Introduction: Emergence of resistance to artemisinin-based combination therapy (ACT) is a major threat to combat Plasmodium falciparum malaria. Regular therapeutic studies to monitor treatment efficacy is essential, and genotyping of molecular makers is useful for mapping development and spread of resistance. Aims: The study aims are to assess efficacy of artesunate-amodiquine (ASAQ) and prevalence of molecular markers of drug resistance in Zanzibar in 2017. Methods: Treatment efficacy of the clinical trial conducted in 2017 was compared with efficacies in 2002 and 2005. A total of 142 samples were genotyped for single nucleotide polymorphisms (SNPs) in the P. falciparum chloroquine resistance transporter gene (pfcrt) gene, the P. falciparum multi drug resistance 1 (pfmdr1) gene, and in the P. falciparum Kelch 13 (PfK13) propeller region. Prevalence of SNPs were assessed during the period 2002-2017. Results: Cure rate was 100% in 2017, compared to 94% and 96%, in 2002-2003 and 2005, respectively. Day 3 fever clearance rate were also high 93% (2002-3), 99% (2005) and 98% (2017) in all studies. Prevalence of pfcrt 76T, pfmdr1 86Y, 184Y and 1246Y and pfmdr1 (86Y, 184Y and 1246Y) YYY haplotypes were significantly decreased between 2002-3 and 2017 (p &lt; 0.001). No SNP in the PfK13 gene related to artemisinin resistance was identified. Conclusion: Efficacy of ASAQ remains high after fourteen years as first-line treatment, despite the wide-scale use of ASAQ, and there is no evidence of selection of resistance markers in Zanzibar. Continuous monitoring of drug efficacy and resistance markers is recommended. / <p>This master thesis is a collaboration project between Institutionen för kvinnors och barns hälsa, Department of Women's and Children's Health, Uppsala Universtiy and Anders Björkman group, Department of Microbiology, Tumor and Cell Biology (MTC), C1, Karolinska Institutet. Laboratory examinations were mainly conducted at MTC house, Karolinska Institutet.</p>

Significant advancements in genome sequencing techniques and other high-throughput initiatives have resulted in the availability of complete sequences of genomes of a large number of organisms, which provide an opportunity to study detailed biological information encoded therein. Identification of functional roles of proteins can aid in comprehension of various cellular activities in an organism, which is traditionally achieved using techniques pertaining to the field of molecular biology, protein chemistry and macromolecular crystallography. The established experimental methods for protein structure and function determination, although accurate and resourceful, are laborious and time consuming. Computational analyses of sequences of gene products and exploration of evolutionary relationships can give clues on protein structure and/or function with reasonable accuracy which can be used to direct experimental studies on proteins of interest, effectively. Moreover, with growing volumes of data, there has been a growing disparity in the number of well-characterized and uncharacterized proteins, further necessitating the use of computational methods for investigating evolutionary and structure-function relationships. The remarkable progress made in the development of computational techniques (Chapter 1) has immensely contributed to the state-of-the-art biological sequence analysis and recognition of protein structure and function in a reliable manner. These methods have largely influenced the exploration of protein sequence-structure-function space. One of the relevant applications of computational approaches is in the understanding of functional make-up of human pathogens, their complex interplay with the host and implications in pathogenesis. In this thesis, sensitive profile-based search procedures have been utilized to address various aspects in the context of three pathogens- Mycobacterium tuberculosis, Plasmodium falciparum and Trypanosoma brucei, which are causative agents of potentially life- threatening diseases. The existing drugs approved for the diseases, although of immense value in controlling the disease, have several shortcomings, the most important of them being the emergence of drug resistance that render the current treatment regimens futile. Thus, the identification of practicable targets and new drugs or new combination therapies become an important necessity. Analyses on structural and functional repertoire of proteins encoded in the pathogenic genomes can provide means for rational identification of therapeutic intervention strategies. This thesis begins with the computational analyses of proteins encoded in M. tuberculosis genome. M. tuberculosis is a primary aetiological agent of tuberculosis in humans, and is o responsible for an estimated 1.5 million deaths every year. The complete genome of the pathogen was sequenced and made available more than a decade ago, which has been valuable in determination of functional roles of its gene products. Yet, functions of many M. tuberculosis proteins remain unknown. Computational prediction of protein function is an on- going process based on ever growing information made available in public databases as well as the introduction of powerful homology recognition techniques. Hence, a continuous refinement is essential to make the most of the sequence data, ensuring its accuracy and relevance. With the use of multiple sequence and structural profile-based search procedures, an enhanced structural and functional characterization of M. tuberculosis proteins, totalling to 95% of the genome was achieved (Chapter 2). Following are the key findings. o Domain definitions were obtained for a total of 3566 of 4018 proteins. Amino acid residue coverage of >70% was achieved for 2295 proteins which constitute more than half of the proteome. o Domain assignments were newly identified for 244 proteins with domain-unassigned regions. Structure prediction for these proteins corroborated all the remote homologyrelationships recognized using profile-based methods, enhancing the reliability of the predictions. o Comparison on domain compositions of proteins between M. tuberculosis and human host, revealed presence of pathogen-specific domains that are not homologous to proteins in human. Such proteins in M. tuberculosis are mainly virulence factors involved in host-pathogen interactions such as immune-dominance and aiding entry and survival in human host macrophages, hence forming attractive targets for drug discovery. o Putative structural and functional information for proteins with no recognizable domains were inferred by means of fold recognition and an iterative profile-based search against sequence database. o Attributing putative structures and functions to 955 conserved hypothetical proteins in M. tuberculosis, 137 of which are reportedly essential to the pathogen, provide