Glutamate dehydrogenase a (GDHa) has been suggested as a potential drug target against the malaria parasite Plasmodium falciparum. GDHa knockout cell line was generated and needed a phenotypic description by means of molecular biology and biochemistry. The knockout cell line was tested for higher oxidative stress sensitivity, levels of relevant proteins and gene transcripts were quantified. Furthermore, concentrations of two key molecules enabling redox homeostasis, glutathione and NADPH, were attempted to quantify. Finally, we attempted to disrupt a gene of another glutamate dehydrogenase, gdhb, which did not result in formation of viable parasites. In conclusion, GDHa is not a suitable drug target and GDHb needs to be further elucidated.

Orientadores: Rolf Dieter Illg, Nicolas Delabays / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-21T13:43:54Z (GMT). No. of bitstreams: 1 Magalhaes_PedroMelillode_D.pdf: 6355659 bytes, checksum: 2d95ce72d59eb7cfb1d57fb1e4c5399c (MD5) Previous issue date: 1996 / Résumé: Le paludisme est aujourd'hui encore l 'une des grandes causes de mortalité et de maladie dans le monde. Au Brésil, i l peuty avoir jusqu'à 500 mille cas de paludisme par ano La maladie, provoquée par le protozoaire Plasmodium, s'est aggravée depuis que l es especes de Plasmodium ont acqui s une rési stance aux remedes traditionnels, chloroquine et mefloquine. L'étude de nouveaux produits a abouti à la découverte des propriétés anti paludéennes d'une lactone sesquiterpenique, appelée Qinghaosu ou Artemisinine, extraite des feuilles de l 'Artemisia annua L., de l a famille dês Asteraceae. Dês composés derives de l 'artémisinine sont encore plus efficaces et sont commercialisés en Chine. Ainsi, la technologie de production et la validation de ces médicaments en Occident sont d'un grand intérêt. L'objectif principal des recherches qui ont composé cette these a été de contribuer à la production des principes actifs de l'espece A. annua L., favorisant ainsi l 'obtention dês médicaments anti-paludéens. Les recherches réalisées à MEDIPLANT CSuisse) et au CPQBA-UNICAMP CBrésil) ont inclus: des aspects de la reproduction, la selection et l¿obtention d¿hybrides pour la culture au Brésil, la fertilisation nitrogén''e et dês tentatives d¿extraction aqueuse de l 'artémisinine. Les résultats ont indiqués que l 'espece, bien que possédant des fleurs completes, présente un taux d'alogamie supérieur à 80%. Ainsi, à travers des croisements controles entre des génotypes sélectionnés dans les populations de base originaires de (hine et du Vietnam, a-t-on obtenu l 'homogénéité des populations et un gain génétique significatif. L'exploitation de la variabilité en biomasse et en teneur d'artémisinine, cette derniére étant connue par controle analytique, a permis de passer de 5Kg d'artémisinine lha pour les populations de base, à 30Kg d'artémisinine lha pour lês popul at i ons amél i orées. La propagat i on végétat i ve, pa r cul ture de tissus ou par boutures, a également été utilisée pour lê programme d'amélioration génétique, essentiellement pour l 'entretien dês progéniteurs et l a fixation des caracteres souhaitables. Deux génotypes, Pl .39 Et P 1 . V , ont été sélectionnés pour la culture en photopériode courte (11 à 12 heures de lumiere), conformément aux conditions du Brésil. Les teneurs en artémisinine et les productions de racines et de feuilles, dans les lignées cultivées en phytotron, ont été plus grandes pour des températures variant entre 22°( et 26°(, par rapport aux rendements obtenus pour des températures variant entre 25°( et 31°(. Le développement des raciness a été directement proportionnel aux teneurs d'artemisine dans les feuilles d'A. annua L.. Les premiers travaux de sélection, menés en des endroits ou les conditions de photopériode n'induisaient pas la floraison de l'espece, a permis la sélection de génotypes plus productifs en biomasse du fait qu'ils expriment tout leur potentiel végétatif. Au Srésil, 1 'évaluation de 1 'hybride Ch. x Viet 55 a attei nt des rendements approximatifs e 30 kg d'artémisinine/ha et par récoltes, deux récoltes par an étant possibles sur une même aire. La culture de cet hybride a été techniquement viable et hautement compétitif grâce aux rendements btenus. L'application de fertilisants azotes àune dose de 90Kg d'azote/ha a augmenté de 28 à 40% es rendements d'artémisinine en fonction de l 'augmentation de la biomasse. La