A research report submitted to the faculty of Medicine, University of the Witwatersrand, Johannesburg, in part fulfillment of the requirements for the degree of Master of Medicine (in the branch of Haematology) Johannesburg 1992. / The observation by Haldane in 1949 that the distribution of malaria and certain thalassaemias were similar and that the former disease must be a selective force tor the continued existence of the latter by preservation of the heterozygotes. This theory which later became known as lithe malaria hypothesis" has been applied to other inherited conditions such as G6PD deficiency, membranopathies, certain blood group polymorphisms, other heamoglobinopathies such as sickle cell disease, blood group polymorphisms and more recently HLA phenotypes. It has been shown that the Duffy blood group antigens are the receptors for. Plasmodium vivax and since these antigens are lacking in most black Africans this species of malaria is virtually absent in Africa. It has also been shown that the glycophorins are at least in part the receptors for Pfalciparum. Several variants of the glycophorins exist and the biochemistry and, where known, the molecular mechanisms by which these arise is reviewed. Experimental work is carried out to establish the growth characteristics of Pfalciparum in an in vitro culture system using cells with glycophorin variants on their membranes. Three such variants were compared to normal cells and two (S~s-U-and Dantu) were found to be partially resistant to invasion by Pfalciparum merozoites whereas the third (Henshaw) was found to be no different to controls. / MT2018

A DISSERTATION SUBMITTED TO THE FACULTY OF MEDICINE, UNIVERSITY OF THE WITWATERSRAND, JOHANNESBURG, FR THE DEGREE OF MASTER OF SCIENCE IN MEDICINE. / The influence of iron on the outcome of various infections have been extensively reviewed. Clinical observations suggests that iron deficiency may be protective against malaria. Various researchers have shown that certain iron chelators blocked the proliferation of plasmodium falciparum in vitro and in vivo. (Abbreviation abstract) / Andrew Chakane 2018

A dissertation submitted to the Faculty of Medicine University of the Witwatersrand, Johannesburg For the degree of Master of Science in Medicine / Chloroquine-resistant Plasmodium falciparum accumulate significantly less chloroquine than susceptical parasites, and this is thought to be the basis of their resistance ( Fitch, 1970 ). Martin et. al ( 1987 ), recently demonstrated that in the presence of verapamil, a calcium channel blocker, chloroquine-resistant P falciparum becomes chloroquine-sensitive, with an increase in the chloroquine accumulation.The mechanism of such reversal has yet to be elucidated / IT2018

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-10-18T16:10:43Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_MalariaMulheresIdade.pdf: 4024160 bytes, checksum: 025c0bc2483afa0356362d19ecf10361 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-12-01T14:07:06Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_MalariaMulheresIdade.pdf: 4024160 bytes, checksum: 025c0bc2483afa0356362d19ecf10361 (MD5) / Made available in DSpace on 2017-12-01T14:07:06Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_MalariaMulheresIdade.pdf: 4024160 bytes, checksum: 025c0bc2483afa0356362d19ecf10361 (MD5) Previous issue date: 2010 / A malária na gestação representa uma ameaça à vida da mãe e do concepto além de influenciar na evolução da gravidez. Visando esclarecer aspectos da malária que acomete gestantes de áreas hiperendêmicas na Amazônia, este estudo se propõe avaliar aspectos clínico-epidemiológicos da doença em mulheres na idade reprodutiva, com ênfase em gestantes internadas em hospital público de referencia para a região de Itaituba. O estudo foi conduzido no HMI de Itaituba no período de 2005 a 2007, através do levantamento de prontuários de mulheres hospitalizadas e com diagnóstico de malária confirmado pela gota espessa, e da análise documental baseada nos dados do SIVEP-Malária. Os resultados evidenciaram que no Pará, em 2007, mais de 51% dos casos notificados foram oriundos de apenas nove municípios, dois deles (Itaituba e Jacareacanga), pertencentes à microrregião de Itaituba, área onde foi registrada maior Incidência Parasitária Anual (IPA). Os dados dos prontuários de 30 pacientes (sete, gestantes) revelaram, em sua maioria, que eram procedentes da área rural do município de Itaituba e haviam sido infectadas por P. falciparum; que as gestantes eram as mais jovens (p<0,05); e que o tempo de internação foi similar entre gestantes e não-gestantes. As intercorrências sobre o curso gestacional foram um óbito fetal (malária por P. vivax, segundo trimestre) e um parto prematuro (malária por P. falciparum, terceiro trimestre). Concluiu-se, a partir dessas observações, que casos graves de malária podem ocorrer