a basis to re-investigate their involvement in pathogenesis and survival in the host. Proteins with no detectable homologues were recognized as M. tuberculosis H37Rv-specific, which can serve as promising drug targets. An attempt was made to identify porin-like proteins in M. tuberculosis, considering MspA porin from M. smegmatis as a template. The difficulty in recognition of putative porins in M. tuberculosis is indicative of novel outer membrane channel proteins, not characterized yet, or high representation of ion-channels, symporters and transporters to compensate for the functional role of porins. In addition, MspA-like proteins were not readily recognized in other slow-growing mycobacterial pathogens that are known to infect human host, apart from M. tuberculosis. This indicates probable acquisition of physiological adaptations, i.e. absence of porins, to confer drug-resistance, in the course of their co-evolution with human hosts. Evolutionary relationships recognized between sequence (Pfam) and structural (SCOP) families aided in association of potential structures and/or functions for 55 uncharacterized Pfam domains recognized in M. tuberculosis. Such associations deliver useful insights into the structure and function of a protein housing the uncharacterized domain. The functional inferences drawn for M. tuberculosis proteins based on the predictions can provide valuable basis for experimental endeavours in understanding mechanisms of pathogenesis and can significantly impact anti-tubercular drug discovery programmes. An interesting outcome benefitted from the exercise of exploring relationships between Pfam and SCOP families, was the identification of evolutionary relationship between a Pfam domain of unknown function DUF2652 and class III nucleotidyl cyclases. A detailed investigation was undertaken to assess this relationship (Chapter 3). Nucleotidyl cyclases synthesize cyclic nucleotides which are critical second messengers in signalling pathways. The DUF2652 family predominantly comprises of bacterial proteins belonging to three lineages- Actinobacteria, Bacteroidetes and Proteobacteria. Thus, recognition of evolutionary relationship between these bacterial proteins and nucleotide cyclases is of particular interest due to the indispensability of cyclic nucleotides in regulation of varied biological activities in bacteria. Use of fold recognition program suggested presence of nucleotide cyclase-characteristic topological motif (βααββαβ) in all the members of the DUF2652 family. Detailed analyses on structural and functional features of the uncharacterized set of bacterial proteins corresponding to 50 bacterial genomes, using profile- based alignments, revealed presence of key features typical of nucleotidyl cyclases, including metal-binding aspartates, substrate-specifying residues and transition-state stabilizing residues. Depending on the features, 20 proteins of Actinobacteria lineage, predominantly mycobacteria, of unknown structure and function were identified as putative nucleotide cyclases, 23 proteins of Bacteroidetes lineage were associated with guanylyl cyclases, while 8 uncharacterized proteins of Proteobacteria were recognized as nucleotide cyclase-like proteins (7 adenylyl and one guanylyl cyclase). Sequence similarity-based clustering of the predicted nucleotide cyclase-like proteins with established nucleotide cyclases indicated the apparent evolutionarily distinctness of the subfamily of class III nucleotidyl cyclases predicted. Furthermore, analysis of evolutionarily conserved gene clusters of the predicted nucleotide cyclase-like proteins indicated functional associations that support the predictions on their participation in cellular signalling events. The inferences made can be experimentally investigated further to ascertain the involvement of the uncharacterized bacterial proteins in signalling pathways, which can help in understanding the pathobiology of pathogenic species of interest. The next objective was the recognition of biologically relevant protein-protein interactions across M. tuberculosis and human host (Chapter 4). M. tuberculosis is well known for its ability to successfully co-evolve with human host in terms of establishing infection, survival and persistence. The current knowledge on the mechanisms of host invasion, immune evasion and persistence in the host environment can be attributed, and is limited, to the experimental studies pursued by numerous groups. Chapter 4 presents an approach for computational identification of biologically feasible protein-protein interactions across M. tuberculosis and human host. The approach utilizes crystal structures of intra-organism protein-protein complexes which are transient in nature. Identification of homologues of host and pathogen proteins in the database of known protein-protein interactions, formed the initial step, followed by identification of conserved interfacial patch and integration of information on tissue-specific expression of human proteins and subcellular localization of human and M. tuberculosis proteins. In addition, appropriate filters were used to extract biologically feasible host-pathogen protein-protein interactions. This resulted in recognition of 386 interactions potentially mediated by 59 M. tuberculosis proteins and 90 human proteins. A predominance of host-pathogen interactions (193 protein-protein interactions) brought about by M. tuberculosis proteins participating in cell wall processes, was observed, which is in concurrence with the experimental studies on immuno-modulatory activities brought about by such proteins. These set of mycobacterial proteins were predicted to interact with diverse set of host proteins such as those involved in ubiquitin conjugation pathways, metabolic pathways, signalling pathways, regulation of cell proliferation, transport, apoptosis and autophagy. The predictions have the potential to complement experimental observations at the molecular level. Details on couple of interesting cases are presented in the chapter, one of which is the probable mechanism of immune evasion adopted by M. tuberculosis to inhibit lysozyme activity in macrophages, and second is the mechanism of nutrient uptake from host. The set of M. tuberculosis proteins predicted to mediate interactions with host proteins have the potential to warrant an experimental follow-up on probable mechanisms of pathogenesis and also serve as attractive targets for chemotherapeutic interventions. proteins known to participate in P. falciparum metabolism. Pathway holes, where evidence on metabolic step exists but the catalysing enzyme is not known, have also been addressed in the study, several of which have been suggested to play an important role in growth and development of the parasite during its intra-erythrocytic stages in human host. A subsequent objective was