INTRODUÇÃO: 50-86% dos pacientes recidivam a hepatite autoimune (HAI) após a suspensão do tratamento imunossupressor. A manutenção da imunossupressão em longo prazo diminui o risco de recidiva, entretanto é necessário ajuste da dose/suspensão do tratamento em 10-30%, em razão do maior risco de neoplasias e infecções. O difosfato de cloroquina (CQ) é droga imunomoduladora que foi utilizada anteriormente em monoterapia para manutenção da remissão da HAI com diminuição da recidiva quando comparada com controle histórico. O objetivo desse estudo foi investigar a eficácia e segurança do CQ na manutenção da remissão em estudo duplo cego e randomizado e avaliar se há um subgrupo com maior benefício ao seu uso. METODOLOGIA: 61 pacientes com diagnóstico provável ou definitivo de HAI em remissão histológica, 90,1% HAI tipo 1; 23% com reatividade do anti-SLA/LP, 56,6% com fibrose avançada (F3/4) à inclusão no estudo, mas com doença hepática compensada, foram randomizados de forma duplo cego e aleatória para receber CQ 250 mg/d ou placebo, durante 36 meses ou até recidiva da doença. No primeiro mês a droga foi utilizada em combinação com a imunossupressão que induziu remissão; com posterior desmame semanal da prednisona, suspensão imediata da azatioprina e manutenção do CQ/placebo até 36 meses. As curvas de sobrevida livre de recidiva foram estimadas pelo método de Kaplan-Meyer e comparadas pelo teste de Log-Rank; as razões de risco e seus respectivos intervalos de confiança foram estimados por regressão simples de Cox. Na regressão múltipla foram avaliadas co-variáveis clinicamente relevantes para recidiva. Para investigar o subgrupo com maior benefício, as interações entre a droga e reatividade de autoanticorpos e perfil de HLA foram analisadas por regressão múltipla de Cox. As variáveis categóricas foram comparadas pelo teste exato de Fisher e as quantitativas pelo teste-t ou teste de Mann-Whitney. Foi considerado estatisticamente significante um valor de p < 0,05. RESULTADOS: 31 pacientes receberam CQ e 30 placebo. Não houve diferenças entre os grupos em relação aos achados clínicos, laboratoriais, histológicos e perfil de HLA. A sobrevida livre de recidiva foi significativamente maior no grupo CQ quando comparada ao placebo (59,3% X 19,9%, p=0,039). Após a suspensão da medicação ao término do estudo, houve 41,6% de recidiva no grupo CQ e 0% no placebo. Na regressão simples de Cox, os fatores associados com recidiva da HAI foram uso placebo, reatividade do anticorpo anti-SLA/LP, perfil de HLA DR3 e DR8. Na regressão múltipla, o uso de placebo (razão de risco de 2,4[IC 95%:1,05- 5,5], p=0,039) e reatividade do anticorpo anti-SLA/LP (razão de risco= 5.4 [IC 95%:1,91-15,3], p=0,002) associaram-se a maior risco de recidiva. Não foi possível definir subgrupo de maior benefício com uso de CQ no que se refere à reatividade do anti-SLA/LP ou perfil de HLA, embora a recidiva tenha ocorrido em 100% dos pacientes anti-SLA/LP(+) no grupo placebo e 50% no grupo CQ. No grupo CQ, 54,8% apresentaram efeitos colaterais, com suspensão da droga em 19,3%. Os efeitos colaterais mais comuns foram prurido e hiperpigmentação cutânea. CONCLUSÕES: O CQ reduziu com segurança o risco de recidiva de HAI, mas não foi possível definir subgrupo com maior benefício com essa medicação / INTRODUCTION: 50-86% of patients relapse autoimmune hepatitis (AIH) after immunosuppressive treatment withdrawal, with a higher risk of progression to liver cirrhosis, death due to liver disease and liver transplantation. The maintenance of long-term immunosuppression decreases the risk of relapse, however, treatment dose adjustment and/or interruption is required in 10-30%, with increased risk of neoplasias and infections. Chloroquine diphosphate (CQ) is an immunomodulatory drug used previously in monotherapy to maintain AIH remission with a decrease risk in relapse rates when compared to a historical control. The aims of this study were to investigate the efficacy and safety of CQ in the maintenance of remission in a double-blind and randomized study, and to evaluate if there is a subgroup with a greater benefit of its use. METHODS: 61 patients with probable or definitive diagnosis of AIH in histological remission, 90.1% type 1; 23% with anti-SLA / LP seropositivity, 56.6% with advanced fibrosis [F3 / 4] at inclusion in the study and with compensated liver disease were randomized double-blindly to receive either CQ 250 mg/day or placebo for 36 months or until relapse. In the first month, the drug was used in combination with the immunosuppressive regimen that induced the remission; with subsequent weekly weaning of prednisone, immediate withdrawal of azathioprine and maintenance of CQ/placebo for up to 36 