tanto associados à espécie vivax como falciparum fazendo-se necessário constante vigilância epidemiológica, especialmente no município de Itaituba, onde está concentrado o maior número de casos da doença. As medidas de vigilância epidemiológica a serem adotadas devem privilegiar o diagnóstico precoce e tratamento imediato das pacientes, sobretudo das gestantes, já que estão sujeitas a maior risco de complicações com sérias conseqüências para o concepto. / A malária na gestação representa uma ameaça à vida da mãe e do concepto além de influenciar na evolução da gravidez. Visando esclarecer aspectos da malária que acomete gestantes de áreas hiperendêmicas na Amazônia, este estudo se propõe avaliar aspectos clínico-epidemiológicos da doença em mulheres na idade reprodutiva, com ênfase em gestantes internadas em hospital público de referencia para a região de Itaituba. O estudo foi conduzido no HMI de Itaituba no período de 2005 a 2007, através do levantamento de prontuários de mulheres hospitalizadas e com diagnóstico de malária confirmado pela gota espessa, e da análise documental baseada nos dados do SIVEP-Malária. Os resultados evidenciaram que no Pará, em 2007, mais de 51% dos casos notificados foram oriundos de apenas nove municípios, dois deles (Itaituba e Jacareacanga), pertencentes à microrregião de Itaituba, área onde foi registrada maior Incidência Parasitária Anual (IPA). Os dados dos prontuários de 30 pacientes (sete, gestantes) revelaram, em sua maioria, que eram procedentes da área rural do município de Itaituba e haviam sido infectadas por P. falciparum; que as gestantes eram as mais jovens (p<0,05); e que o tempo de internação foi similar entre gestantes e não-gestantes. As intercorrências sobre o curso gestacional foram um óbito fetal (malária por P. vivax, segundo trimestre) e um parto prematuro (malária por P. falciparum, terceiro trimestre). Concluiu-se, a partir dessas observações, que casos graves de malária podem ocorrer tanto associados à espécie vivax como falciparum fazendo-se necessário constante vigilância epidemiológica, especialmente no município de Itaituba, onde está concentrado o maior número de casos da doença. As medidas de vigilância epidemiológica a serem adotadas devem privilegiar o diagnóstico precoce e tratamento imediato das pacientes, sobretudo das gestantes, já que estão sujeitas a maior risco de complicações com sérias conseqüências para o concepto.

The repeated emergence of drug resistance in Plasmodium falciparum underscores the importance of understanding the genetic architecture of current resistance pathways, as well as any associated fitness costs. Why resistance emerges in particular regions of the world has been linked to particular genetic backgrounds that better tolerate resistance-associated polymorphisms; this is likely to play a key role in driving the epidemiology of drug resistance, however is infrequently studied at a large scale in a laboratory setting. The first results chapter establishes a barcoding approach for P. falciparum with the aim of tracking parasite growth in vitro. The strategy used was adapted for P. falciparum by using a pseudogene (PfRh3) as a safe harbour to insert unique molecular barcodes. These libraries of barcoded P. falciparum vectors were also used as a readout of transfection efficiency. The second chapter establishes a proof of principle for phenotyping by barcode sequencing, using a panel of barcoded parasites generated in different genetic backgrounds that comprise sufficient genetic diversity to pilot the method. These were grown in the presence and absence of antimalarial compounds, and growth phenotypes were measured in parallel using BarSeq. The third results chapter studies the contribution of mutations in Pfkelch13, a molecular marker of artemisinin resistance, to parasite fitness. Combining CRISPR/Cas9-based genome editing and high throughput sequencing, the impact of Pfkelch13 alleles on fitness in the context of particular strain backgrounds is revealed. In particular, the impact of genetic background in the emergence and spread of drug-resistant lineages (referred to as KEL1) in Southeast Asia carrying a Y580 Pfkelch13 allele. Overall, given the current pace of genome sequencing of pathogenic organisms such as P. falciparum, it will be important to increase the scale of experimental genetics, in order to tackle in real-time natural variation that might be under constant selection from drugs, thus anticipating the emergence of drug resistance in changing parasite populations. Through this work, tools were developed to facilitate parallel phenotyping by measuring in vitro growth using high-throughput sequencing. The work also develops novel approaches to address the importance of genetic background and a potential role for positive epistasis in a lineage responsible for the recent outbreak of drug-resistant malaria in Southeast Asia.