forme nitrate s'est révél ée l a pl us favorabl e. Un possible déplacement de l ' ét a t de plus forte concentration de l 'artémisinine est suggéré. Des extractions aqueuses à partir de feuilles riches en artémi si ni ne présentent une fracti on contenant 50% du pri nci pe actif. On suggere que ce procédé puisse être employé comme pré processus pour des extractions industrielles, ou même servir de base à de nouvelles recherches sur l 'activité anti ¿paludéenne d'infusions. Les études de l'Artemisia annua ont eu pour consequence l ' établ i ssement d'un programme d'amélioration génétique au CPQSA / Resumo: A malária é ainda uma das grandes causas de mortalidade e morbidade no mundo. No Brasil, chegam a ocorrer 500 mil casos de malária por ano. A doença, causada pelo protozoário Plasmodium, tem sido agravada desde que espécies de Plasmodium passaram a adquir resistência às drogas tradicionais, cloroquina emefloquina. O estudo de novos medicamentos resultou no descobrimento das propriedades antimaláricas de uma lactona sesquiterpênica, chamada Qinghaosu ou Artemisinina, extraída das folhas da Artemísia annua L., da família Asteraceae. Compostos derivados da artemisinina são ainda de maior eficácia e vem sendo comercializados na China. Assim, a tecnologia de produção e a validação destes medicamentos no ocidente, são de grande interesse. O objetivo principal das pesquisas que compuseram esta Tese foi de contribuir para a produção dos princípios ativos da espécie Artemisia annua L., favorecendo a obtenção dos medicamentos antimalájicos. As pesquisas realizadas na MEDIPLANT (Suíça) e no CPQBA-UNICAMP (Brasil) envolveram: aspectos da biologia da reprodução; seleção e obtenção de híbridos para cultivo no Brasil; fertilização nitrogenada e ensaios de extração aquosa da artemisinina. Os resultados indicaram que a espécie, embora possua flores completas, apresenta taxa de a 1 oogamia superior a 80%. Assim, através de cruzamentos controlados entre genótipos selecionados nas populações de base, originais da China e do Vietnam, obteve-se a homogeneidade das populações e significativo ganho genético. A exploração da variabilidade em biomassa e em teores de artemisinina, esta última conhecida por monitoramento analítico, possibilitou passar de 5Kg de artemisinina /há nas populações de base, para aproximadamente 30Kg de artemisinina/ha nas populações melhoradas. A propagação vegetativa, por cultura de teci dos ou por estacas, foi também utilizada no programa de melhoramento genético, principalmente para a manutenção dos progenitores e a fixação de caracteres desejáveis. Dois genótipos, Pl.39 e Pl. V, foram selecionados para cultivo em fotoperíodo curto, (11-12 horas de luz) conforme as condições do Brasil. Os teores de artemisinina e as produções de raízes e de folhas, em estirpes cultivadas em fitotrons, foram maiores na faixas de temperatura ambiente de 22 a 26°C, em comparação com os rendimentos obtidos na faixa entre 25 a 31°C. A produção de raízes foi diretamente relacionada com os teores de artemisinina nas folhas de A annua L.. Os primeiros trabalhos de seleção, conduzidos em local onde as condições de fotoperíodo não induzi am o florescimento da espécie, permitiu a seleção de genótipos mais produtivos em biomassa por expressarem todo seu potencial vegetativo. A avaliação, no Brasil, do híbrido Ch. x Viet 55, atingiu rendimentos de aproximadamente 30Kg de artemisinina /há /corte, sendo possível a realização de 2 cultivos por ano, na mesma área o cultivo deste híbrido foi tecnicamente viável e altamente competitivo, devido aos rendimentos alcançados. A aplicação de fertilizantes nitrogenados na dose de 90Kg de nitrogênio / há proporcionou um aumento dos rendimentos de artemisinina que variou entre 28 e 40%, em função de maior biomassa. A forma nitrato foi a mais favorável. Um possível deslocamento do estádio de maior concentração de artemisinina é sugerido. Extrações aquosas, a partir de folhas ricas em artemisinina, apresentaram fração contendo 50% do princípio ativo. Sugere-se que tal procedimento possa servir de base para novas pesquisas farmacológicas e toxicológicas sobre a atividade anti-malárica de infusões. Os estudos com a A. annua L. resultaram no estabelecimento de um programa de melhoramento genético no CPQBA / Abstract: Malaria is still one of the greatest causes of mortality in the world; in Brazil there are over 500.000 reported cases per