectuer les interventions. Avec plus de 1,3 milliard d’habitants et un territoire couvrant différentes zones climatiques, les enseignements tirés de l’élimination du paludisme en Chine pourraient servir de référence à d’autres pays. Selon les initiatives Belt and Road, la Chine participera plus globalement aux activités liées à la gouvernance. Les partenaires et les parties prenantes de la campagne mondiale pour l’élimination du paludisme s'intéressent aux opportunités futures et aux domaines potentiels qui impliqueraient une expertise chinoise. 1.Une analyse récapitulative a été réalisée sur la distribution à l'échelle nationale des vecteurs du paludisme, leurs caractéristiques bionomiques, leurs mesures de contrôle et les études connexes. La distribution en Chine des principaux vecteurs du paludisme a été réduite. Anopheles sinensis, qui avait déjà été signalé comme étant moins efficace dans la transmission du paludisme, est en train de devenir l’espèce prédominante dans le sud-ouest de la Chine. Les résultats de l'échantillonnage sur le terrain ont indiqué l'existence de vecteurs du paludisme hautement efficaces, par ex. An. minimus et An. harrisoni à la frontière Sino-Birmane. De plus, des taux élevés de piqûres humaines, des densités élevées d'adultes et de larves et des taux de parous ont été observés chez An. sinensis et An. harrisoni, qui révèlent une très grande réceptivité et un risque élevé de réintroduction du paludisme le long de la frontière Sino-Birmane. 2. L'analyse comparative des cas importés de paludisme signalés dans d'anciennes zones non endémiques et d'anciennes zones endémiques en Chine a montré que toutes les anciennes zones non endémiques signalaient désormais des cas importés de paludisme. 3. Une analyse résumée des progrès accomplis en matière d'élimination du paludisme depuis 2010 a été réalisée, avec un accent particulier sur la cartographie des foyers de paludisme résiduels et la répartition des cas de paludisme en Chine. L'incidence du paludisme contracté localement a fortement diminué, parallèlement à la diminution concomitante des zones d'endémie palustre de 762 comtés signalant le paludisme en 2010 à seulement deux comtés adjacents aux zones frontalières (Province du Yunnan: Chine-Myanmar et Tibet, Chine-Inde). En 2017, la Chine a atteint pour la première fois zéro cas de paludisme autochtone. En conclusion, la Chine est sur la voie de l’élimination du paludisme d’ici 2020. Le risque de réintroduction lié à l’émergence de cas importés de paludisme et à la présence de vecteurs du paludisme hautement efficaces présents dans le pays reste la cible de la surveillance du paludisme. Afin de maintenir l'élimination du paludisme, une collaboration internationale intensifiée, axée sur les zones transfrontalières et la population mobile / migrante, est appelée à prendre des mesures. Les études pilotes sur la manière de présenter les leçons tirées de l'élimination du paludisme en Chine et l'expertise Chinoise sont sur la liste des mesures à prendre, qui harmoniseraient l'aide de la Chine à l'élimination du paludisme dans les pays cibles. / Malaria has occurred in 80% of the counties in China in the past. After several decades of effort, malaria prevalence decreased drastically and China is currently approaching elimination throughout the country. Information on malaria vectors is still found not well documented, which could hinder the development of appropriate surveillance strategies and WHO certification. The major risk to malaria elimination is the re-introduction of the disease from imported malaria cases. There are an increasing number of imported malaria cases caused by Chinese citizens returning from malaria-affected countries recently. The information about their characters, drivers and route of introduction in different areas will provide evidence-based data to policy makers where and when to carry out the interventions. This will in turn allow them to develop efficient guidelines for risk-assessment of malaria re-introduction and for allocating appropriate resources. As a country with over 1.3 billion population and a territory covering different climatic zones, lessons learnt from malaria elimination in China that could serve as references to other countries. According to the Belt and Road initiatives, China will participate more globally to governance related activities. Partners and stakeholders within Global malaria elimination campaign are interested to the future opportunity and potential fields that would involve Chinese expertise. 1.A summary analysis was conducted on the nationwide distribution of malaria vectors, their bionomic characteristics, control measures and related studies. The distribution in China of the principal malaria vectors was found reduced, in particular for Anopheles lesteri and Anopheles dirus s.l., including the two main malaria vector species, An. dirus and An. baimaii, which nearly disappeared after several years of malaria control effort. Anopheles sinensis, which was previously reported to be less efficient in malaria transmission, is becoming the predominant species in Southwestern China. The field sampling results indicated the existence of high efficient malaria vectors, e.g. An. minimus and An. harrisoni at the China-Myanmar border. In addition, elevated human-biting rates, high adult and larval densities, and parous rates were found in both An. sinensis and An. harrisoni, which reveal a very high receptivity and risk of malaria re-introduction along the China–Myanmar border. 2.The comparative analysis of imported malaria cases reported from former non-endemic areas and former endemic areas in China showed that all former non-endemic areas are now reporting imported malaria cases. 3. A summary analysis on malaria elimination progress since 2010, was carried out with specific focus on mapping the residual malaria foci and the distribution of malaria cases in China. The incidence of locally acquired malaria has declined sharply along with the concomitant decrease of malaria-endemic areas from 762 counties reporting malaria in 2010 to just two counties adjacent to border areas (Yunnan Province: China-Myanmar and Tibet, China-India) in 2016. In 2017, China achieved zero indigenous malaria case report for the first time. In conclusion, China is on the track to achieve malaria elimination by 2020. The risk of re-introduction caused by the emergence of imported malaria cases and the occurrence of highly efficient malaria vectors present in the country is still the target of malaria surveillance. To maintain malaria elimination, intensified international collaboration with specific focus on cross-border areas and mobile/migrant population is called to take actions. The pilot studies on how to introduce the lessons learned from malaria elimination in China and the Chinese expertise are on the list to take action in future, which would harmonize the China aid to malaria elimination in the target countries.