The most virulent malaria parasite, Plasmodium falciparum, makes use of extensive antigenic diversity to maximise its transmission potential. Parasite genomes contain several highly polymorphic gene families, whose products are the target of protective immune responses. The best studied of these are the PfEMP1 surface proteins, which are encoded by the var multi-gene family and are important virulence factors. During infection, the parasite switches expression between PfEMP1 variants in order to evade adaptive immune responses and prolong infection. On the population level, parasites appear to be structured with respect to their var genes into non-overlapping repertoires, which can lead to high reinfection rates. This non-random structuring of antigenic diversity can also be found at the level of individual var gene repertoires and var genes themselves. However, not much is known about the evolutionary determinants which select for and maintain this structure at different ecological scales. In this thesis I investigate the mechanisms by which multi-scale immune selection and other ecological factors influence the evolution of structured diversity. Using a suite of theoretical frameworks I show that treating diversity as a dynamic property, which emerges from the underlying infection and transmission processes, has a major effect on the relationship between the parasite’s transmis- sion potential and disease prevalence, with important implications for monitoring control efforts. Furthermore, I show that an evolutionary trade-off between within-host and between-host fitness together with functional constraints on diversification can explain the structured diversity found at both the repertoire and parasite population level and might also account for empirically observed exposure-dependent acquisition of immunity. Together, this work highlights the need to consider evolutionary factors acting at different ecological scales to gain a more comprehensive understanding of the complex immune-epidemiology of P. falciparum malaria.

The protozoan parasite Plasmodium falciparum is responsible for the deadliest form of malaria, which causes 584,000 fatalities annually and whose complications include coma, anemia, respiratory distress, and renal failure. Although malaria eradication efforts were hindered by the rise of chloroquine (CQ) resistance (CQR), CQ continues to be clinically deployed in resistance-free regions. CQR is primarily mediated by mutations in the P. falciparum chloroquine resistance transporter (pfcrt) gene, which also modulates parasite susceptibility to first-line artemisinin-based combination therapies (ACTs). In certain geographical regions (e.g. Africa), mutant pfcrt alleles display considerable fitness costs and have undergone attrition in the absence of CQ pressure. Surveillance of resistant field isolates presently centers on the PfCRT mutation K76T, ubiquitous among CQ-resistant parasites and always accompanied by ≥3 additional mutations. Despite the global adoption of K76T as a molecular marker of CQR, the contributions of this and other mutations to P. falciparum drug resistance versus fitness had not been previously defined. AIMS: We aimed to address the following: (1) Do PfCRT mutations beyond PfCRT K76T directly contribute to CQR? (2) Do PfCRT mutations contribute to parasite fitness during the pathogenic asexual blood stage? (3) Are there predictable mutational paths in the evolution of pfcrt-mediated drug resistance? (4) How do PfCRT mutations impact current antimalarials, including the first-line ACTs? APPROACH: Using zinc finger nucleases, we generated isogenic, pfcrt-modified blood-stage P. falciparum parasites encoding wild-type (CQ-sensitive) or variant PfCRT haplotypes. Variants included a combinatorial library of alleles harboring 1-4 mutations comprising the simplest CQ-resistant haplotype (Ecu1110). Additional genetic dissections of full-length or partial pfcrt alleles encompassed the most common variants found in Africa and Asia, including