year. Malaria, caused by the protozoa Plasmodfum, has been aggravated by the increasing resistance of Plasmodfum to the traditional drugs cloroquine and mefloquine. The study of new drugs resulted in the identification of antimalarial activities of an endoperoxide sesquiterpene lactone, called Qinghaosu or Artemisinin, extracted from the leaves of A. annua L., from the Asteraceae family. Compounds derived from artemisinin are highly efficient and are being commercialized in China. The production technology and validation of these drugs in the occidental world are of great interest. The main objective of this thesis was to contribute to the production of the chemical compounds of the species A. annua L., in order to allow the production of anti malarial drugs. The research work developed at MEDIPLANT (Switzerland) and CPQBA-UNICAMP (Brazil) involved: biological aspects of reproduction; selection of hybrids for production in Brazil; nitrogen fertilization and assays of extraction in water of artemisinin. The results indicated that the species, although having complete flowers, presents an alogamy rate superior to 80%. Through controlled hybridization between selected genotypes from China and Vietnam, population uniformity and genetic gain was obtained. Through the analysis of biomass and artemisinin contents (estimated by analytical monitoring), it was possible to increase the artemisinin production of 5Kg/ha for the base population to approximately 30Kg/ha for the genetically bred population. Vegetative propagation, through tissue culture ar stakes, was also utilized in the genetic breeding program, mainly for maintenance of progenitors and fixation of desirable qualities. Two genotypes, Pl.39 and Pl.V were selected for cultivation under short day photoperiod (11-12 hours of light), according to Brazilian conditions. The artemisinin content and the production of roots and leaves in strains cultivated in fitotrons were higher under temperatures ranging from 22 to 26°C, when compared to those cultivated between 25 and 31°C. The root production was directly related to the contents of artemisinin in the leaves of A. annua L.. The first breeding trial, conducted where local photoperiodic conditions did not induce flowering, also allowed the selection of more productive genotype in biomass. In the evaluation for Brazil, the hybrid Ch. X Viet 55 produced approximately 30Kg of artemisinin /ha /cut, with 2cuts possible per year. The cultivation of this hybrid was technically viable end highly competitive, due to the production obtained. The application of Nitrogen fertilizers (90Kg/ha) increased the artemisinin contents in the range of 28 to 40% due to increase in biomass. The nitrate form was the most favorable. It is possible that the period of highest concentration of artemisinin had been delayed. The water extraction of leaves rich in artemisinin produced a fraction containing 50% of active compound. It is suggested that this procedure can be utilized as a base for new pharmacology and toxicology investigations on the antimalarial activity of infusions. The studies with A. annua L. resulted in the establishment of a genetic breeding program at CPQBA / Doutorado / Doutor em Ciências

Introdução: A malária autóctone no Estado de São Paulo (ESP) caracteriza-se por surtos esporádicos na região oeste e transmissão persistente na região leste onde ocorrem casos oligossintomáticos com baixa parasitemia pelo Plasmodium vivax. Objetivos: Analisar a completitude das fichas de notificação de malária autóctone; estimar a tendência da incidência de casos autóctones no ESP de 1980 a 2007; analisar o comportamento clínico e epidemiológico dos casos em duas regiões de autoctonia neste período. Métodos: Foi realizado um estudo descritivo com 19 variáveis das fichas de notificação de malária do ESP, analisadas em duas regiões e em dois períodos (1980-1993 e 1994-2007). Fontes de dados: SUCEN/SES/SP, SINAN/CVE/SES/SP e DATASUS. Resultados: A completitude foi superior a 85% em 11 variáveis. A tendência da incidência de malária autóctone no ESP foi decrescente. Foram notificados 821 casos de autoctonia, 91,6% na região leste, predominando P. vivax. A infecção assintomática teve maior porcentagem no segundo período (p<0,001). Discussão: A completitude das informações foi satisfatória. As diferenças clínicas encontradas merecem atenção da vigilância epidemiológica que deve lidar com o desafio