the recognition P. falciparum proteins potentially capable of remodelling erythrocytes to suit their niche (Chapter 7). Exploitative mechanisms are brought about by the parasite to remodel erythrocytes for growth and survival during intra-erythrocytic stages of its life-cycle, the understanding of which is limited to experimental studies. To achieve physicochemically viable protein-protein interactions potentially mediated by proteins of human erythrocytes and P. falciparum proteins, a structure-influenced protocol, similar to the one demonstrated in Chapter 4, was employed. Information on subcellular localization and protein expression is crucial especially for parasites like P. falciparum, which reside in One of the major shortcomings with current treatment regimen for tuberculosis is the emergence of multidrug (MDR) and extensively drug-resistant (XDR) strains that render first-line and second-line drug treatments futile. This entails a need to explore target space in M. tuberculosis as well as explore the potential of existing drugs for repurposing against tuberculosis. A drug repurposing strategy i.e. exploring within-target-family selectivity of small molecules, has been implemented (Chapter 5) to contribute towards time and cost-saving anti-tubercular drug development efforts. With the use of profile-based search procedures, evolutionary relationships between targets (other than proteins of M. tuberculosis) of FDA-approved drugs and M. tuberculosis proteins were investigated. A key filter to exclude drugs capable of acting on human proteins substantially reduced the chances of obtaining anti-targets. Thus, total of 130 FDA-approved drugs were recognized that can be repurposed against 78 M. tuberculosis proteins, belonging to the functional categories- intermediary metabolism and respiration, information pathways, cell wall and cell processes and lipid metabolism. The catalogue of structure and function of M. tuberculosis proteins and their involvement in host-pathogen protein-protein interactions compiled from chapters 2 and 4 served as a guiding tool to explore the functional importance of targets identified. Many of the potential targets identified have been experimentally shown to be essential for growth and survival of the pathogen earlier, thus gaining importance in terms of pharmaceutical relevance. Polypharmacological drugs or drugs capable of acting of multiple targets were also identified (92 drugs) in the study. These drugs have the potential to stand tolerance against development of drug resistance in the pathogen. Comparative sequence and structure-based analysis of M. tuberculosis proteins homologous to known targets yielded credible inferences on putative binding sites of FDA-approved drugs in potential targets. Instances where information on binding sites could not be readily inferred from known targets, potentially druggable sites have been predicted. Comparison with earlier experimental studies that report anti-tubercular potential of several approved drugs enhanced the credibility of 74 of 130 FDA-approved drugs that can be readily prioritized for clinical studies. An additional exercise was pursued to identify prospective anti-tubercular agents by means of structural comparison between ChEMBL compounds and 130 FDA-approved drugs. Only those compounds were retained that showed considerably high structural similarity with approved drugs. Such compounds with minor changes in terms of physicochemical properties provide a basis for exploration of compounds that may exhibit higher affinities to bind to M. tuberculosis targets. The set of approved drugs recognized as repurpose-able candidates against tuberculosis, in concert with the structurally similar compounds, can significantly impact anti-tubercular drug development and drug discovery. The next part of the thesis focuses on Plasmodium falciparum, an obligate intracellular protozoan parasite responsible for malaria. The parasite genome features unusual characteristics including abundance of low complexity regions and pronounced sequence divergence that render protein structure and function recognition difficult. The parasite also manifests remarkable plasticity in its metabolic organization throughout its developmental stages in two hosts-human and mosquito; thus obtaining an exhaustive list of metabolic proteins in the parasite gains importance. Considering the utility of multiple sensitive profile-based search approaches in enhanced annotation of M. tuberculosis genome, a similar exercise was employed to recognize potential metabolic proteins in P. falciparum (Chapter 6). A total of 172 metabolic proteins were identified as participants of 78 metabolic pathways, over and above 609heterogeneous environmental conditions at different stages in their lifecycle. Inclusion of such data aided in extraction of 208 biologically relevant protein-protein interactions potentially mediated by 59 P. falciparum proteins and 30 erythrocyte proteins. Host-parasite protein-protein interactions were predicted pertaining to several major strategies spanning intra-erythrocytic stages in P. falciparum pathogenesis including- gaining entry into the host erythrocytes (category: RBC invasion, protease), redirecting parasitic proteins to erythrocyte membrane (category: protein traffic), modulating erythrocyte machinery (category: rosette formation, putative adhesin, chaperone, kinase), evading immunity (category: immune evasion) and eventually egress (category: merozoite egress) to infect other uninfected erythrocytes. Elaborate means to analyse and evaluate the functional viability of a predicted interaction in terms of geometrical packing at the interfacial region, electrostatic complementarity of the interacting surfaces and interaction energies is also demonstrated. The protein-protein interactions, thus predicted between human erythrocytes and P. falciparum, have the potential to provide a useful basis in understanding probable mechanisms of pathogenesis, and indeed in pinning down attractive targets for antimalarial drug discovery. The emergence of drug resistance against all known antimalarial agents, currently in use, necessitates discovery and development of either new antimalarial agents or unexplored combination of drugs that may not only reduce mortality and morbidity of malaria, but also reduce the risk of resistance to antimalarial drugs. In an attempt to contribute towards the same, Chapter 8 explores the established concept of within-target-family selectivity of small molecules to recognize antimalarial potential of the approved drugs. Eighty six FDA-approved drugs, predominantly constituted by antibacterial agents, were identified as feasible candidates for repurposing against 90 P. falciparum proteins. Most of the potential parasite targets identified are known to participate in housekeeping machinery, protein