months. Recurrence-free survival curves were estimated by the Kaplan-Meyer method and compared by the Log-Rank test; the hazard ratios and their respective confidence intervals were estimated by simple Cox regression. Clinically relevant covariables for relapse were re-evaluated bymultiple Cox regression. To investigate the existence of a subgroup with a greater benefit, interactions between the drug and autoantibody reactivity and HLA profile were analyzed by the Cox multiple regression. Categorical variables were compared by Fisher\'s exact test and the quantitative by the t-test or Mann- Whitney test. A p value of < 0.05 was considered statistically significant. RESULTS: 31 patients received CQ and 30 placebo. There were no differences between the two groups in relation to clinical, laboratory, histological and HLA profiles. Relapse-free survival was significantly higher in the CQ group when compared to placebo (59.3% X 19.9%, p=0.039). After antimalarial withdrawal at the end of the study, there was 41.6% relapse in the CQ group and 0% in the placebo. In the Cox simple regression, factors associated with AIH relapse were placebo use, anti-SLA/LP seropositivity, and HLA DR3 and DR8 profiles. In multiple regression, placebo use (Hazard Ratio = 2.4 [95% CI: 1.05-5.5], p = 0.039) and anti-SLA/LP seropositivity (Hazard Ratio = 5.4 [95% CI: 1.91-15.3], p = 0.002) were associated with a higher risk of relapse. It was not possible to define a subgroup with a greater benefit of CQ with respect to anti-SLA/LP positivity or HLA profile, although all anti-SLA/LP(+) patients in placebo group relapsed, compared to 50% in CQ group. In the CQ group, 54.8% had side effects, but 19.3% had drug withdrawal. The most common side effects were pruritus and cutaneous hyperpigmentation. CONCLUSIONS: Chloroquine safely reduced the risk of relapse of AIH, but it was not possible to define a subgroup with greater benefit with medication use

Orientador: Lin Chau Ming / Banca: Ari de Freitas Hidalgo / Banca: Maria Christina de Mello Amorozo / Resumo: A malária persiste como uma das principais doenças negligenciadas em todo o mundo. Anualmente três milhões de pessoas a contraem e cerca de um milhão morrem por causa dela. A região do mundo mais afetada é a África Subsariana e, no Brasil, a região amazônica é a mais afetada. As populações locais aprenderam a retirar do meio ambiente os recursos necessários para sobreviver e são atualmente a principal esperança na descoberta de novos medicamentos antimaláricos frente ao parasita da malária que apresenta resistência aos fármacos existentes. Neste trabalho estudou-se o manejo das plantas antimaláricas utilizadas por populações locais de Barcelos, Amazonas. Durante o primeiro semestre de 2013 foram entrevistadas 52 pessoas de sete comunidades rurais e da zona urbana de Barcelos, reconhecidas localmente por seu conhecimento sobre o uso de plantas medicinais. Foram indicadas 118 plantas, seis animais e dois minerais para o tratamento da malária e para outros oito sintomas reconhecidos pelos entrevistados. Foram indicadas 58 espécies para o tratamento da malária, 70% dessas são nativas da região amazônica e 15 plantas não possuem nenhuma referência na literatura com esta indicação de uso. Também foram indicadas 27 plantas para febre, 51 plantas para o tratamento do fígado, 17 plantas para o tratamento da anemia e 15 plantas para dores de cabeça. Foram descritas 12 diferentes formas de preparo das partes utilizadas. 47% das plantas indicadas têm suas folhas utilizadas e 24% sua casca e entrecasca. Das 118 plantas indicadas 46% são arbóreas, 30% herbáceas, 15% arbustivas e 9% trepadeiras. 65% têm origem amazônica, 26% são exóticas do Brasil e as demais são nativas do Brasil, mas não ocorrem naturalmente na região amazônica. De todas estas plantas 45% são cultivadas, 8% podem ser cultivadas ou favorecidas, 17% são favorecidas e as demais não têm nenhum tipo de ... / Abstract: Malaria remains one of the major diseases around the world. Every year three million people contract it and about a million died because of it. The most affected region of the world is sub-Saharan Africa and in Brazil the Amazon region is the most affected. Local people have learned how to survive just with the enviroment and are currently the main hope in the discovery of new antimalarial drugs against the malaria. This work studied the management of antimalarial plants used by local populations of Barcelos, Amazonas. During the first half of 2013 52 people from 7 rural communities and the urban area of Barcelos, locally recognized for their knowledge on the use of medicinal plants were interviewed. 118 plants , six animals and two minerals are used to treat malaria and eight other symptoms. 