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.

Notre étude s’est proposée d’analyser l’incidence du paludisme et son évolution dans le temps et dans l’espace, ainsi que de rechercher les facteurs de risque d’accès palustres chez les enfants d’un village du Moyen-Oyapock (Camopi), peuplé d’Amérindiens wayampi et émerillon, d’une part, et d’un village du Haut-Maroni (Antecume Pata), peuplé d’Amérindiens wayana, d’autre part. L’approche a été multiple avec, pour chacun des deux sites d’étude :- Une analyse de survie (modèle de Cox) à partir des accès palustres confirmés biologiquement dans une cohorte d’enfants de moins de sept ans régulièrement suivis, ainsi qu’un questionnaire de type Connaissances, Attitudes et Pratiques (CAP), puis les caractéristiques des habitats et la description de leur environnement immédiat.- Une analyse spatiale avec une classification de l’occupation du sol à partir d’images satellites SPOT 5, l’extraction de variables environnementales d’intérêt, l’étude de leur effet sur la transmission du paludisme et la mise au point d’une méthode objective de sélection d’un rayon d’observation autour des habitations pour la caractérisation de l’environnement.- Une étude de séries temporelles (ARIMA) afin de déterminer l’effet des évènements climatiques et hydrologiques sur le paludisme, aux niveaux local et plus global (El Niño).Les taux d’incidence d’accès palustres sur la période 2001-2009 se sont révélés particulièrement élevés chez les jeunes enfants, notamment à Camopi avec une moyenne de 773‰ par année. Une diminution brutale de l’incidence a eue lieu en 2007 sur le Haut-Maroni et ce phénomène est observé à Camopi depuis 2010. Une prémunition se développe assez rapidement au cours de la vie (2-3 ans), surtout contre les reviviscences à Plasmodium vivax. Les facteurs environnementaux se sont avérés être les plus nombreux et les plus fortement liés à l’incidence palustre. En effet, le dégagement des alentours du carbet de toute végétation et une certaine distance de celui-ci à la forêt sont des facteurs protecteurs. La composante géographique est également apparue essentielle à Camopi avec une incidence qui variait selon le fleuve d’habitation et en fonction de la distance au hameau principal. Les facteurs météorologiques locaux (température et niveau du fleuve) se sont également révélés être liés à l’incidence du paludisme à court terme (0-3 mois). Par ailleurs, nos résultats ont permis d’émettre un certain nombre d’hypothèses quant à la transmission et au(x) vecteur(s) local(ux), et notamment de suggérer la participation d’un vecteur autre qu’An. darlingi dans la transmission du paludisme à Camopi. Nous avons également prouvé par ce travail que la télédétection et les systèmes d’information géographique sont très prometteurs pour la prise en compte de la dimension spatiale et environnementale dans l’étude des maladies transmissibles, notamment dans les zones d’accès difficile de Guyane. / The aim of our study was to analyze the incidence of malaria in children and its evolution through time and space, as well as to search for risk factors in a village in Mid-Oyapock (Camopi), populated by Amerindians Wayampi and Emerillon, on the one hand, and a village in Upper-Maroni (Antecume Pata), populated by Amerindians Wayana, on the other hand. The approach was multiple with, for both study sites:- A survival analysis (Cox modelling) completed out of biologically confirmed malaria attacks in a cohort of children under seven, as well as a Knowledge, Attitudes, Practices and Behavior (KAPB) questionnaire, and also the characteristics of the houses and the description of their immediate environment.- A spatial analysis with a land cover classification from SPOT 5 satellite images, the extraction of environmental variables, the study of their effect on malaria transmission and the development of an objective method for picking the proper observation horizon around houses in order to characterize the environment.- A time series study (ARIMA) to determine the effect of climatic and hydrological events on malaria at local and global (El Niño) scales.The incidence rates of malaria attacks during the period 2001-2009 were particularly high among young children, especially in Camopi with an average of 773‰ by year. A sharp decline in incidence occurred in 2007 on the Upper Maroni and this phenomenon has been observed in Camopi since 2010. An acquired immunity develops quite rapidly during the life (2-3 years old), especially against P. vivax relapses. Environmental factors were found to be the most strongly associated with malaria incidence. Indeed, living in a hut cleared from the surrounding vegetation and at a larger distance from the forest are protective factors. The geographic component also appeared essential in Camopi with an incidence which varied with the river of living and with the distance from the main hamlet. The local meteorological factors (temperature and river level) also proved to be linked to malaria incidence in the short term (0-3 months). Moreover, our results have allowed issuing a number of assumptions about the transmission and the local vector(s), in particular to suggest the involvement of another vector than An. darlingi in the malaria transmission in Camopi. We also proved by this work that remote sensing and geographic information systems hold great promise for the inclusion of the spatial and environmental dimensions in the study of transmitted diseases, especially in areas of difficult access in French Guiana.