biosynthesis, metabolic pathways and cell growth and differentiation, and thus are pharmaceutically relevant. During intra-erythrocytic growth of P. falciparum, the parasite resides within the erythrocyte, within a protective encasing, known as parasitophorous vacuole. Hence a drug, intended to target a parasite protein residing in an organelle, must be sufficiently hydrophilic or hydrophobic to be able to permeate cell membranes and reach its site of activity. On the basis of lipophilicity of the drugs, a physical property determined experimentally, 57 of 86 FDA-approved drugs were recognized as feasible candidates for use against P. falciparum during the course of blood-stages of infection, which can be prioritized for antimalarial drug development programmes. The final section of the thesis focuses on the protozoan parasite Trypanosoma brucei, a causative agent of African sleeping sickness (Chapter 9). This disease is endemic to sub-Saharan regions of Africa. Despite the availability of completely sequenced genome of T. brucei, structure and function for about 50% of the proteins encoded in the genome remain unknown. Absence of prophylactic chemotherapy and vaccine, compounded with emergence of drug-resistance renders anti-trypanosomal drug discovery challenging. Thus, considering the utility of frameworks established in earlier chapters for recognition of protein structure, function and drug-targets, similar steps were undertaken to understand functional repertoire of the parasite and use drug repurposing methods to accelerate anti-trypanosomal drug discovery efforts. Structures and functions were reliably recognized for 70% of the gene products (5894) encoded in T. brucei genome, with the use of multiple profile-based search procedures, coupled with information on presence of transmembrane domains and signal peptide cleavage sites. Consequently, a total of 282 uncharacterized T. brucei proteins could be newly coined as potential metabolic proteins. Integration of information on stage-specific expression profiles with Trypanosoma-specific and T-.brucei-specific proteins identified in the study, aided in pinning down potential attractive targets. Additionally, exploration of evolutionary relationships between targets of FDA-approved drugs and T. brucei proteins, 68 FDA-approved drugs were predicted as repurpose-able candidates against 42 potential T. brucei targets which primarily include proteins involved in regulatory processes and metabolism. Several targets predicted are reportedly essential in assisting the parasite to switch between differentiation forms (bloodstream and procyclic) in the course of its lifecycle. These targets are of high therapeutic relevance, hence the corresponding drug-target associations provide a useful resource for experimental endeavours. In summary, this thesis presents computational analyses on three pathogenic genomes in terms of enhancing the understanding of functional repertoire of the pathogens, addressing metabolic pathway holes, exploring probable mechanisms of pathogenesis brought about by potential host-pathogen protein-protein interactions, and identifying feasible FDA-approved drug candidates to repurpose against the pathogens. The studies are pursued primarily by taking advantage of powerful homology-detection techniques and the ever-growing biological information made available in public databases. Indeed, the inferences drawn for the three pathogenic genomes serve an excellent resource for an experimental follow-up. The set of protocols presented in the thesis are highly generic in nature, as demonstrated for three pathogens, and can be utilized for genome-wide analyses on many other pathogens of interest. The supplemental data associated with the chapters is provided in a compact disc attached with this thesis.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-15T12:09:54Z No. of bitstreams: 1 nicollibellottidesouza.pdf: 1508500 bytes, checksum: 7150363e1a046cd4741555cb142f6f21 (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-09-26T20:18:43Z (GMT) No. of bitstreams: 1 nicollibellottidesouza.pdf: 1508500 bytes, checksum: 7150363e1a046cd4741555cb142f6f21 (MD5) / Made available in DSpace on 2016-09-26T20:18:43Z (GMT). No. of bitstreams: 1 nicollibellottidesouza.pdf: 1508500 bytes, checksum: 7150363e1a046cd4741555cb142f6f21 (MD5) Previous issue date: 2011-02-22 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A malária é causada por protozoários do gênero Plasmodium e é responsável por 250 milhões de casos e 1 milhão de mortes anualmente. Um dos principais empecilhos para o controle da doença é o desenvolvimento de resistência do parasito aos fármacos comumente usados, o que torna urgente a pesquisa por novos antimaláricos. Nesse contexto, análogos de cloroquina acoplados a 6-mercaptopurina e a alquil aminas e complexos de platina foram avaliados quanto a atividade antimalárica utilizando o teste supressivo descrito por Peters em modelo murino de infecção por Plasmodium berghei NK65. Tais análogos exibiram altos valores de supressão da parasitemia, entre 60% e 94% em comparação com o controle não tradado. Considerando o papel imunossupressor de derivados de purina, o análogo de cloroquina acoplado a 6-mercaptopurina MPQUI foi avaliado quanto a aspectos imunológicos (contagem de leucócitos específicos, dosagem de TNF-α e IL-10), não interferindo na resposta imune tendo como base os parâmetros analisados. Portanto, esses análogos devem ser objetos de futuras pesquisas, podendo fornecer novos antimaláricos, já que apresentaram-se promissores e não influenciaram a resposta imune nos parâmetros analisados. / Malaria is caused by protozoan parasites of the genus Plasmodium and is responsible for 250 million cases and 1 million deaths annually. One of the main obstacles for the disease control is the development of resistance by the parasite to the commonly used antimalarials, what makes the research for new ones urgent. In this context, chloroquine analogs attached to 6-mercaptopurine and to alkyl-amines and platinum complexes were evaluated for their antimalarial activity using the 4-day suppressive test described by Peters which was carried out in mice infected with Plasmodium berghei NK65. These analogs exhibited high values of parasitemia suppression, which ranged from 60% to 94% in comparison to untreated control. Considering the immune suppressor role of purine derivates, the chloroquine analog attached to 6-mercaptopurine MPQUI was evaluated for immunological aspects (specific leukocytes count, TNF-α and IL-10 measurements in sera of mice), revealing no interference in the immune response considering these parameters. Therefore, these analogs may be objects of further research, aiming at new antimalarials, since they were shown to be promising and did not influence the immune response in the parameters analyzed.