a unique fitness-neutral mutant allele (Cam734) that has undergone expansion in Southeast Asia. Parasite antimalarial drug susceptibility was determined using IC50-based (cytostatic) assays or parasite survival-based (cytocidal) assays and was combined with data from flow cytometric parasite growth competition assays to computationally model mutant pfcrt evolution. To further define the biochemical impacts of PfCRT mutations, our studies leveraged metabolomic, heme fractionation, and drug transport studies. RESULTS: Key findings emerging from our studies included the following: (1) PfCRT K76T is insufficient for CQR and an inaccessible first mutational step in pfcrt evolution; (2) Alongside proliferation rates, parasite resistance gains dictate a constrained pfcrt mutational landscape and predict important roles for the active metabolites of CQ and amodiaquine in guiding pfcrt evolution; (3) To various degrees, PfCRT polymorphisms beyond K76T increase the potency of both the artemisinin and partner drug components of first-line ACT regimens; (4) Emerging PfCRT mutations (e.g. A144F) directly contribute to the enhanced fitness of pfcrt alleles and are necessary for multidrug resistance, independent of K76T. CONCLUSIONS: Our studies uncovered multiple pleiotropic contributions of PfCRT mutations to antimalarial drug resistance, countering earlier dogma that non-K76T mutations are merely compensatory. Evolutionary modeling revealed parasites’ ability to navigate constrained mutational landscapes and evolve drug resistance via rare mutational bursts. These results collectively highlight the capacity of PfCRT to acquire novel mutations that successfully balance parasite multidrug resistance with the essential role of PfCRT in maintaining digestive vacuole physiology. Our studies are of direct relevance to the regional recommendations of antimalarials, whose activity is influenced by, and in certain cases enhanced against, pfcrt-mutant parasites.

Malaria affects over half of the world's population and causes half a million deaths annually, especially in Sub-Saharan Africa. Four species of the apicomplexan Plasmodium parasite (P. falciparum, P. ovale, P. malariae and P. vivax) are responsible for malaria in Africa. Both parasite and host factors contribute to heterogeneity in the risk of developing malaria, clinical manifestation of the disease as well as the number of treatments required to clear parasites. The epidemiology of the different species, and the role of exposure to mixed-species Plasmodium co-infections in generating heterogeneity remains poorly studied. Being an obligate intracellular parasite the blood-stage life cycle of the Plasmodium parasite takes place in the erythrocytes of the human host. The surfaces of these erythrocytes are the medically important ABO blood group antigens that have been reported to influence the susceptibility or otherwise of an individual developing severe malaria. In this thesis I have considered the contributions of the species of Plasmodium parasites and the ABO blood group of the host in driving heterogeneity in human malaria. The aims of this thesis were to determine: (i) the seroepidemiology of the different Plasmodium species in two mesoendemic African populations (Zimbabwe and Sudan); (ii) to determine if heterogeneity in clinical presentations of malaria (history of fever, body temperature and parasitaemia) and response to drug treatment is related to exposure to single vs. mixed-Plasmodium species infection; (iii) the spatial and temporal dynamics of malaria prevalence and Plasmodium species distribution in a mesoendemic village in eastern Sudan; (iv) gene expression changes in 3D7 P. falciparum parasites as they infect erythrocytes of different ABO blood group donors. For aims (i to iii) I developed an enzyme-linked immunosorbent assay using antigens derived from Plasmodium merozoite surface protein 1, also known as MSP-119, to detect IgG antibodies to all four malaria parasite species in Zimbabwean and Sudanese populations. In