The experiment discussed in the next chapter was to confirm the aforementioned bystander effect. In the first experiment we separated hosting and non-hosting mRBCs by the percol purification method and then measured the corner frequencies of them. The mean fc of the distribution is almost the same, and this confirms the effect of the parasite on the non-hosting mRBC. In the next experiment, we have incubated nRBCs in the spent media and measured the corner frequency at six-hours intervals to see how the fc changed with the incubation time. The results showed that within 24 hours, the fc of the incubated nRBCs increases to the level of the iRBCs. The fact that nRBCs are getting affected by the spent media indicates that some substances must be released in the spent media which alter the physical properties of the nRBCs. This kind of effect on non-host mRBCs was previously observed by some earlier works [Dondorp97, Sabolovic91a, Bambardekar08]. It has also been recently shown that the rosetting of the host mRBCs to the non-host mRBCs is also activated by the substances released in the medium [Handunnetti89, Wahlgren89], which are also somewhat similar to the bystander effect observed by us. In addition to this, there are reports which suggest that sickle cell disease also shows binding properties [Roseff08, Zhang12] which may be due to the substances released in the medium. So it was already observed that the released substances induced changes in the properties of RBCs, but our study gives a direct confirmation of the same. The next study was to find out the released substances which were responsible for the observed changes above. We incubated infected and uninfected RBCs in different drugs. Then, we measured them to see what kind of changes occur in the corner frequency of the incubated RBCs. The corner frequency of normal RBCs incubated in db-cAMP shows the maximum change. So the released substance that is responsible for the bystander effect may be due to the db-cAMP. All the experiments above were done using samples cultured only in the lab. Since the environment of the blood taken directly from the patient may differ from the one that is cultured in the lab, it is natural to find out if similar kinds of changes can be observed in the clinical sample or not. The study in chapter 6 was targeted to find out the same. We took clinical samples from BMRI for patients having a confirmed malaria infection by both P. falciparum and P. vivax. This also provided us the opportunity to work with the P. vivax infected sample as it is very difficult to culture them in the lab. The results shown in this chapter clearly indicate that similar kinds of changes occur in the clinical sample also. It is worth noting that even though P. vivax infects only immature RBCs (reticulocytes), changes were also observed in P. vivax samples. This gives us another strong confirmation about the previously observed bystander effect. This also indicates that this technique can be used as a tool to diagnose malaria. Although we cannot differentiate between P. falciparum and P. vivax, this technique combined with other well established techniques can give us more confirmation. So, in all the experiment above we have shown an easy and novel technique which can be used to differentiate between normal and malaria-infected RBCs. We have also observed the bystander effect and tried to find out the released substances which are responsible for this effect. We have shown that this technique can use the bystander effect of malaria to identify malaria. It has also been shown that the RBCs taken from the patient sample also show the same changes as the cultured samples, which gives us the possibility that this technique can be used as a diagnostic tool combined with other technique. This technique can also be used in experiments like the effects of drugs and to find out drugs for diseases like malaria. Future outlook 1. We have observed the changes only for malaria. There may be other diseases like sickle cell anemia which can also alter the corner frequency of the distribution of RBCs. We have to find out the specificity of the observed changes. 1 We can directly measure the elasticity of RBCs using dual traps in optical tweezers to find out the effect of different infections and drugs on the rigidity of RBCs and compare the with the data above. 2 We can also study other cells using the same method to see if we can find out any difference between healthy and unhealthy cells.