57 species are used for the treatment of malaria, 70 % of these are native from the Amazon region and 11 of them don't have any reference in the literature. 27 plants are used for fever, 51 plants for the treatment of liver, 17 plants for the treatment of anemia and 15 plants for headaches are also indicated. 12 different preparation of the plants were described. 47 % of listed plants have their leaves used and 24 % used their bark. Of the 118 plants indicated 46 % are woody herbaceous 30 % , shrub 15 % and 9 % climbing plants. 65 % have Amazonian origin , 26 % are exotic in Brazil and the rest are native to Brazil , but do not occur naturally in the Amazon region. 45 % are grown, 8 % can be grown or favored, 17 % are favored and the other does not have any kind of conscious management. 4 % of the plants are only acquired in trade. 26 % of plants are propagated by seeds directly, 17 % by vegetative parts of plants and 12% of seedlings that are transplanted. The use of antimalarial plants is important in the study region even with the use of allelochemicals medicine, but preventive methods , and plants used ... / Mestre

De acordo com a Organização Mundial da Saúde, AIDS,malária e tuberculose são as três maiores doenças infectantes do mundo, atingindo principalmente crianças. Regiões paupérrimas e de clima tropical, como a África sub-saariana, são as mais atingidas. Este quadro agrava-se com a disseminação de cepas do Plasmodium falciparum resistentes à cloroquina e multi-resistentes.Além disso, alguns fármacos utilizados na terapêutica da malária apresentam vários efeitos adversos, comprometendo o tratamento. Trata-se de um grande desafio e o seu enfrentamento requer estratégias. O desenvolvimento de novos quimioterápicos deve fundamentar-se em diferenças bioquímicas e morfológicas entre as células do hospedeiro e do parasita. A biossíntese de fosfolipídeos de membrana em parasitas do grupo Apicomplexa é de extrema importância para a maturação e a reprodução do parasita e constitui-se em bom alvo para novos antimaláricos, uma vez que é encontrada somente em parasitas. Hemácias infectadas têm sua absorção modificada em relação aos eritrócitos não-infectados, conferindo seletividade a substâncias como lipídeos. O trabalho em questão propõe a síntese de antimetabólitos da serina, visando à inibição das enzimas fosfatidilserina síntase e serina descarboxilase, fundamentais para a biossíntese de fosfolipídeos de membrana desses parasitas.. Cinco derivados heterocíclicos da serine foram sintetizados: derivados diidroimidazólico, diidroxazólico, diidroxazínico, diidropirimidínico e diidrooxatiólico. Também, o transportador fosfolipídico com o ácido esteárico foi sintetizado. Os antimetabólitos serão acoplados a esse e outros fosfolipídeos, obtendo-se fármacos dirigidos específicos direcionados seletivamente a eritrócitos infectados. / According to the World Health Organization, Aids, malaria and tuberculosis are the three greatest infectious diseases in the world. Children are the most involved in those diseases. Extremely poor regions, as sub-Saharan, Africa, are the most affected. In the worst case scenario, one of the parasites that causes malaria, Plasmodium falciparum, become resistant to chloroquine and the current therapy. Besides, some drugs used in the mataria chemotherapy are very toxic, showing many side effects, and compromising the treatment. This is a big challenge and facing it requires new strategies.. The development of chemotherapeutic has been inspired in biochemical differences between the parasite and the host. Plasmodíum falciparum needs to biosynthesize phospholipids for their membrane. These phospholipids are very important to the maturation and reproduction of the parasite and occur only in it.. This makes the phospholipids biosynthesis a good target for new and specific antimalarial drug design. Infected red blood cell shows modified permeation, allowing the lipids to be freely transported, what is not usual in the non-infected red blood cells. This said, in the present work the design and synthesis of serine metabolic inhibitors, using the bioisosteric strategy, have been proposed. The inhibition of the phosphatidylserine biosynthesis, an important phospholipid, is expected. These inhibitors will be linked to phospholipids, to promote the selective permeation to the infected red-blood cell.. These inhibitors will be linked to phospholipids, to promote the selective permeation to the infected redblood cell. In the present work five heterocyclic serine inhibitors: diidroimidazolic, diidroxazolic, diidroxazinico, diidropyriminic and diidroxatiolic. Also synthesized a phospholipid to be connected to the heterocyclic inhibitors.