da infecção assintomática por Plasmodium. / Introduction: Autochthonous malaria in São Paulo State is characterized by sporadic outbreaks in the west region and by persistent transmission with oligoassymptomatic cases in the east region, with low parasitemia by Plasmodium vivax. Objectives: To assess the completeness of autochthonous malaria reporting forms; to estimate autochthonous malaria incidence trends in São Paulo State from 1980 to 2007; to analyze the clinical and epidemiological patterns in two distinct regions of autochthony in this period. Methods: This was a descriptive study that analysed 19 report form variables, comparing the east and the west in two periods (1980-1993 and 1994-2007). Sources of secondary data: SUCEN/SES/SP, SINAN/CVE/SES/SP e DATASUS. Results: The completeness was over 85% on 11 variables. The autochthonous malaria incidence trend was decreasing. There were 821 cases of autochthony, 91.6% occurred in the east, predominantly caused by P. vivax. The asymptomatic infection had higher percentage in the second period (p <0.001). Discussion: The completeness of the information was satisfactory. The clinical differences observed deserve attention from surveillance that must deal with the challenge of asymptomatic infection by Plasmodium.

A malária é a doença parasitaria que mais mata no mundo. Metade da população mundial está sob risco de contrair a doença, que mata mais de 1 milhão de pessoas por ano, principalmente crianças com menos de 5 anos de idade. A periodicidade das febres é o sintoma característico da doença. A febre é resultado da lise dos eritrócitos mediada pela saída do parasita, para infectar novas células. Esse evento é sincronizado, com os parasitas rompendo as células ao mesmo tempo. Para que isso aconteça, os eventos celulares que são necessários para a maturação do parasita dentro do eritrócito tem que ser finamente regulada. O hormônio circadiano melatonina é o sinal que sincroniza o ciclo intraeritrocítico do Plasmodium. Neste trabalho, reportamos que a sincronia observada na maioria das espécies do parasita pode ser usada por ele como meio de evadir o sistema imune do hospedeiro e assegurar a continuidade da infecção. Quando dessincronizamos o desenvolvimento do parasita P. chabaudi utilizando luzindol, um antagonista da melatonina, foi observado que o uso de uma dose sub-otima do antimalárico cloroquina aumenta a sobrevida de camundongos infectados. Reportamos também que P. berghei, parasita de roedor que possui uma infecção não sincronizada, não percebe o hormônio. Diferentemente do que é observado com espécies que possuem sincronia, a melatonina falhou em induzir aumento de cálcio intracelular ou sincronizar o ciclo de vida do parasita in vitro. No trabalho também é relatado a construção de vetores para nocauteamento de genes em Plasmodium, afim de se conhecer a função das genes alvo por análise de fenótipo. / Malaria is the most killing parasitic disease in the world. Half of the world population is at risk of contracting the disease, which kills over 1 million people, being children under 5 the most affected. The fever periodicity is the characteristic symptom of the disease. The fever is a result of the burst of the erythrocyte when the parasite leaves the host cell to infect other ones. This event is highly synchronous, with the parasites going out of the cells at the same time. For this to happen, the cellular events that are necessary for parasite growth have to be very well regulated. The circadian hormone melatonin is the signal that synchronizes the intraerythrocytic cycle of Plasmodium. In this work, we report that this synchrony, observed in the majority of the parasites species, could be used as a way to evade the immune system, assuring the continuity of the infection. When we disrupt this synchrony with luzindole, a melatonin antagonist, we observe that a suboptimal dose of the antimalarial chloroquine increases the survival of the infected mice. We also report that P. berghei, rodent parasite that possess and unsynchronized infection, cant perceive the hormone. Unlike what is observed in species that have a synchronous infection, melatonin fail to induce intracellular calcium increase or promote cell cycle synchronization in vitro. Here we also report the construction of knockout vector for Plasmodium, to be used to investigate the functions of the target genes by phenotype analysis.