Malaria accounts for 198 million cases worldwide; with a high mortality rate. 584000 deaths were reported in 2013. Malaria is a re-emerging disease globally due to drug resistance, parasite recrudescence and non-availability of a vaccine. Chloroquine, quinine and antifolates served as frontline antimalarial drugs for decades. Development of resistance to chloroquine and antifolates, and the decreased efficacy of mefloquine, and even quinine, in malaria-endemic regions, has led to artemisinin derivatives evolving as frontline drugs. Artemisinin is a potent antimalarial compound and clears around 104 parasites per cycle. Despite being a potent antimalarial, artemisinin derivatives suffer from poor pharmacokinetic properties and short half lives. This has led to the development of artemisinin-based combination therapies (ACTs) using a partner drug with a longer half-life. However, resistance to ACTs has been reported in the last few years, perhaps due to lack of adherence to prescribed regimens or suboptimal treatment and the use of counterfeit drugs. Therefore there is an urgent need to develop an alternative ACT which overcomes these limitations. This thesis entitled “Adjunct therapy with curcumin for the treatment of malaria: studies in a murine model” describes the antimalarial activity of curcumin and artemisinin and the adjunct role of curcumin in the prevention of parasite recrudescence and cerebral malaria. The thesis is divided into three chapters: The first chapter entitled “Introduction: Malaria and anti-malarial drugs” consists of a brief introduction of malaria, the parasite life cycle and currently known antimalarial drugs. During the course of infection, the Plasmodium undergoes sporogony in the mosquito, and merogony and schizogony in the human host. All these life cycle stages are briefly described with depictions. A major part of this chapter is dedicated to describe antimalarial compounds under the following headings 1. Quinoline derivatives 2. 4-aminoquinolines 3. Antifolates 4. Artemisinin derivatives 5. Antibiotics and 6. Curcumin. The second chapter is aimed at examining the ability of curcumin-arteether (a synthetic derivative of artemisinin) combination therapy in preventing parasite recrudescence in a murine model through immunomodulation employing various immunological, molecular biological, and biochemical techniques. The use of suboptimal doses of antimalarial drugs leads to recrudescence or relapse of malaria (reappearance of the parasite in blood after antimalarial regimen). In the present study we have addressed this issue by the use of curcumin as an adjunct molecule with α,β arteether (a synthetic derivative of artemisinin). We have studied recrudescence in a Swiss mice model. A suboptimal dose was standardized by the use of different doses of α,β arteether (AE) ranging from 250µg to 1500 µg. We found 750 µg to be a suboptimal dose and studied the adjunct nature of curcumin when animals were treated with AE suboptimal dose or AE+curcumin (AC) combination treatment and monitored the survival of animals. Our results clearly demonstrate that ~95% of animals treated with the suboptimal AE dose died of recrudescent malaria but there was almost 100% survival of AC-treated animals; these animals were under observation for at least 3 months. We have studied the effect of curcumin in a recrudescence model at the molecular level. Curcumin by itself has antimalarial activity, but only in combination with α,β arteether prevented recrudescence. Our results indicate that curcumin has immunomodulatory activity. Serum cytokine analysis and spleen mRNA analysis for proinflammatory and anti-inflammatory mediators indicate that AC treatment effectively reduced both mRNA and serum cytokine levels of IFNγ, TNFα, IL-12 and effectively increased both mRNA and serum levels IL-10 and antibodies of the IgG subclass. Using TLR2 and IL-10 knockout animals, we have conclusively demonstrated that TLR2 is involved in the production of IL-10, and IL-10 is required for the AC-mediated protection of animals during the recrudescence period. We conclude that curcumin is able to prevent parasite recrudescence essentially by switching the Th1 response to a Th2 response. The third chapter deals with the study the effect of areether-curcumin (AC) combination therapy in the prevention of Experimental Cerebral Malaria. Although malaria mortality rates have decreased by an impressive 47% between 2000 and 2013, it is still a major affliction of mankind (WHO 2014). Plasmodium falciparum infection causes human cerebral malaria (HCM). The mortality rate in HCM is unacceptably high (15–20%), despite the availability of artemisinin-based therapy. HCM is characterized by a rapid progression from headache, general malaise, and prostration to hemiparesis, ataxia, unrousable coma, and death. Paediatric HCM deaths are mostly due to respiratory arrest. Alternatively, death may be due to parasite-mediated injury to a sensitive location; a small lesion due to parasite in brain stem can cause sudden respiratory arrest. In HCM, cytoadherence of pRBCs in brain microvasculature has been implicated as a major contributing factor for CM pathology. The failure of a large number of adjunct therapies in HCM demands the development of new intervention strategies. An effective adjunct therapy is urgently needed. Experimental Cerebral Malaria (ECM) in mice manifests many of the neurological features of HCM. In this study, we have demonstrated the efficacy of curcumin and PLGA nanocurcumin in the treatment of Experimental Cerebral Malaria (ECM), using the Plasmodium berghei ANKA-infected mouse model (C57BL/6). Curcumin/PLGA nanocurcumin alone can prevent the onset of ECM. We have shown that curcumin/PLGA nanocurcumin can prevent CD8+ T cell, CXCR3+ CD8 T cell and parasite-infected RBC (pRBC) sequestration in the brain. These are also the essential parameters underlying HCM. We have also demonstrated that curcumin effectively inhibits T cell proliferation in spleen. We have explained the anti-inflammatory effects of curcumin by showing the inhibition of NF-B in both brain and spleen, which is a plausible explanation. But, curcumin/PLGA nanocurcumin treated animals died later due to build up of parasitemia in blood and subsequent anemia. Moreover, a combination therapy with arteether and curcumin given even after the onset of neurological symptoms can completely cure and protect the animals against mortality. We have tested AC-combination after the onset of symptoms to mimic patient conditions in HCM, since the murine regimens reported were not successful in the treatment of HCM. Our results clearly demonstrate that AC treatment even after the onset of symptoms ensures 100% survival. Since the bioavailability of curcumin is reported to be poor, we have also tested the efficacy of PLGA nanocurcumin and find that it is superior to native curcumin in terms of therapeutic effects. It is concluded that curcumin would be an ideal adjunct drug to be used with the artemisinin derivatives to treat malaria, including cerebral malaria.