A maioria das cepas de Plasmodium falciparum mostra-se resistente à cloroquina (CQ), considerado o antimalárico ideal. A busca por novos agentes terapêuticos bem como a restauração do efeito antimalárico de fármacos disponíveis, através da associação a agentes moduladores da resistência (AMR), vem sendo enfatizada. Entretanto, ausência de efeito modulador da resistência e efeito antiplasmódico intrínseco foram observados quando AMR clássicos, como o fenotiazínico clorpromazina e o iminodibenzílico desipramina, foram ensaiados in vitro em isolados brasileiros de P. falciparum resistentes à CQ. Sabe-se que a ação antiparasitária de compostos de natureza tricíclica, como os fenotiazínicos, foi descrita há mais de um século e continua a ser de interesse. Em adição, vale notar a ocorrência de farmacóforo comum, formado por sistema heteroaromático, ligado a átomo de nitrogênio, secundário ou terciário, por cadeia lateral de três a quatro átomos de carbono, entre compostos quinolínicos antimaláricos, fármacos psicotrópicos e AMR. Este trabalho teve como objetivo estudar, por emprego da modelagem molecular, características estereo-eletrônicas e lipofílicas e a interação a nível molecular de compostos de natureza tricíclica (fenotiazínicos e iminodibenzílicos) com a hematina (provável sítio de ação da CQ) comparando ao antimalárico CQ. Semelhanças estéreo-eletrônicas e lipofílicas puderam ser visualizadas entre as moléculas da CQ e dos compostos tricíclicos. No entanto, algumas distinções ausência de planaridade e maior densidade eletrônica sobre os anéis tricíclicos dos anéis heterocíclicos quando comparadas à CQ. Tais características se fizeram refletir na interação com a hematina, como demonstrado nos estudos de ancoramento como também nos estudos de UV-VIS e de Raman Ressonante. / The most Plasmodium falciparum strains show resistance to chloroquine (CQ), yet considered the ideal antimalaric agent. The search for new therapeutic compounds and the restoration of the antimalarial effect of available drugs through the association with modulating agents has been emphasized. However, lack of modulating effect and intrinsic antiplasmodial activity were observed when classic modulating agents, such phenothiazine chlorpromazine and iminodibenzylic desipramine, were tested in vitro against Brazilian isolated resistant of P. falciparum to CQ. The antiparasitic action of tricyclic compounds as the phenotiazine class has been described for more than a century and continues to be of interest. In addition, it was noted the occurrence of common pharmacophore, formed by a heteroaromatic system, a secondary or tertiary nitrogen atom, linked by a side chain of three to four carbon atoms, present among the quinoline antimalarials, the psychotropic drugs and the modulating agents of chloroquine resistance. The goal of this work was by using molecular modeling to study stereo-electronic features and lipophilic characteristics and the interaction on molecular level of tricyclic compounds (phenothiazines and iminodibenzylics) with hematin (probable site of action of CQ) in comparison to antimalaric CQ. In results, similarities stereo-electronic and lipophilic could be viewed between the molecules of CQ and tricyclic compounds. However different features could be noticed such as absence of planarity and a higher electronic density on the tricyclic rings when compared to CQ. These features shown to be relevant to interaction with the &#181;-oxo dimer of hematin, as observed in docking studies and UV-VIS and Resonance Raman.

De acordo com a Organização Mundial da Saúde, AIDS,malária e tuberculose são as três maiores doenças infectantes do mundo, atingindo principalmente crianças. Regiões paupérrimas e de clima tropical, como a África sub-saariana, são as mais atingidas. Este quadro agrava-se com a disseminação de cepas do Plasmodium falciparum resistentes à cloroquina e multi-resistentes.Além disso, alguns fármacos utilizados na terapêutica da malária apresentam vários efeitos adversos, comprometendo o tratamento. Trata-se de um grande desafio e o seu enfrentamento requer estratégias. O desenvolvimento de novos quimioterápicos deve fundamentar-se em diferenças bioquímicas e morfológicas entre as células do hospedeiro e do parasita. A biossíntese de fosfolipídeos de membrana em parasitas do grupo Apicomplexa é de extrema importância para a maturação e a reprodução do parasita e constitui-se em bom alvo para novos antimaláricos, uma vez que é encontrada somente em parasitas. Hemácias infectadas têm sua absorção modificada em relação aos eritrócitos não-infectados, conferindo seletividade a substâncias como lipídeos. O trabalho em questão propõe a síntese de antimetabólitos da serina, visando à inibição das enzimas fosfatidilserina síntase e serina descarboxilase, fundamentais para a biossíntese de fosfolipídeos de membrana desses parasitas.. Cinco derivados heterocíclicos da serine foram sintetizados: derivados diidroimidazólico, diidroxazólico, diidroxazínico, diidropirimidínico e diidrooxatiólico. Também, o transportador fosfolipídico com o ácido esteárico foi sintetizado. Os antimetabólitos serão acoplados a esse e outros fosfolipídeos, obtendo-se fármacos dirigidos específicos direcionados seletivamente a eritrócitos infectados. / According to the World Health Organization, Aids, malaria and tuberculosis are the three greatest infectious diseases in the world. Children are the most involved in those diseases. Extremely poor regions, as sub-Saharan, Africa, are the most affected. In the worst case scenario, one of the parasites that causes malaria, Plasmodium falciparum, become resistant to chloroquine and the current therapy. Besides, some drugs used in the mataria chemotherapy are very toxic, showing many side effects, and compromising the treatment. This is a big challenge and facing it requires new strategies.. The development of chemotherapeutic has been inspired in biochemical differences between the parasite and the host. Plasmodíum falciparum needs to biosynthesize phospholipids for their membrane. These phospholipids are very important to the maturation and reproduction of the parasite and occur only in it.. This makes the phospholipids biosynthesis a good target for new and specific antimalarial drug design. Infected red blood cell shows modified permeation, allowing the lipids to be freely transported, what is not usual in the non-infected red blood cells. This said, in the present work the design and synthesis of serine metabolic inhibitors, using the bioisosteric strategy, have been proposed. The inhibition of the phosphatidylserine biosynthesis, an important phospholipid, is expected. These inhibitors will be linked to phospholipids, to promote the selective permeation to the infected red-blood cell.. These inhibitors will be linked to phospholipids, to promote the selective permeation to the infected redblood cell. In the present work five heterocyclic serine inhibitors: diidroimidazolic, diidroxazolic, diidroxazinico, diidropyriminic and diidroxatiolic. Also synthesized a phospholipid to be connected to the heterocyclic inhibitors.