Malaria is one of the most serious tropical infectious diseases affecting mankind. The prevention of the disease is hampered by the increasing resistance of the parasite to existing chemotherapy and -prophylaxis drugs. The need for novel therapeutic targets and drugs is therefore enormous and the understanding of the biochemistry of the parasite is imperative. The aim of this study was the identification and molecular characterisation of the eDNA of one such metabolic target protein, ornithine decarboxylase (ODC), in the human malaria parasite P. falciparum. The P. falciparum ODC eDNA was isolated by means of a modified RT-PCR technique, RACE. No sequence data were available and the primers used were based on consensus areas identified in the protein sequences from other related organisms. The isolation and identification of the eDNA with degenerate primers was successful in 3' -RACE, but necessitated the optimisation of the eDNA synthesis protocol and the use of total RNA as starting material. The sequence obtained facilitated the application of 5' -RACE with ODC-specific primers based on the 3' -RACE sequence data. The full-length ODC eDNA sequence was obtained by overlap-alignment of various segments. A novel suppression PCR technology was applied during the 5' -RACE in order to create an uncloned eDNA library of amplified cDNAs representing only the mRNA population. The P. falciparum ODC eDNA contains an open reading frame of ---2847 bp and translates to a large 939 amino acid protein. The protein contained large internal insertions and was extended by '""273 N-terminal residues compared to ODCs from other organisms. Several possible signature motifs were identified for phosphorylation, glycosylation and transamidation. The P. falciparum ODC protein seems to contain more hydrophilic and a-helix forming residues. These characteristics should be further investigated after expression of the recombinant protein. The isolation of the P. falciparum ODC eDNA facilitates the validation of this protein as an antimalarial target. / Dissertation (MSc)--University of Pretoria, 1998. / gm2014 / Biochemistry / unrestricted

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.

El documento digital no refiere asesor / Determina la eficacia del esquema de tratamiento antimalárico combinado de artesunato más mefloquina contra la malaria por Plasmodium falciparum no complicado en la región de Loreto, Perú. Determina la respuesta parasitológica y clínica de pacientes con malaria por P. falciparum no complicada al nuevo esquema de tratamiento artesunato más mefloquina. Determina el nivel de resistencia inicial del P. falciparum a las drogas artesunato y mefloquina. Busca conocer la tasa del efecto gametocida del artesunato en el esquema de tratamiento artesunato más mefloquina en la región Loreto. / Trabajo académico