Der Überträger von Malaria, Anopheles gambiae, fordert jedes Jahr das Leben von etwa einer halben Million Menschen. Dennoch haben Mücken Mechanismen entwickelt, die die Ausbreitung von Krankheitserregern einschränken können. Der NF-κB-ähnliche Signalweg REL2 wurde als Schlüsselmechanismus für die Abtötung von Plasmodium falciparum in Mücken beschrieben, der sie bei experimenteller Aktivierung resistent gegen Plasmodium-Parasiten macht. Der Weg blieb jedoch schlecht charakterisiert. Zur Charakterisierung des REL2-Signalwegs wurden Loss-of-Function-Mutanten des Gens das Transkriptionsfaktor REL2 kodiert verwendet. Hier wurde die Fitness der Mücken mit REL2-Mangel, ihre Anfälligkeit für eine Infektion mit P. falciparum und Veränderungen in ihren mikrobiellen Gemeinschaften untersucht. Es wurde eine entscheidende Rolle des REL2-Signalwegs bei der Kontrolle der Proliferation des Mitteldarmmikrobioms und folglich des Überlebens der Mücken identifiziert. Es konnte kein konsistenter Phänotyp in der Anfälligkeit von REL2-Mutanten gegenüber Plasmodium beobachtet werden. Die Interaktion des Signalwegs und des Parasiten war indirekt, als Folge der REL2- und Mikrobiom-Interaktion. Gewebespezifische Transkriptomsequenzierung wurde verwendet um REL2-Genziele zu identifizieren. Die Ergebnisse zeigten die gewebespezifische Funktion des REL2-Signalwegs, wobei der Mitteldarm der Hauptort seiner Aktivität ist. Die identifizierten REL2-Genziele gehörten zu verschiedenen Domänen der Mückenphysiologie, einschließlich des Stoffwechsels. Mit weiteren Experimenten wurde die Rolle des REL2-Signalwegs bei der Blutmahlzeitverdauung und dem Management von Zucker- und Lipidressourcen beobachtet. Diese Studie ist die erste, die die Hauptgewebe der REL2-Aktivität und die Genziele des Signalwegs in Mücken identifiziert. Seine Hauptfunktion ist die Regulierung der mikrobiellen Gemeinschaften der Mücken im Mitteldarm, aber der Signalweg ist auch an der Feinabstimmung von Stoffwechselprozessen beteiligt. / Malaria vector, Anopheles gambiae, claims lives of around half a million people each year. But mosquitoes have developed an array of mechanisms that can limit pathogen proliferation. NF-κB-like signaling pathway REL2 has been described as a key mechanism for Plasmodium falciparum killing in mosquitoes, rendering them resistant to Plasmodium parasites when experimentally activated. However, the pathway remained poorly characterized. With an aim to characterize the REL2-dependent Plasmodium killing, loss-of-function mutants of the gene encoding the NF-κB-like transcription factor REL2 were used. The fitness of the REL2 deficient mosquitoes, their susceptibility to P. falciparum infection, and changes in their microbial communities were investigated. A critical role of REL2 pathway in controlling the midgut microbiome proliferation, and consequently the mosquito survival, has been identified. Interestingly, no consistent phenotype could be observed in the susceptibility of REL2 mutants to Plasmodium. Rather, the interaction of the pathway and parasite seems to be indirect, as a consequence of the REL2 and microbiome interaction. Furthermore, tissue-specific transcriptome sequencing was used to identify REL2 gene targets. The sequencing results revealed the tissue-specific function of the REL2-pathway, with the midgut being the main site of its activity. Identified REL2 gene targets belonged to various domains of mosquito physiology, including metabolism. With further experiments, the role of the REL2 pathway in blood meal digestion and management of sugar and lipid resources has been observed. Taken together, this study is the first to identify the main tissues of the REL2 activity and also gene targets of the pathway in mosquitoes. Its main function was found to be the regulation of mosquito microbial communities in the midgut, but the pathway also seems involved in fine-tuning of metabolic processes.

Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes (IEs) is a common property of field isolates associated with severe disease (Pain, Ferguson et al. 2001). Platelet receptors CD36 (Pain, Ferguson et al. 2001), P-Selectin (Wassmer, Taylor et al. 2008) and gC1qR (Biswas, Hafiz et al. 2007) mediate clumping. To characterize the molecular specificities of the clumping phenotype, I cloned clumping parasite line IT/C10 by limiting dilution. I characterized var gene expression in the IT/C10 clones using generic primers for the DBL tag region (Bull, Berriman et al. 2005). Clumping assays were conducted in the presence of specific reagents to delineate host factors hypothesized to contribute to development of the clumping phenotype. Finally, I conducted a clinical study with isolates from children with malaria in Kilifi, Kenya. This study shows that in parasite line IT/C10, platelet-mediated clumping is associated with Itvar30 suggesting a prominent role for the PfEMP-1 encoded by this var gene in development of platelet-mediated clumping. For IT/C10 parasites, platelet activation appears to be involved in platelet-mediated clumping. Platelet P-Selectin appears to mediate clumping using lectin-dependent interactions. To further elucidate the mechanisms that mediate clumping by host platelets, I have used a panel of platelet antagonists to delineate specific platelet activation pathways. Our results show that platelet activation plays an important role in platelet-mediated clumping. Finally, in this study, platelet-mediated clumping was associated with parasitaemia, but not with disease severity.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-10-18T12:20:57Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AtividadeAntiplasmodialModelagem.pdf: 2404946 bytes, checksum: f50c8e09794716ea2356ae20dc9b113b (MD5) / Approved for entry into archive by Irvana Coutinho (irvana@ufpa.br) on 2017-11-03T14:56:45Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AtividadeAntiplasmodialModelagem.pdf: 2404946 bytes, checksum: f50c8e09794716ea2356ae20dc9b113b (MD5) / Made available in DSpace on 2017-11-03T14:56:45Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_AtividadeAntiplasmodialModelagem.pdf: 2404946 bytes, checksum: f50c8e09794716ea2356ae20dc9b113b (MD5) Previous issue date: 2008 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A malária é uma infecção causada pelo Plasmodium sp. e pode ser grave, se não tratada precocemente. Ela acomete importante fração da humanidade e tem profundo impacto sanitário mundial. Estima-se que 3,3 bilhões de pessoas estejam expostas ao risco de transmissão. Um dos problemas inerentes à infecção é o progressivo aumento da resistência do parasito aos antimaláricos. Nesse contexto, fazem-se necessários estudos para o desenvolvimento de novas alternativas quimioterápicas. Muitas substâncias têm atividade antiplasmodial comprovada, como as chalconas. Entretanto, as propriedades fisico-químicas dessas moléculas, que são importantes para suas ações biológicas, não estão bem estabelecidas. Neste trabalho, foi realizada a modelagem molecular e avaliada a atividade antiplasmodial de duas chalconas (HBR1 e LH2) e quatro derivados de chalconas (GH3, IV4, LCH1 e LCH3). Para isso, foram determinadas: as concentrações inibitórias em 50% do crescimento do P. falciparum in vitro e as propriedades físico-químicas das substâncias, como HOMO, LUMO, potencial eletrostático, C log P, energia de hidratação, polarizabilidade e volume molecular, através de cálculos virtuais. Os resultados dos valores calculados foram correlacionados com a atividade biológica, a fim de se identificar parâmetros químicos que possam influenciar a ação antiplasmodial. As concentrações inibitórias em 50% do crescimento do P. falciparum variaram de 0,2 a 1,7 μM, sendo que estes valores foram menores do que os descritos na literatura. O estudo da correlação entre as atividades biológicas e as propriedades fisico-químicas mostraram parâmetros determinantes de atividade biológica, como LUMO, potencial eletrostático, C log P e energia de hidratação, que podem auxiliar na seleção de moléculas mais ativas contra o P. falciparum. Assim, essas propriedades moleculares podem ser utilizadas no planejamento racional de novas chalconas e/ou derivados com atividade antiplasmodial. / Malaria is an infection caused by Plasmodium sp. and It can be serious, if not treated precociously. It affects significant fraction of humanity and has profound health impact worldwide. It is estimated that 3.3 billion people are exposed to the risk of transmission. One of the problems of the infection is the growing emergence of parasite resistance to antimalarial drugs. In this context, studies are needed to develop new alternative chemotherapy. Many substances, such as the chalcones, have had their antiplasmodial activity proven. However, the physicochemical properties of these molecules, which are important for biological actions, are not well established. In this work, molecular modeling was performed and the antiplasmodial activity was evaluated of two chalcones (HBR1, and LH2) and four derivatives of chalcones (GH3, IV4, LCH1, and LCH3). For that, we determined the drug concentration inhibitory of 50% of the growth of P. falciparum in vitro as well as the physicochemical properties of derivatives of chalcones as HOMO, LUMO, electrostatic potential, C log P, hydration energy, polarizability and molecular volume through virtual calculations. The results of the calculated values were correlated with the biological activity in order to identify chemical parameters that can influence the antiplasmodial action. The inhibitory concentrations in 50% of the growth of P. falciparum ranged from 0,2 to 1,7 M, and these values were smaller than described them in the literature. The study of the correlation between the biological activities and the physicochemical properties showed determinating parameters for the biological activity, as LUMO, electrostatic potential, C log P and hydration energy, which may help in the selection of molecules more active against P. falciparum. Thus, these molecular properties can be used in the rational planning of new chalcones and/or derivatives with antiplasmodial activity.