Orientador: Fabio Trindade Maranhão Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T06:37:45Z (GMT). No. of bitstreams: 1 Lopes_StefanieCostaPinto_M.pdf: 23177514 bytes, checksum: 4171205441f1112d18413fd5eed95ac3 (MD5) Previous issue date: 2008 / Resumo: A violaceína é um pigmento violeta extraído da bactéria Gram-negativa Chromobacterium violaceum. Diversos trabalhos atribuíram à violaceína diferentes atividades biológicas tais como bactericida, antiviral, fungicida e antitumoral. Os efeitos da violaceína também foram observados contra protozoários patogênicos, como Leishmania sp e Trypanossoma sp. Neste trabalho, avaliamos a atividade antimalárica da violaceína in vitro e in vivo contra Plasmodium humano e murino, respectivamente. Neste sentido, a violaceína se mostrou tão eficiente quanto o quinino em eliminar P. falciparum (3D7), mas três vezes menos eficiente que a cloroquina. Além disso, não encontramos diferença na sua atividade contra formas jovens e maduras, mostrando uma atuação similar nos diferentes estágios de desenvolvimento do parasita. Os experimentos in vivo utilizando camundongos infectados com PcchAS tratados por 11 dias consecutivos (0-10) revelaram uma potente atividade desta droga, inibindo o desenvolvimento parasitário em até 86% no pico da parasitemia. Também foi encontrado 60% de inibição quando o tratamento iniciou-se 5 dias após o estabelecimento da infecção. Quando administrada em animais infectados com uma cepa murina letal (PcchAJ), a violaceína protegeu 80% dos animais, em contraste à 100% de mortalidade dos animais não tratados. A comparação do ED50 demonstrou que a violaceína é tão eficiente quanto o artesunato e 2 vezes mais ativa que a cloroquina, sob as mesmas condições de tratamento. Finalmente, animais naive tratados com a violaceína durante 11 dias consecutivos não mostraram alterações na densidade de glóbulos vermelhos e no peso. Também não foi observado nenhum dano morfológico nos cortes histológicos. Coletivamente, estes resultados demonstram claramente o potencial antimalárico da violaceína e abre perspectivas para o entendimento dos mecanismos envolvidos na inibição parasitária por este composto. / Abstract: Violacein is a violet pigment extracted from the bacteria Gram-negative Chromobacterium violaceum. Growing bodies of evidences have implicated violacein as an antimicrobial, antifungal, antitumoral, and a moderate trypanocidal and leishmanial activity has also been observed. Herein, we evaluated the anti-malarial activity of violacein against murine and human-derived Plasmodium. Indeed, violacein showed to be efficient in killing P. falciparum (3D7 strain) as much as quinine, but 3 fold less pronounced than chloroquine. Moreover, this anti-Plasmodial activity detected was direct against both young and mature stages of this human parasite. In vivo experiments in P. c. chabaudi AS (PcchAS) infected mice treated during 10 consecutive days after infection (0-10) revealed a powerful activity of this drug, by inhibiting parasite burden up to 86%. Also, 60% of inhibition was noticed when violacein was administrated 5 days after infection establishment. When administrated in mice infected with a lethal strain of murine-derived Plasmodium (PcchAJ) violacein protected 80% of these mice, in contrast to 100% of mortality reported to the non-treated animals. ED50 comparisons demonstrated that violacein was efficient as much as artesunate and twice more active than chloroquine. Finally, naïve mice inject with violacein during 10 consecutive days did not display alterations on red blood cell density; weight and neither morphological damages were noticed in spleen and liver histological sections. Collectively, these data clearly demonstrated the anti-malarial effect of violacein and open perspectives to understating the mechanisms involved in killing this parasite by this compound. / Mestrado / Imunologia / Mestre em Genética e Biologia Molecular

Although Malaria rates are on the decline due to the efforts of the World Health Organization and other organizations dedicated to the eradication of this disease, a relaxed attitude towards the development of new antimalarial entities would be flawed. Due to the emergence of resistance in the parasite, the almost 50% world-wide reduction in malarial death rates that have been produced over the past 15 years are threatening to be lost New drugs are urgently needed and our approach focuses on the re-evaluation and optimization of the historic antimalarial ICI 56,780. Due to its causal prophylactic activity, along with its ability to prevent transmission and potent blood schizonticidal activities, it was revisited with the hopes of first understanding which functionalities were responsible to the