the Zimbabwean study, plasma samples from 100 individuals each (aged 5-18 years) from three villages (Burma Valley, Mutoko and Chiredzi) were screened for exposure to Plasmodium parasites. In Daraweesh, Sudan, plasma samples from 333 individuals (aged 1-74 years) who had experienced a first malaria episode between 1990 and 2000 were recruited into the study. For study aim (iv) I cultured a single clone of 3D7 P. falciparum parasite using erythrocytes of individuals of different ABO blood group types, harvested parasite RNA and sequenced it to determine gene expression changes in the different hosts. I showed that human IgG antibodies to MSP-119 antigens of the four Plasmodium species are species-specific and do not cross-react. In both study populations almost all antibody responses involved P. falciparum, and single-species responses were almost exclusively directed against P. falciparum antigens. Mixed-species responses accounted for more than a third of responses, and were associated with chloroquine treatment failure, with significantly high proportion of individuals with mixed-species infections requiring repeated treatment with chloroquine/sulfadoxine-pyrimethamine for parasite clearance. This finding highlights the need for a sensitive method for detecting mixed-species malaria infections to enable the assessment of the true prevalence and magnitude of the disease burden caused by the non-falciparum species in endemic populations. Drug treatment failures associated with mixed species infections have significant impact on malaria morbidity and mortality. Treatment failure or partial parasite clearance has the potential to allow dormant liver stages of P. vivax and P. ovale to become a source of parasite reservoir for onward transmission. Furthermore, untreated low-grade chronic infections caused by P. malariae have been reported to cause systemic diseases many years after the primary infection. Spatial analysis of malaria epidemiology showed that malaria parasite transmission in Daraweesh was focal, and that infections are not randomly distributed in the village. Two space-time clusters of significantly increased malaria risk were identified (1993- 1999, and 1998-1999) with marked variations between households, but little or no variation in the species of Plasmodium over time. Similarly, multiple significant clusters were identified for the parasite species; three for P. falciparum, two for P. vivax and P. malariae, and one for P. ovale. These clusters had overlapping time frames, with some of the species significantly infecting the same households. This suggests that even in a small geographic area malaria transmission shows heterogeneity, and that such data can provide useful information to guide malaria control efforts. Finally, I demonstrated that 3D7 P. falciparum parasite growth was similar in the erythrocytes of different blood group donors, and provide preliminary data to show that the non-coding RNA gene, PF3D7_1370800, is differentially expressed in blood group A donors relative to blood groups B and O donors. Further research is needed to better understand the role of this gene in malaria pathology. All together, these findings will aid malaria researchers and other stakeholders in making informed choices about tools for diagnosing Plasmodium species, and control programmes targeting eradication of malaria caused by all Plasmodium species, as is the case of incorporating these findings into current malaria research in Sudan.