Ambient mass spectrometry (MS) is a new and growing sub-field in MS which has opened new research avenues, particularly for applications relating to the analysis of solid samples. Results on the implementation and application of ambient MS techniques including: desorption electrospray ionization (DESI) and direct analysis in real time (DART) indicated that these techniques could serve as complementary tools for the rapid qualitative screening of pharmaceuticals, allowing up to two orders of magnitude improvement in throughput compared to traditional methods such as liquid chromatography MS. The selectivity of DESI could be enhanced by performing the experiment in the reactive mode. In this mode, complexation reactions between reagents added to the spray solvent and analytes on the sample surface resulted in analyte stabilization, inhibiting fragmentation. They also resulted in a concomitant enhancement in the analyte surface activity, facilitating their evaporation from secondary droplets culminating in an improvement in sensitivity. Also for drug tablets analysis, the analyte signal dependency on DESI geometrical set-up variables could be mitigated following the careful and controlled addition of an isotopically labeled internal standard (IS) to the sample or by spraying samples with a pair of reagents with different affinities for the analyte. Either of these approaches resulted in an analyte-to-IS signal ratio (in the former) or an analyte complex ratio (in the later), which was largely independent of DESI experimental variables allowing quantitative analysis using this technique. DESI MS was also observed to be a very powerful tool for determining the 2-D distribution of various pharmaceutically important compounds on tablet and tissue surfaces. The ability to map the distribution of molecules of interest by DESI MS has very great implications in drug tablet quality control and in determining the role of chemical signals presented on tissue surfaces. DESI was observed to be limited to ionizing molecules of medium to high polarities without much limitation in terms of mass range, whereas DART was better suited for the analysis of molecules within a broader range of polarities, but within a more limited mass range (up to 800 Da approximately). These limitations were circumvented by implementing a novel multimode ambient ion source, desorption electrospray/metastable-induced ionization (DEMI), which combines various aspects of DESI and DART. Initial experiments with the DEMI ion source demonstrated its ability to enable the simultaneous analysis of molecules within a broader range of polarities and masses than DESI and DART alone.

The disease malaria afflicts more than a billion people and kills almost one to three million of them every year. Of the four species of Plasmodium affecting man viz., P. falciparum, P. vivax, P. ovale and P. malariae, Plasmodium falciparum is the deadliest as it causes cerebral malaria. The situation has become worse with the continuous emergence of drug resistance in the parasite. Therefore, improving existing drugs and deciphering new pathways for drug development are the need of the hour. The discovery of the type II fatty acid biosynthesis pathway in Plasmodium falciparum (Surolia and Surolia, 2001) has opened up new avenues for the development of new antimalarials as this pathway is entirely different from the human host in which type I pathway exists. Although many biochemical pathways such as the purine, pyrimidine and carbohydrate metabolic pathways, and the phospholipid, folate and heme biosynthetic pathways operate in the malaria parasite and are being investigated for their amenability as antimalarial therapeutic targets, no antimalarial of commercial use based on the direct use of these biochemical pathways as targets has emerged so far. This is due to the fact that the structure and function of the targets of these drugs overlaps with that of the human host. A description of such pathways forms the Chapter 1 of the thesis. This is followed by a description of the discovery and the importance of fatty acid biosynthesis pathway and the available literature on the various enzymes that are targets of potential antimalarials. Three isoforms are known for condensing enzymes - FabH which functions in initiation, and FabB and FabF which function in elongation. These isoforms differ in their biochemical properties and have unique roles to play in deciding the membrane composition of any organism. This aspect is also discussed in this chapter. Cloning and expression of -ketoacyl-ACP synthase, FabB/F from Plasmodium