Ce travail est consacré à la recherche et au développement de nouvelles molécules à viséepharmacologique en série naphtoquinonique en utilisant des réactions par transfert monoélectroniquede type SRN1 et des cyclisations radicalaires oxydatives induites par l'acétate de manganèse(III). Lapremière partie décrit l’étude de la réactivité SRN1 de la 2-(chlorométhyl)-3-méthoxynaphtoquinoneavec divers anions nitronates conduisant à la formation de produits de C-alkylation avec de bonsrendements. Ces derniers ont fait l’objet d’une réaction de réduction-cyclisation permettant la synthèsede nouveaux dérivés benzo[g]indol-5(3H)-ones. Dans la seconde partie, une nouvelle réactiond’oxydation initiée par l’acétate de manganèse(III) a été développée sur la 2-hydroxy-3-méthylnaphtoquinone dans des conditions opératoires douces. En effet, la réactivité originale de la 2-hydroxy-3-méthylnaphtoquinone avec divers alcènes aromatiques en présence de Mn(OAc)3 et dedioxygène, a permis pour la première fois en série naphtoquinonique, l’obtention de nouveaux dérivésoriginaux dihydronaphto[2,3-c][1,2]dioxine-5,10(3H,10aH)-diones sous forme d’un mélange dediastéréoisomères à potentialités antipaludiques. Un mécanisme réactionnel original a été proposé pourla formation de ces produits. / This work is focused on the research and development of new pharmacologicalmolecules in naphthoquinonic series, synthesized by single electron transfer reaction SRN1 ormanganese(III) acetate catalyzed oxidative radical cyclization. The first part describes the SRN1reactivity of 2-(chloromethyl)-3-methoxynaphthoquinone with various nitronate anions leading to theC-alkylation products. The reduction-cyclization reaction of the latter derivatives allowed us to obtainnew benzo[g]indol-5(3H)-one derivatives. In the second part, a new reaction initiated by Mn(OAc)3 on2-hydroxy-3-methylnaphthoquinone was developed under mild conditions. Indeed, the original reaction of2-hydroxy-3-methylnaphthoquinone with various aromatic alkenes in presence of dioxygen led to newdihydronaphtho[2,3-c][1,2]dioxine-5,10(3H,10aH)-dione derivatives as a mixture of diastereoisomerswith antimalarial potential. An original mechanism was proposed in order to explain the formation ofthese products.

Malaria is caused by a protozoan parasite, Plasmodium falciparum, which is responsible for over 500,000 deaths per year worldwide. Although malaria medicines are working well in many parts of the world, antimalarial drug resistance has emerged as one of the greatest challenges facing malaria control today. Since the malaria parasites are once again developing widespread resistance to antimalarial drugs, this can cause the spread of malaria to new areas and the re-emergence of malaria in areas where it had already been eradicated. Therefore, the discovery and characterization of novel antimalarials is extremely urgent. A previous drug screen in Dr. Chakrabarti's lab identified several natural products (NPs) with antiplasmodial activities. The focus of this study is to characterize the hit compounds using Principal Component Analysis (PCA) to determine structural uniqueness compared to known antimalarial drugs. This study will compare multiple libraries of different compounds, such as known drugs, kinase inhibitors, macrocycles, and top antimalarial hits discovered in our lab. Prioritizing the hit compounds by their chemical uniqueness will lessen the probability of future drug resistance. This is an important step in drug discovery as this will allow us to increase the interpretability of the datasets by creating new uncorrelated variables that will successively maximize variance. Characterization of the Natural Product inspired compounds will enable us to discover potent, selective, and novel antiplasmodial scaffolds that are unique in the 3-dimensional chemical space and will provide critical information that will serve as advanced starting points for the antimalarial drug discovery pipeline.

Deuxième pays le plus endémique pour le paludisme au monde, la république démocratique du Congo (RDC) a adopté officiellement l’initiative Faire Reculer le Paludisme en 2001 et depuis, aligne sa politique de lutte contre le paludisme sur les directives de l’OMS dont les orientations les plus récentes consistent en 3 stratégies :[1] assurer aux malades un accès rapide à un traitement efficace et abordable, [2] assurer aux personnes exposées au risque, notamment les jeunes enfants et les femmes enceintes, l’association la plus adaptée de mesures de protection au niveau personnel et communautaire et [3] donner l’accès, aux femmes enceintes exposées au risque, à des traitements préventifs.<p>Afin de contribuer à l’amélioration de la lutte antipaludique en RDC, le travail a été réalisé en 3 parties :une portant sur la pertinence du traitement préventif intermittent à la Sulfadoxine-Pyriméthamine (TPI-SP) chez la femme enceinte en contexte d’augmentation de la résistance du parasite, une 2e sur les facteurs déterminants l’adhésion des prestataires de soins, des vendeurs des médicaments et des patients à association Artesunate-Amodiaquine (AS-AQ) et une 3e décrivant le paludisme sévère chez l’enfant.<p>La 1ère partie du travail portant sur le TPI-SP a été réalisée en 2 temps. La première approche a consisté en une analyse rétrospective des poids de naissance des nouveau-nés en fonction de la prise du TPI-SP par les mères dans 3 maternités en 2007 où les niveaux de résistance à la SP étaient différents. La deuxième approche, comparait l’effet du traitement préventif intermittent à la Sulfadoxine-Pyriméthamine en 1998/1999 et en 2007 à la prophylaxie à base de chloroquine en 1998/1999 à l’Est de la RDC dans un milieu où la résistance à la Sulfadoxine-Pyriméthamine avait accru considérablement.