compound's activity. Secondly, we wanted to optimize the substituents in the 3, 6 and 7-positions. Finally and most importantly, we wanted to address the cross-resistance problem of the ICI 56,780 scaffold. Initial, analogues showed the importance of the ester in facilitating the convergence of the RI towards 1. Although those analogues lost activity in W2, TM90-C2B, and Pb, they were our first glimpse at this important trend that was later exploited in our 3-halo-6-butyl-7-(2-phenoxyethoxy)quinolin-4(1H)-one and 3-halo-6-butyl-2-methyl-7-(2-phenoxyethoxy)quinolin-4(1H)-ones which showed RI values of < 5 for our best analogues. Although our lead compound 3-bromo-6-butyl-2-methyl7-(2phenoxyethoxy)quinolin-4(1H) one possessed decreased activities as compared to ICI 56,780 at 2.60 nM for W2, 12.2 nM for TM90-C2B and 2.12 nM for Pb, it had 100% inhibition of parasite development on day 6 PE in our scouting assay and 61% inhibition on day 6in our Thompson model, increased from the < 2% value given by the ICI 56,780. Solubility and unfavorable in vivo stability were still major issues for this scaffold. Therefore, a series of piperazinyl 4(1H)-quinolones with greatly enhanced solubility were designed and tested in detailed structure activity relationships and structure property relationship studies. Initial results showed that 7-piperazinyl-4(1H)-quinolones possessed greatly increased solubilities when compared to ICI 56,780 analogues. Primarily, the linker length and the piperazine core was probed. This showed that compounds with a single carbon spacer were most active. Further testing of the 6-position gave methyl 6-methyl-4-oxo-7-((4-phenylpiperazin-1-yl)methyl)-1,4-dihydroquinoline-3-carboxylate with W2 and TM90-C2B values of 0.435 nM and 147 nM respectively. Substitution on the piperazinyl phenyl gave the most active compounds however the RI of >1500 was unacceptable. Because of this, 3-halo substituents were added to these quinolones with promising results. With RIs of < 3, the compounds were promising, however they were not active in vivo. However, methyl 6-methoxy-4-oxo-7-((4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)methyl)-1,4-dihydroquinoline-3-carboxylate and methyl 6-methyl-4-oxo-7-((4-(4-(trifluoromethyl)phenyl)piperazin-1-yl)methyl)-1,4-dihydroquinoline-3-carboxylate both gave cures in our in vivo Thomson model. These studies highlight the potential in using detailed structural activity and structural property studies to re-evaluate and optimize historic antimalarials. These studies have introduced a new generation of soluble 4(1H)-quinolones with high potency against P. falciparum.

Magister Public Health - MPH / Malaria is a health, social and economic burden in Nigeria and consistently ranks amongst the four most common causes of childhood deaths. Treatment of malaria is usually started at home; care is only sought from the health facility when the treatment is ineffective (McCombie, 1996). Patent medicine vendors (PMVs) have been identified as a widely patronized source for drugs used in the home treatment of malaria (Breiger et al, 2001; Goodman, et al, 2007; Salako et al, 2001). Inadequate or poor knowledge and practices in the use of anti-malaria drugs (AMDs) increases morbidity and mortality, undermines therapeutic efficacy, and promotes the emergence and spread of drugresistant malaria. Aim: The aim of the study was to describe and quantify the knowledge and self-reported practices of PMVs in the use of antimalarials, particularly artemisinin-based combination therapies (ACTs), in a poor urban community in Lagos state, Nigeria. / South Africa

Magister Public Health - MPH / Despite their high level of exposure, vulnerability and uniquely itinerant culture, the local knowledge of the nomadic Fulani population is not taken into account in the development of Nigeria’s home management of malaria policy. Programme-relevant information for extending access to an ethnographic study of factors that nomads use for presumption of malaria in children was collected from dry-season campsites in Demsa Local Government Area of Northeastern Nigeria. Mothers of under-five children with previous experiences at presumptive malaria management from 9 randomly selected nomadic Fulani camps were interviewed. The obtained information was used to develop a guide for key informant interviews of nomadic Fulani cultural consultants and elders, health service providers and policymakers. Findings indicate that nomads presume malaria when a child has “hot body” or lack appetite. Nomads believe that fever accumulates in the body as one steps on wet grounds during the rains. The nascent disease is triggered by the consumption of fruits that resemble the colour of urine such as the light