falciparum is described in Chapter 2. PfFabB/F is coded by the nuclear genome and is targeted to the apicoplast. The gene is coded by the locus MAL6P1.165 and the putative amino acid sequence of the protein exists in PlasmoDB. All apicoplast targeted proteins have a characteristic bipartite leader sequence consisting of a signal and a transit peptide sequence (Waller et al., 1998). Since the mature protein start site was not known and none of the software packages could predict the site, I aligned the PfFabB/F sequence with the sequences of other -ketoacyl-ACP synthases. On the basis of similarity with E. coli synthases and the mature protein start site of plant synthases, I cloned the first construct of PfFabB/F. The sequence was amplified by PCR and ligated in pET as well as pGEX vector. Expression in various hosts under different temperature and induction conditions could not solubilize the protein in significant quantities and most of the protein was found in inclusion bodies. Next I expressed the sequence with five more amino acids towards the N-terminal and expressed it as an N- terminal NusA fusion. The protein was purified by single step Ni-NTA affinity chromatography. Along with the full length protein (108 kDa), a truncated version of the protein was also obtained. The identity of the protein was confirmed by western blotting using anti-His antibody and anti-FabB/F antibody. In Chapter 3, the substrate specificity of PfFabB/F has been elucidated. PfFabB/F condenses malonyl-ACP with a range of acyl-ACPs. In vivo, acyl carrier protein (ACP) shuttles the acyl substrates between various enzymes of the fatty acid biosynthesis pathway. Enzymes of the pathway other than synthases can accept substrate analogs like acyl-CoA and acyl-NAC’s also in vitro. Acyl-ACPs are not very stable species and thus are not commercially available. Therefore, they have to be synthesized. Since malonyl-ACP could not be synthesized by chemical means, enzymatic synthesis of acyl-ACPs was done. Acyl-ACP synthetase (Aas) or holo-ACP synthase (ACPS) can be used for enzymatic synthesis. Aas is specific only for longer chain substrates; therefore, I decided to use holo-ACP synthase, an enzyme responsible for converting apo-ACP to holo-ACP in the presence of CoA in vivo (Lambalot and Walsch, 1995). When acyl-CoAs are supplied in place of CoA, acyl-ACP is produced. Malonyl-ACP and acyl-ACPs (C4-C16:1) were thus synthesized using holo-ACP synthase from E. coli. The reaction went to almost 95% completion, indicating broad substrate specificity of this enzyme. Bacterial or plant acyl-ACPs of different chain lengths can be resolved by Conformation Sensitive PAGE (Heath and Rock, 1995, Post- Beittenmiller et al., 1991). However, Pfacyl-ACPs synthesized using ACPS did not show any significant shift on CS-PAGE. Therefore I resorted to MALDI-TOF (Matrix Assisted Laser Desorption and Ionization- Time Of Flight) for monitoring the PfFabB/F condensation reactions. PfFabB/F condensed C4-C12-ACPs with malonyl-ACP to their corresponding -ketoacyl-ACP products, with C6, C8 and C10-ACPs being most readily elongated. C14-ACP was very sluggishly elongated, and C16 and C16:1-ACPs were not elongated at all. The condensation reaction was also followed by autoradiography using14C labeled malonyl-ACP, exploiting the clear mobility shift between malonyl-ACP and the other acyl-ACPs. The inhibitory effect of cerulenin, a known inhibitor of condensing enzymes was also checked. PfFabB/F also exhibited malonyl decarboxylase activity resulting in the production of acetyl-ACP in the absence of any significant condensation activity. All the enzymes of fatty acid synthesis pathway required to complete a cycle were assembled together for the in vitro reconstitution of Plasmodium fatty acid synthesis cycle which is described in Chapter 4. Earlier studies of Surolia & Surolia have shown that C12 and C14 fatty acids are the major constituents of Plasmodium lipids. One of my objectives was to determine the maximum length of the acyl ACP product that is synthesized when all the functionally active enzymes of fatty acid synthesis are put together (Kapoor et. al, 2001, Sharma et al., 2003, Karmodiya and Surolia, 2006). Condensing enzymes have a deterministic role in the fatty acid composition as they catalyze the only irreversible step in fatty acid biosynthesis. By analyzing products of the elongation cycle by mass spectrometry it was apparent that C14-ACP is the longest species formed. As already mentioned, PfFabB/F readily elongates C12-ACP but C14-ACP is weakly elongated. Thus the end product of the Plasmodium