<p>Dans les régions où la résistance du parasite était de faible (Mikalayi: 1,6% d’échec thérapeutique chez l’enfant) à modéré (Kisangani: 21,7% d’échec thérapeutique chez l’enfant), le TPI-SP réduit le risque de faible poids à la naissance, à Kisangani (OR ajusté :0,15; IC95%, 0,05-0.46) et à Mikalayi (OR ajusté :0,12; IC95%, 0,01-0,89). Dans ces 2 sites, le poids de naissance moyen des Nouveau-nés était plus élevé pour les mères ayant reçu 2 doses par rapport à celui des Nouveau-nés des mères en ayant reçu une seule ou aucune (P<0.001). A Rutshuru où la résistance était élevée (60,6% d’échec thérapeutique chez l’enfant), l’effet du TPI-SP semble moindre: en 2007 - en comparaison dose-dépendante (2 doses Vs 0-1 dose) du poids à la naissance des nouveau-nés en 2007 pour les mères sous TPI-SP – on observait un effet bénéfique chez les primigestes et non chez les multigestes et une légère régression du gain pondéral [(53,9g ( P=0,027) pour les nouveau-nés des mères sous SP en 2007 Vs 70,2g (P=0,003) pour ceux des mères sous SP en 1998/1999 par rapport à ceux des mères sous chloroquine en 1998/1999. Néanmoins, le TPI-SP reste efficace en 2007 comme en 1998-1999 par rapport à la prophylaxie à base de chloroquine en 1998/1999 (poids moyen supérieur et réduction du risque de PPN pour les 2 groupes sous SP). <p>La 2e partie, traitait des déterminants de l’adhésion des prestataires et des patients au nouveau médicament recommandé par la politique nationale pour la prise en charge des cas de paludisme simple ( AS-AQ ) en 2 enquêtes transversales :une étude préliminaire qui a été réalisée dans les CS fonctionnels de Kisangani en avril 2008 et une étude étendue à 3 des 10 districts sanitaires de la province Orientale de mars à juin 2009. Ces 2 enquêtes ont ensuite servi à une analyse systémique des facteurs d’adhésion des prestataires de soins à l’AS-AQ basée sur le modèle de diagnostic de Green et Kreuter et les étapes de changement de comportement de PROCHASKA pour la planification des interventions de promotion.<p>Le recours à l’AS-AQ pour le traitement du paludisme simple progresse (41% et 69% des prescriptions dans les services sanitaires respectivement 3 et 4 ans après le changement de politique national de lutte antipaludique), mais reste inférieur à la cible d’au moins 80% visée par la politique nationale. Malgré la croyance en l’efficacité du nouveau traitement, son utilisation effective rencontre comme principaux obstacles, selon l’avis des prestataires de soins, des vendeurs de médicaments et des patients, sa faible disponibilité, son coût élevé, la présence sur le marché d’antipaludiques retirés du protocole national de traitement et de l’AS-AQ de mauvaise qualité à un coût moindre et la crainte des effets indésirables. Tandis que les facteurs incitatifs à son utilisation sont, l’efficacité thérapeutique perçue de l’AS-AQ, la présence du médicament dans les formations sanitaires, la recommandation de son utilisation par les directives du Ministère de la Santé (notamment sous forme de guides techniques), la formation et la supervision des prestataires, l’intention de prescrire l’AS-AQ aux patients ou d’en prendre soi-même, une plus longue durée de consultation, le fait de fournir des explications aux patients, de travailler dans le milieu rural.<p>La dernière partie du travail consistait en une étude prospective menée du 1er janvier 2010 au 28 février 2011 décrivant le diagnostic et la prise en charge du paludisme grave chez les enfants admis dans 2 HGR de Kisangani.<p>Le paludisme constitue un des principaux motifs d’hospitalisation des enfants en RDC (37,0% à Kisangani) dont l’évaluation est souvent incomplète (53,6% avec goutte épaisse négative ou sans, insuffisamment explorés et traités comme paludisme grave) et le traitement parfois inadéquat (outre les affections non palustres probables non traitées, il y avait notamment surutilisation des produits sanguins exposant les enfants aux risques infectieux transfusionnels). Ainsi, les limites du plateau technique des HGR et l’organisation du circuit des malades semblent entraîner une sous-estimation, entre autre, des complications métaboliques du paludisme grave et des autres infections graves du jeune enfant et par conséquent des écarts au protocole de prise en charge préjudiciables aux patients.<p>Ces résultats mettent en lumière la nécessité :<p>•d’inscrire parmi les priorités du Programme National de Lutte contre le Paludisme, des recherches pour évaluer une option alternative au traitement préventif intermittent avec 2 doses de SP (traitement préventif intermittent avec d’autres antipaludiques) et à l’est du pays le recours préférentiel à la moustiquaire imprégnée à longue durée, en particulier chez la multigeste. <p>•de retenir, pour la promotion de l’utilisation de l’AS-AQ pour le traitement du paludisme simple, comme priorités :<p> o mettre à profit les opportunités actuelles de financement dans le domaine de la lutte antipaludique pour améliorer la disponibilité de l’AS-AQ à un coût accessible et à améliorer l’approvisionnement aussi bien des formations sanitaires publiques et privées que des officines pharmaceutiques ;<p> o assainir le secteur pharmaceutique de manière à endiguer la circulation d’antipaludique de mauvaise qualité à bas prix qui alimente les habitudes d’automédication courante dans les ménages ;<p> o élaborer des programmes de promotion de l’utilisation de l’AS-AQ, en élargissant la cible des interventions autant aux prestataires de soins du secteur privé qu’aux vendeurs de médicaments ;<p> o Adapter les programmes de promotion de l’AS-AQ au stade de changement de comportement auquel se trouve les acteurs après analyse des déterminants de leurs comportements notamment, les facteurs prédisposant (aussi bien les connaissances que les croyances et les intentions des prescripteurs), les facteurs potentialisant (notamment la disponibilité de l’AS-AQ de bonne qualité, son accessibilité financière aux patients) et les facteurs renforçant (supervision, contrats de performance).<p>•de renforcer le plateau technique des HGR et y améliorer le circuit des patients pour leur permettre de jouer pleinement leur rôle dans la prise en charge des formes graves du paludisme. /<p><p>Second most endemic country for malaria in the world, the Democratic Republic of Congo (DRC) has officially adopted the Roll Back Malaria (RBM) in 2001, since then aligns its malaria control policy on WHO guidelines which last orientations are based on three major strategies [1] prompt access to effective treatment and affordable for the patients [2] association of the most appropriate measures to protect the persons at risk both at individual and community level, including young children and pregnant women, [3] and access to preventive treatments to pregnant women at risk.