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-12-02T14:39:23Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EstudoFitoquimicoAvaliacao.pdf: 4226562 bytes, checksum: 7edac4fb3f72b9175767c12c0d78fa0a (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-12-02T16:31:44Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EstudoFitoquimicoAvaliacao.pdf: 4226562 bytes, checksum: 7edac4fb3f72b9175767c12c0d78fa0a (MD5) / Made available in DSpace on 2014-12-02T16:31:44Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_EstudoFitoquimicoAvaliacao.pdf: 4226562 bytes, checksum: 7edac4fb3f72b9175767c12c0d78fa0a (MD5) Previous issue date: 2013 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / SCTIE - Secretaria de Ciência, Tecnologia e Insumos Estratégicos do Ministério da Saúde do Brasil / DECIT - Departamento de Ciência e Tecnologia da Secretaria de Ciência, Tecnologia e Insumos Estratégicos do Ministério da Saúde do Brasil / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / FAPESPA - Fundação Amazônia de Amparo a Estudos e Pesquisas / Parahancornia fasciculata (Poir.) Benoist (Apocynaceae), também conhecida como Parahancornia amapa (Hub.) Ducke, é uma espécie vegetal empregada popularmente no tratamento da malária, infecções no útero, gastrite, anemia, problemas respiratórios, entre outros. Os objetivos do presente trabalho foram realizar o estudo fitoquímico, avaliar a toxicidade oral aguda e a atividade antimalárica in vitro e in vivo de extratos, frações e substância isolada obtidas a partir de cascas do caule de P. fasciculata. Foram realizados dois tipos de extrações com o pó das cascas de P. fasciculata, por maceração / percolação, com etanol 96°GL e diclorometano, esta última tendo sido realizada a com o pó das cascas alcalinizado com hidróxido de amônio, obtendo-se os extratos secos EEPF e EDAPF, respectivamente. Uma terceira extração foi realizada a partir do EEPF por aquecimento sob refluxo, sucessivamente, com Hex:DCM (1:1), AcOEt:DCM (1:1) e AcOEt. EEPF foi, também, submetido a fracionamento por extrações ácido-base resultando nas frações de neutros (EEPFN) e de alcalóides (EEPFA). A prospecção fitoquímica realizada com o EEPF foi desenvolvida por CCD em cromatoplacas de sílica gel tendo sido detectada a presença de triterpenos, esteróides, heterosídeos flavônicos, saponinas, polifenóis, taninos, heterosídeos antracênicos e heterosídeos cardiotônicos. EDAPF foi submetido à cromatografia em coluna de sílica gel. Foram recolhidas 30 frações sendo que as frações Fr1-3, Fr4, Fr5-7 e Fr11 concentraram a maior parte da massa do extrato cromatografado. Da Fr5-7 foi isolada uma mistura de ésteres do lupeol que representam os componentes majoritários do EDAPF. Esta fração passou por um processo de hidrólise alcalina e o produto obtido (Fr5-7Hid) foi analisado por espectrometrias no IV, RMN de 1H e 13C e foi identificado como o triterpeno lupeol. A fração insolúvel em AcOEt obtida a partir do EEPF, por aquecimento sob refluxo, apresentou resultado positivo para o teste de proantocianidinas e foi submetido a doseamento desta classe de metabólitos. Os resultados foram expressos em porcentagem dos teores para a amostra não diluída (10,46±0,3419%), amostra diluída a 1:10 (9,94± 0,1598%) e amostra diluída a 1:100 (10,55± 0,9299%). A avaliação da atividade antiplasmódica in vitro em culturas de cepas W2 de Plasmodium falciparum foi realizada pelo teste da Proteína II Rica em Histidina (HRP-II) tendo sido testados EEPF, EEPFN, EEPFA, Fr1-3, Fr4, Fr5-7(ésteres do lupeol), Fr11 e o Fr5-7Hid (lupeol). Os melhores resultados obtidos foram para EEPF, EEPFA E EEPFN (CI50= ~ 50 μg/mL) sendo considerados moderadamente ativos. As demais amostras apresentaram CI50 > 50 μg/mL e foram consideradas inativas. Realizou-se também a avaliação da atividade antimalárica in vivo em camundongos fêmeas suíços infectados com cepas ANKA de P. berghei com o EEPF e o EEPF-HEX:DCM (1:1) em concentrações de 500, 250 e 125mg/kg de peso. EEPF foi parcialmente ativo, somente no 8° dia, em todas as concentrações. Já EEPF-HEX:DCM (1:1) foi parcialmente ativo na dose de 500mg/kg de peso e nas demais doses foi inativo. O teste de toxicidade oral aguda foi realizado em camundongos fêmeas suíços, pelo método da dose fixa (5.000mg/kg), com EEPF e não apresentou nenhum sinal de toxicidade evidente, o que foi confirmado pela ausência de alterações nos exames anátomohistopatológicos realizados. / Parahancornia fasciculata (Poir.) Benoist (Apocynaceae), also known as Parahancornia amapa (Hub.) Ducke is a species used in the