FAB pathway is influenced by the substrate specificity of PfFabB/F. This confirms the role of PfFabB/F as a decisive enzyme in determining the length of fatty acids synthesized. The inhibition of the cycle by cerulenin and triclosan is also described in this chapter. Chapter 5 describes the ability of the PffabB/F gene to complement for the mutation of condensing enzymes in CY244 cells (fabBtsfabF-, Yasuno et al., 2004). CY244 cells were transformed with pBAD alone or PfFabB/F cloned in pBAD vector (pBADPffabB/F) and the growth was monitored at non-permissive temperature. The product of PfFabB/F could rescue the growth of mutant cells at high temperature but only in the presence of oleic acid. FabB and FabF are the isoforms of condensing enzymes involved in elongation of the fatty acid synthesis cycle but they have a unique role to play (Garwin et al., 1980). FabB is responsible for unsaturated fatty acid synthesis, and fabB-mutants require oleic acid supplementation for growth. FabF is utilized in temperature regulation of membrane fluidity and E. coli FabF elevates the level of C18:1 or cis-vaccenic acid at lower growth temperature but FabF-mutants have no growth phenotype (Ulrich et al., 1983). Rescue of CY244 cells in the presence of oleic acid supplementation indicated that the PffabB/F gene behaves like FabF and not FabB. Analysis of the fatty acid composition of membrane lipids of CY244 cells transformed with pBAD vector or pBADPffabB/F by GC-MS demonstrated no elevated levels of cis-vaccenic acid in transformed cells. This observation is in agreement with the in vitro determined substrate specificity data which shows that PfFabB/F does not elongate C16:1ACP. The thesis ends with a summary of the findings in Chapter 6 in the context of FabB and FabF enzymes known from other sources. 2, 4, 4’-Trichloro-2’hydroxydiphenylether, commonly known as triclosan, has been used as a topical antibacterial agent for decades. I determined its efficacy in treating acute systemic bacterial infection in mouse model. Triclosan, as compared to other well known antibiotics, could extend the survival time of mice by 48 hours. This work is described in Appendix I. (Sharma et al., 2003)

Petroleum ether, dichloromethane, and 80% ethanol extracts of 15 plant species collected in Nigeria were screened for in vitro antibacterial, anti-inflammatory and antimalarial activities. Antibacterial activity was tested using the agar diffusion method, while the minimum inhibitory concentrations (MIC) of the active extracts were determined using the microtitre serial dilution method. Most antibacterial activity detected was against Gram-positive bacteria with Staphylococcus aureus being the most susceptible. The highest activity was found in petroleum ether and dichloromethane leaf extracts of Mallotus oppositifolius; petroleum ether, dichloromethane and ethanolic root extracts of Newbouldia laevis; and ethanolic root extracts of Morinda lucida and Canthium subcordatum. Against the Gram-negative bacterium Escherichia coli, the highest activity was found in dichloromethane leaf extracts of Newbouldia laevis, ethanolic root extracts of Phyllanthus amarus, Mallotus oppositifolius, and Canthium subcordatum. A total of 60 plant extracts were screened for antiplasmodial activity. A chloroquine sensitive strain of Plasmodium falciparum (D10) was used. In the assay, the parasite lactate dehydrogenase (pLDH) activity was used to measure parasite viability. About 11 extracts showed promising activity with an IC₅₀ ranging from 2.5 to 13.4 µg/ml. The petroleum ether leaf extract of Hyptis suaveolens had the highest activity (IC₅₀ = 2.5 µg/ml). The cyclooxygenase (COX-1 and COX-2) assays were used to test for anti-inflammatory activity. All the plant species, with the exception of Hedranthera barteri and Picralima nitida showed anti-inflammatory activity. Apart for a few ethanolic extracts, all the activities were recorded with petroleum ether and dichloromethane extracts. Employing bioassay-guided activity fractionation, an antibacterial anthraquinone identified as emodin was isolated from ethanolic root extract of Senna occidentalis. Although this compound had been isolated from other sources, this was the first report of isolation from Senna occidentalis. Using a similar approach a novel antimalarial diterpenoid was isolated from the petroleum ether leaves extract of Hyptis suaveolens. It had IC₅₀ of 0.1 µg/ml. This new compound is worthy of further investigation and may act as an important lead compound for future antimalarial drugs. / Thesis (Ph.D.)-University of KwaZulu-Natal, 2005.