<p>In order to contribute in improving malaria control in DRC, the study was carried out in three parts: the first one on the relevance of IPT with SP in pregnant women in the context of increased parasite resistance, the second one on the determinants of adherence of healthcare providers, drugs sellers and patients to AS-AQ, the last one describing severe malaria in children.<p>The first part of the study on IPTp-SP was performed in 2 stages. We had firstly carried out a retrospective analysis of birth weight comparing newborns whom mothers had received 2 SP doses to those whom mothers had received one or none, in three maternity hospitals in 2007 where levels of SP resistance were different. Then we evaluated the effect of IPTp-SP in 1998/1999 and 2007 compared to prophylaxis with chloroquine in 1998/1999 in eastern DRC in a region where resistance to SP was significantly increased. <p>In areas where parasite resistance was low (Mikalayi: 1.6% of therapeutic failure in children) our moderate (Kisangani: 21.7% of therapeutic failure in children), the IPTp-SP reduced the risk of LBW in Kisangani (OR adjusted 0.15, 95% CI, 0.04-0.58) and Mikalayi (adjusted OR, 0.12, 95% CI, 0.01-0.89). In both sites, the average birth weight was higher for mothers having received two rather than one or no SP doses (P<0.001). While in Rutshuru, where resistance was high (60.6% treatment failure), the effect of IPT-SP seems lower. In 2007, IPTp-SP had an effect only in primigravidae (dose-response comparison: 2 doses vs. 0-1dose). It was also observed in a slight decrease in body weight gain [(53.9 g (P = 0.027) for mothers having received SP in 2007 vs. 70.2 g (P = 0.003) for those mothers who had received SP in 1998/1999 compared to mothers who had received chloroquine in 1998/1999.<p>In the second part of the thesis, two cross-sectional surveys were carried out to identify determinants of the adherence of healthcare providers, drugs sellers and patients to the new drug recommended by the national policy for the treatment of uncomplicated malaria (AS-AQ). A preliminary survey was conducted in functional health centre in Kisangani in April 2008 and a second survey extended to three out of 10 health districts in the Eastern Province from March to June 2009. <p>The use of AS-AQ for the treatment of uncomplicated malaria progresses (from 41% to 69% prescriptions in health services between 2008 and 2009, 3 and 4 years respectively after the malaria control policy change), but still below the target of at least 80% pursued by national policy. Despite the perceived efficacy of AS-AQ by both healthcare providers and drugs vendors, its use was limited due to its low availability, high cost, mistrust on the quality of the available product, availability of inexpensive antimalarial drugs withdrew from national politicy and the fear for adverse effects. While the incentives for its use were, the therapeutic efficacy, availability, directives of the ministries of health (technical guidelines), training and supervision of healthcare providers, the intention to prescribe AS-AQ to patients or to use oneself, lengthy consultations, providing explanations to patients, working in rural areas.<p>The last part of the thesis was a prospective study conducted from January 2010 to February 2011 that included all children admitted for severe malaria with at least one of the criteria for severe malaria according to WHO.<p>Malaria is one of the main reasons for hospitalization of children in the DRC (37.0% in Kisangani) whose evaluation is often uncompleted (53.6% no or negative blood smear that are insufficiently explored and treated as severe malaria) and sometimes inadequately treated (in addition to non-malarial severe diseases treated as severe malaria, there was overuse of blood products conducting probably to exposition of children to risks of infection through transfusion). Thus, poor technical support and inadequate organization of the patient circuit seem to lead to underestimation, among others, of metabolic complications of severe malaria and in the non-recognition of other serious infections early childhood, problems that are detrimental to the patients, even when effective drugs are available.<p>These results highlight the need: <p>•to include among the priorities of the National Malaria Control Program, research to evaluate an alternative option to 2 doses IPTp-SP (IPT using other antimalarial drugs) and in the east of the country preferential use of MILD, especially in multigravidae. <p>•for the use of AS-AQ for the treatment of uncomplicated malaria primarily promote:<p> o the use of current funding opportunities in the field of malaria control to improve the availability of the AS-AQ at an affordable cost and to improve the supply of both public and private health facilities as well as pharmacies;<p> o to clean up the pharmaceutical sector in order to stem the flow of poor quality cheapest antimalarial drugs that feeds the habits of self-medication common in households;<p> o to develop communication programs, training and supervision of healthcare providers to promote the use of AS-AQ, expanding the target of interventions to both healthcare providers in the private sector and drugs sellers;<p> o To adapt AS-AQ promoting programs to the stage of behavior change after analysis of determinants of actors behavior, in particular, the predisposing factors (knowledge as well as beliefs and intentions of the prescriber), the potentializing factors (including the availability of the good quality AS-AQ, affordability to patients) and reinforcing factors (supervision, performance contracts).<p>•to strengthen the technical support of general hospitals and to improve the organization of the patient circuit in order to enable them to play their full role in the management of severe malaria.<p> / Doctorat en Sciences de la santé publique / info:eu-repo/semantics/nonPublished