complexioned skin of the Fulani. Fever is therefore regarded as natural affliction of the Fulani for which there is no cure. All fevers are referred to as paboje and expected to go away on the third recrudescence. Fever that persists after the third recurrence is called djonte which is treated at home without health facility support. Besides physical accessibility, the unfriendliness and lack of respect of health personnel for nomadic Fulani culture were reasons for avoiding health facilities. These factors encourage home management of djonte with antimalaria on the presumption that they are all malaria-induced. The nomads are willing to participate in interventions that will improve management of fevers and malaria among them. Although this preliminary study provides the foundation for appreciating the basis of home management of malaria among the nomads, complementary quantitative information will be required for holistic understanding of how these factors may interrelate to influence malaria intervention programme for the nomadic Fulani. / South Africa

Magister Pharmaceuticae - MPharm / Plasmodium falciparum malaria continues to be a worldwide health problem, especially in developing countries in Africa and is responsible for over a million fatalities per annum. Chloroquine (CQ) is low-cost, safe and was the mainstay aminoquinoline derived chemotherapeutic agent that has been used for many years against blood-stage malaria. However, today the control of malaria has been complicated by increased resistance of the malaria parasite to existing antimalarial agents such as CQ. The primary cause of resistance is mutation in a putative ATP-powered multidrug efflux pump known as the p-glycoprotein (pGP) pump, and point mutation in P. falciparum CQ resistance transporter (PfCRT) protein. These mutations are responsible for the reduced accumulation of CQ at its primary site of action, the acidic digestive food vacuole of the parasite.To overcome the challenges of CQ resistance in P. falciparum, chemosensitiser offer an attractive approach. Chemosensitisers or reversal agents are structurally diverse molecules that are known to reverse CQ resistance by inhibiting the pGP efflux pump and/or the PfCRT protein associated with CQ export from the digestive vacuole in CQ resistant parasites. Chemosensitisers include the well-studied calcium channel blocker verapamil and antihistaminic agent chlorpheniramine. These drugs have little or no inherent antimalarial activity but have shown to reverse CQ resistance in P. falciparum when co-administered with CQ. Because of the channel blocking abilities of pentacycloundecylamines (PCUs) such as NGP1-01, it is postulated that these agents may act as chemosensitisers and circumvent the resistance of the Plasmodium parasite against CQ. Therefore as a proof of concept we conducted an experiment using CQ co- administered with different concentrations of NGP1-01 to evaluate the ability of NGP1-01 to act as a chemosensitiser.Herein, we report the ability of NGP1-01, the prototype pentacycloundecylamine (PCU), to reverse CQ resistance (> 50 %) and act as a chemosensitiser. NGP1-01 alone exhibited very low intrinsic antimalarial activity against both the resistant and sensitive strain (> 2000 nM), with no toxicity to the parasite detected at 10 µM. A statistically significant (p < 0.05) dose dependent shift was seen in the CQ IC50 values at both 1 µM and 10 µM concentration of co-administeredNGP1-01 against the resistant strain. Based on this finding we set out to synthesise a series of novel agents comprising of a PCU moiety as the reversal agent (RA) conjugated to a CQ-like aminoquinoline (AM) molecule and evaluate the potential of these PCU-AM derivatives as antimalarial- and/or reversed CQ agents. As recently shown by Peyton et al., (2012), the conjugation of a CQ-like molecule with a RA such as the chemosensitiser imipramine and derivatives thereof is a viable strategy to reverse CQ resistance in multidrug-resistant P. falciparum. The novel compounds were obtained by amination and reductive amination reactions. The synthetic procedures involved the conjugation of the Cookson’s diketone with different tethered 4-aminoquinoline moieties to yield the respective carbinolamines and the subsequent imines. This was followed by a transannular cyclisation using sodium cyanoborohydride as reducing agent to yield the desired PCU-AM derivatives. The CQ-like AMderivatives were obtained using a novel microwave (MW) irradiation method. Structure elucidation was done by utilising 1H- and 13C NMR spectroscopy as well as IR absorption spectrophotometry and mass spectrometry. Five PCU-AM reversed CQ derivatives were successfully synthesised and showed significant in vitro