Early diagnosis and prompt treatment is one of the malaria control strategies used to minimize malaria morbidity and mortality. One of the mechanisms to implement early diagnosis and prompt treatment is community access to diagnostic services and effective antimalarial drugs. However, in Ethiopia the health system is underdeveloped and much of the rural population has limited access to modern health services. Therefore, the Ethiopian government introduced the Health Extension Programme(HEP) which is a community-based health care delivery system aimed at accessing essential health services through its health extension workers (HEWs). Involvement of the HEWs in prescribing and dispensing antimalarial drugs is shown to have improved community access to antimalarial drugs. However, there is insufficient knowledge of HEWs compliance to malaria treatment guidelines and patient adherence of patients treated by HEWs. The objectives of this study has been to describe the HEWs practice in malaria treatment, to evaluate adherence of patients to antimalarial drugs, to explore the factors influencing the HEWs malaria treatment practice and patient adherence, and to develop the guidelines to support the HEWs in malaria treatment practice. A qualitative study design was used to study the HEWs practice in malaria treatment along with patient adherence. Data were collected using in-depth face-to-face interviews, focus group discussion and patient medical record review and were analysed according to Tesch’s steps. The study revealed that the HEWs adequately comply with malaria treatment guidelines during diagnosis of malaria, as well as during the prescribing and dispensing of antimalarial drugs. However, there are some factors influencing the performance of HEWs. These are: shortage of diagnostic kit/RDT, shortage of antimalarial drugs, patient pressure to obtain coartem, work load, and community beliefs with regard to antimalarial drugs effectiveness. This study also revealed that the HEWs follow up after treatment of patients and good community support systems improved patient adherence to antimalarial drug use. Factors negatively influencing patient adherence were identified to include: forgetfulness, fear of shortage of drugs, adverse drug effects, duration of treatment, rapid relief of malaria symptoms and inadequate awareness of the consequence of incomplete dosage. Guidelines were developed to support the HEWs in malaria treatment practice with the aim to improve patient adherence to antimalarial drugs / Health Studies / D. Litt. et Phil. (Health Studies)

Le paludisme reste l'une des plus redoutables maladies infectieuses avec plus de 200 millions d'infections et près de 430 000 décès chaque année, principalement des enfants de moins de 5 ans vivant en Afrique subsaharienne. L'espèce Plasmodium falciparum est responsable de la grande majorité de la mortalité. Le contrôle de l'endémie palustre reste encore aujourd'hui un problème majeur de santé publique, notamment à cause des résistances aux antipaludiques développées par les parasites. L'apparition de ces résistances s'opère par la pression de sélection médicamenteuse, et leur diffusion progressive se fait principalement via le déplacement des hôtes infectés. Cependant, la dynamique d'émergence, de diffusion et de persistance des parasites résistants résulte d'interactions complexes entre les antipaludiques, l'Homme, le parasite et le vecteur. Le travail présenté ici participe à la démarche de lutte contre le paludisme en proposant tout d'abord un état des lieux de la résistance de Plasmodium aux antipaludiques utilisés au Cameroun, avec des outils moléculaires, phénotypiques et cliniques. Une deuxième partie explore, in vitro, les possibles conséquences d'une utilisation prolongée des dérivés d'artémisinine sur le phénotype de P. falciparum, alors que la résistance à cette molécule est déjà installée. Le modèle in vitro utilisé a permis de mettre en évidence un nouveau profil de pluri-résistance suite à des pressions continues à l'artémisinine. Enfin, une dernière partie de ce travail analyse le rôle du moustique dans l'épidémiologie des résistances et montre que la sporogonie favoriserait la diffusion des allèles minoritaires, résistants ou non, présents chez l'Homme. L'ensemble de ces travaux confirme la multiplicité des facteurs agissants sur la dynamique de résistance et la complexité de leurs interactions rendant toute prévision très spéculative. Même si une meilleure connaissance des phénomènes sociétaux, épidémiologiques, biologiques et pharmacologiques impliqués dans les résistances reste une priorité, la surveillance phénotypique et génotypique régulière sur le terrain apparait à ce jour, le meilleur outil pour adapter au mieux les stratégies de contrôle du paludisme. / Malaria remains one of the most terrible infectious diseases with more than 200 million infections and 430,000 deaths each year, mostly children under five years old in sub-Saharan Africa. Plasmodium falciparum is responsible for the vast majority of malaria mortality cases. Control of malaria still remains a major public health problem, in particular because of resistances to antimalarials that parasites developed. The apparition of these resistances is due to the drug pressure, and their progressive diffusion is mainly via the travelling of infected hosts. However, the dynamics of emergence, diffusion and persistence of resistant parasites result from complex interactions between the antimalarials, the Human, the parasite and the vector. The work presented here participates in the malaria control process by first proposing an inventory of Plasmodium resistance to antimalarials used in Cameroon, thanks to molecular, phenotypic and clinical tools. A second part explores the possible consequences of prolonged use of artemisinin derivatives on the P. falciparum phenotype, in areas where resistance to this molecule is already established. The in vitro model used showed that continuous artemisinin pressures induced a new pluri-resistance profile. Finally, a last part analyses the role of the mosquito in the epidemiology of resistances and shows that the sporogony favours the diffusion of minority alleles, resistant or not, presented in humans. All this work confirms the multiplicity of forces acting on the dynamics of resistances and the complexity of their interactions making any prediction very speculative. Even if better knowledge of the societal, epidemiological, biological and pharmacological phenomena involved in resistances is a priority, regular phenotypic and genotypic surveillance in the field remains the best tool for adapting malaria control strategies.