treatment of malaria, uterus infections, gastritis, anemia, respiratory problems, among other ailments. The objectives of this study were to carry out the phytochemical study of the trunk bark from P. fasciculata, to evaluate the in vitro and in vivo antimalarial activity as well as the acute oral toxicity of extracts and fractions from this plant species. The powder bark of P. fasciculata was submitted to extractions by maceration/percolation with ethanol 96% and with dichloromethane after alkalinization of the bark powder affording the dry extracts EEPF and EDAPF, respectively. EEPF underwent two different re-extractions: 1) acid-base extractions affording the neutral (EEPFN) and alkaloidal fractions (EEPFA) and 2) heating under reflux with different solvents, leading to the fractions EEPF-DCM:HEX (1:1), EEPF-DCM: AcOEt (1:1) and EEPFinsoluble in AcOEt. Phytochemical screening of EEPF by TLC revealed the presence of triterpenes and steroids, flavonoid heterosides, saponins, polyphenols, tannins, anthracene heterosides and cardiotonic heterosides. EDAPF was submitted to chromatography through a silica gel column to give 30 fractions of which Fr1-3, Fr4, Fr5-7 and Fr11 represented most of the extract that was chromatographed. Fr5-7 led to the isolation of a mixture of esters of lupeol which are the major components of this extract. Saponification of this fraction afforded Fr5-7Hid that was analyzed by IV, 1H and 13CNMR and was identified as the triterpene lupeol. The insoluble AcOEt fraction derived from re-extraction of EEPF gave a positive test for proanthocyanidins which were quantitatively determined and the results were expressed in percentage for the content of these metabolites in an undiluted sample (10,46 ± 0,3419 %), a 1:10 diluted sample (9,94 ± 0,1598 %) and a 1:100 diluted sample (10,55 ± 0,9299%). The evaluation of the antiplasmodial activity in vitro was carried out against W2 strains of Plasmodium falciparum by the assay of the Histidine-Rich Protein II (HRPII) with EEPF, EEPFN, EEPFA, Fr1-3, Fr4, Fr5-7 (lupeol esters), Fr11 and Fr5-7Hid (lupeol). The best result was obtained for EEPF, EEPFA, EEPFN (CI50 = ~ 50 μg / mL) that can be considered as moderately active. The remaining samples showed CI50 > 50 μg / mL and were considered inactive. The in vivo antimalarial activity was performed in Swiss female mice infected with ANKA strains of Plasmodium berghei with EEPF and EEPF-DCM:HEX (1:1) at concentrations of 500, 250 and 125mg/kg body weight. EEPF was partially active only on the 8th day in all concentrations tested while EEPF-DCM:HEX (1:1) was partially active at a dosis of 500mg/kg and was inactive in the remaining doses. The acute oral toxicity test was determined for EEPF in Swiss female mice by the method of the fixed dose (5,000mg/kg) when no apparent signs of toxicity were observed what was confirmed by the absence of anatomic and histopathologic changes.

Tam, Hou Si. / 33 in title is subscript. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 174-187). / Abstracts in English and Chinese. / THESIS COMMITTEE --- p.i / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.iii / 摘要 --- p.v / TABLE OF CONTENTS --- p.vii / LIST OF FIGURES --- p.xii / LIST OF TABLES --- p.xvii / LIST OF ABBREVIATIONS --- p.xviii / CHAPTER / Chapter 1. --- INTRODUCTION / Chapter 1.1 --- Malaria --- p.1 / Chapter 1.2 --- Malaria is a public health problem --- p.1 / Chapter 1.3 --- Malarial parasite --- p.3 / Chapter 1.4 --- Life cycle of P. falciparum --- p.3 / Chapter 1.4.1 --- The pre-erythrocytic stage --- p.3 / Chapter 1.4.2 --- The asexual erythrocytic stage --- p.3 / Chapter 1.4.3 --- The sexual transmission stage --- p.6 / Chapter 1.5 --- Chemoprophylaxis and chemotherapy of malaria --- p.7 / Chapter 1.6 --- Drug resistance of malaria parasite --- p.7 / Chapter 1.7 --- The progress for malaria vaccine --- p.10 / Chapter 1.8 --- Vaccine candidates for asexual erythrocytic stage --- p.11 / Chapter 1.9 --- Merozoite Surface Protein-1 (MSP-1) --- p.13 / Chapter 1.9.1 --- Structure of MSP-1 --- p.13 / Chapter 1.9.2 --- The processing of MSP-1 --- p.17 / Chapter 1.9.3 --- MSP-1 as a blood-stage vaccine --- p.19 / Chapter 1.9.4 --- The vaccine potency of MSP-133 --- p.23 / Chapter 1.10 --- Merits of E. coli expression system --- p.25 / Chapter 1.11 --- Aim of study --- p.26 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.30 / Chapter 2.2 --- Methods --- p.39 / Chapter 3. --- EXPRESSION AND PURIFICATION OF RECOMBINANT MSP-l33kv+19 PROTEIN / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Results / Chapter 3.2.1 --- Construction of pET32a/MSP-l33kv+19 expression vector --- p.64 / Chapter 3.2.2 --- SDS-PAGE analysis of the expressed protein --- p.74 / Chapter 3.2.3 --- Western blot analysis of the expressed protein --- p.78 / Chapter 3.2.4 --- Modification of the expression conditions --- p.78 / Chapter 3.2.5 --- Protein purification by IMAC --- p.82 / Chapter 3.2.6 --- Cleavage of fusion partner from the rMSP-133kv+19 protein --- p.82 / Chapter 3.2.7 --- Verification of non-fused recombinant MSPl33kv+19 protein by N-terminal amino acid sequencing --- p.86 / Chapter 3.2.8 --- Separation of target protein from the fusion mixture by IMAC --- p.86 / Chapter 3.2.9 --- Separation of digestion product by Size Exclusion Chromatography --- p.89 / Chapter 3.2.10 --- Conformational test of the purified protein --- p.89 / Chapter 3.2.11 --- Separation of target protein from contaminants by Anion-Exchange Chromatography --- p.92 / Chapter 3.2.12 --- Separation of target protein from contaminants by Immuno-Affinity Chromatography --- p.95 / Chapter 3.3 --- Conclusion --- p.95 / Chapter 4. --- IMMUNOLOGICAL CHARACTERIZATION OF BACTERIAL EXPRESSED rMSP-l33kv+19 / Chapter 4.1 --- Introduction --- p.97 / Chapter 4.2 --- Results / Chapter 4.2.1 --- Immunogenicity of recombinant NfMSP-133kV+19 protein --- p.98 / Chapter 4.2.2 --- Specificity of anti-NfMSP-133kv+19 sera to MSP-l33kv. MSP-l33 and MSP-l19 --- p.98 / Chapter 4.2.3 --- Cross reactivity of anti-MSP-133kv+19 and anti-BVp42 serum --- p.103 / Chapter 4.2.4 --- Competitive ELISA --- p.103 / Chapter 4.2.5 --- Test for the presence of inhibitory B-cell epitopes on rMSP-l33kv+19 --- p.111 / Chapter 4.2.6 --- In vitro parasitic growth inhibition assay --- p.113 / Chapter 4.3 --- Conclusion --- p.115 / Chapter 5. --- EXPRESSION AND PURIFICATION OF RECOMBINANT MSP-l33kc+19 PROTEIN / Chapter 5.1 --- Introduction --- p.116 / Chapter 5.2 --- Results / Chapter 5.2.1 --- Construction of pET32a/MSP-133kv+19 expression vector --- p.117 / Chapter 5.2.2 --- Expression of recombinant MSP-133kc+19 protien (rMSP-133kc+19) --- p.124 / Chapter 5.2.3 --- Purification of rMSP-l33kc+19 by IMAC --- p.127 / Chapter 5.2.4 --- Cleavage of fusion partner from target protein --- p.127 / Chapter 5.2.5 --- Construction of pRSETA/MSP-l3X33kc+19 expression vector --- p.135 / Chapter 5.2.6 --- SDS-PAGE analysis of the protein expression --- p.146 / Chapter 5.3 --- Conclusion --- p.153 / Chapter 6. --- DISCUSSION / Chapter 6.1 --- Expression of rMSP-l33kv+19 --- p.154 / Chapter 6.2 --- Purification of rMSP-l3.3kv+19 --- p.156 / Chapter 6.3 --- Conformational test of rMSP-133kv+19 --- p.157 / Chapter 6.4 --- Biological and immunological activity of NfMSP-133kv+19 --- p.158 / Chapter 6.5 --- Expression of rMSP-133kc+19 --- p.166 / Chapter 6.6 --- Future prospects --- p.167 / REFERENCES --- p.174 / APPENDICES / Chapter 1. --- HiTrap NHS-activated HP for ligand coupling procedure --- p.188 / Chapter 2. --- Reuse of Ni+-NTA Resin procedure --- p.190 / Chapter 3. --- Sequence alignment of MSP-133 (MAD20 & Welcome/Kl alleles) --- p.191 / Chapter 4. --- Nucleotide sequence and amino acid sequence of P. falciparum MSP-l33kv+19 --- p.192 / Chapter 5. --- Nucleotide sequence and amino acid sequence of P. falciparum MSP-l33kc+19 --- p.193 / Chapter 6. --- "Nucleotide sequence and amino acid sequence of P. falciparum MSP-142 (3D7 isolate, MAD20 allele)" --- p.194 / Chapter 7. --- Amino acid sequence of Plasmodium falciparum MSP-l42 --- p.195