antimalarial activity against the CQ sensitive strain (NF54). PCU-AM derivatives 1.1 – 1.4 showed antimalarial IC50 values in the ranges of 3.74 – 17.6 ng/mL and 27.6 – 253.5 ng/mL against the CQ-sensitive (NF54) and CQ-resistant strains (Dd2) of Plasmodium falciparum, respectively. Compound 1.1 presented with the highest antimalarial activity against both strains and was found to be 5 fold more active against the resistant strain than CQ. The reversed CQ approach resulted in improved resistance reversal and a significantly lower concentration PCU was required compared to NGP1-01 and CQ in combination. This may be attributed to the improved ability of compound 1.1 to actively block the pGP pump and/or the increased permeability thereof because of the lipophilic aza-PCU moiety. Compound 1.1 also showed the lowest RMI value confirming that this compound has the best potential to act as a reversed CQ agent in the series. Cytotoxicity IC50 values observed for compounds 1.1 – 1.4 were in the low micromolar concentrations (2.39 – 9.54 µM) indicating selectivity towards P. falciparum (SI = 149 – 2549) and low toxicity compared to the cytotoxic agent emetine (IC50 = 0.061 µM).These results indicate that PCU channel blockers and PCU-AM derived conjugates can be utilised as lead molecules for further optimisation and development to enhance their therapeuticpotential as reversal agents and reversed CQ compounds.

Malaria causes nearly 3 million deaths annually. The parasite species responsible for the most lethal forms of malaria is P. falciparum (Miller et.al., 2002). Its destructive effect is most evident in the developing African countries, which lack the infrastructure and financial support to effectively control the disease. The only viable means of control at this stage is the use of antimalarial chemotherapy and –prophylaxis, but these drugs are losing their effectivity due to parasite resistance. This underlines the need for new, safe, efficient and cheap drugs as a solution to the African malaria problem. Within the validated folate metabolic pathway of P. falciparum, the identification of three new genes has provided new options for drug inhibition (Lee et.al., 2001). One of these genes encodes the bifunctional dihydrofolate synthase-folylpolyglutamate synthase (DHFS-FPGS), which is unique to P. falciparum (Salcedo et.al., 2001). When compared to human folylpolyglutamate synthase (FPGS), the parasite enzyme is an attractive drug target for selective inhibition due to the additional DHFS activity and low sequence similarity. However, to assess the value of DHFS-FPGS as a drug target and rationally design new drugs against the enzyme, large amounts of enzyme are needed for activity studies and structural determination. The heterologous expression of malaria genes often result in low expression levels, due to its high A+T content and codon bias. To circumvent this problem, a modified P. falciparum dhfs-fpgs, adapted to E.coli codon preferences and with a lower A+T content was synthesised in this study for increased expression. A two-step overlap-extension PCR method was optimised for the synthesis of the 1586bp dhfs-fpgs from only 1 pmol each of partially overlapping oligonucleotides. The use of partially overlapping oligonucleotides, lower amounts of starting material and fewer PCR cycles cut the costs of gene synthesis and the optimisation increased PCR efficiency, when compared to other gene synthesis reports (Carpenter et al., 1999; Zhang et al., 2002). The correct sequences could be obtained from the sequencing of as little as three clones. The successfully constructed dhfs-fpgs gene was expressed in a variety of E. coli expression hosts and vector systems. In all the systems, expression levels of the synthetic gene were much higher than for the native P. falciparum gene. Functional complementation of a DHFS-FPGS deficient E. coli strain verified that the DHFS and FPGS activities were encoded by the synthetic gene, that complementation was achieved to a greater extent than for native P. falciparum dhfs-fpgs and that a synthetic tagless and C-terminal Histidine-tagged DHFS-FPGS had the highest levels of DHFS and FPGS activity. Preliminary purification studies for these two constructs were performed for optimised enzyme isolation, to be followed by activity assays. These optimisations will also serve as basis for future large-scale isolation strategies to obtain sufficient amounts of protein for the structural determination of the enzyme, which would be vital to drug target verification, drug development and –design, thus paving the way for a new generation of antifolate malaria therapy. / Dissertation (MSc(Biochemistry))--University of Pretoria, 2006. / Biochemistry / unrestricted