Plasmodium falciparum est un parasite protozoaire responsable de la forme la plus sévère de la malaria. Depuis quelques années, les cas de résistance aux antipaludiques sont devenus de plus en plus fréquents et de plus en plus répandus. En plus de sa résistance aux drogues actuellement disponibles, ce parasite reste jusqu' à aujourd'hui réfractaire aux vaccinations. L’identification de nouvelles approches basées sur l`inhibition spécifique de certaines de ses cibles moléculaires vitales est devenue une nécessité. La recombinase à site spécifique de P. falciparum (Pf-Int) est un enzyme qui a été récemment identifié dans le laboratoire à partir de PlasmoDB. Cette recombinase à site spécifique joue potentiellement un rôle clé dans le système de recombinaison nécessaire à la viabilité du parasite. Cette protéine de 490 acides aminés, soit ~57 kDa, contient une région C-terminale qui porte les résidus conservés du site catalytique des recombinases à tyrosine R-H-K-R-(H/W)-Y. La prédiction montre une région N-terminale qui ressemble à celle de l’intégrase du phage lambda avec un mélange de structures secondaires α et β.Lors de ces travaux, nous avons d’abord montré par RT-PCR que le gène (MAL13P1.42) qui code pour PF-Int est transcrit pendant le cycle intra-érythrocytaire avec un maximum pendant la phase schizont. Nous avons ensuite essayé de montrer l`implication de Pf-Int dans le cycle parasitaire. Ceci a été réalisé grâce à un parasite (KO: knock-out) dont le gène Pf-Int a été invalidé. Ces analyses montrent que Pf-Int n'a aucun impact apparent sur le cycle de développement intra-érythrocytaire du parasite, en particulier sur la durée du cycle et le taux de croissance. Au niveau moléculaire, nous avons également procédé à la production d'anticorps anti-Pf-Int en utilisant le fragment C-162 (Résidus 162-490). La comparaison des profils de marquage, par cet anticorps, des extraits protéiques du KO et du parasite sauvage par la technique de Western blot n'a pas permis d'identifier la protéine endogène dans le parasite sauvage. Dans le but de déterminer la localisation sub-cellulaire de Pf-Int, nous avons réalisé des essais de sur-expression de différentes protéines de fusion dans le parasite. Nous avons essayé de déterminer l’impact de trois codons d’initiation différents ainsi que l’impact de la présence de la région N-terminale (1-190aa) de Pf-Int sur sa localisation subcellulaire en utilisant une chimère entre la partie N-terminale et la protéine GFP. Lors de ces travaux, nous avons réussi à sur-exprimer différentes régions de Pf-Int sous forme recombinante dans E. coli. Nous l’avons d’abord caractérisé par des études biophysiques. Ainsi nous avons pu déterminer, par dichroïsme circulaire (CD), le contenu en structures secondaires de Pf-Int, qui est proche de celui des autres membres de la même famille. Nous avons également démontré sa stabilité par CD couplé à la dénaturation thermique. Le spectre RMN-1D a aussi pu être enregistré. La troisième partie de nos travaux a concerné l’identification des cibles ADN de Pf-Int. Deux stratégies de recherche de cibles par affinité ont été utilisées au laboratoire en utilisant une première bibliothèque de séquences synthétisées chimiquement et une deuxième bibliothèque formée de fragments d’ADN génomique de P. falciparum. Ces deux approches ont permis l’identification de deux séries de cibles ADN. Grace aux cibles ADN identifiées, nous avons pu démontrer l’interaction de différents fragments de Pf-Int avec ces cibles par des expériences de retard sur gel natif (EMSA). Nous avons aussi pu démontrer que les protéines recombinantes sont actives in vitro. En effet, ces dernières sont capables de former des complexes covalents en présence de l’ADN cible. La conservation de la protéine, ainsi que son expression différentielle nous laisse à penser que son rôle est certes loin d’être élucidé, mais que Pf-Int reste une cible potentielle pour P. falciparum. / Plasmodium falciparum is a protozoan parasite responsible for the most severe form of malaria. In recent years, cases of resistance to antimalarial drugs have become increasingly frequent and common. In addition to its resistance to drugs currently available, there is no vaccine available against this parasite till now. The identification of new approaches based on the specific inhibition of some of its molecular targets has become vital.The identification of the Pf-Int site specific recombinase in Plasmodium falciparum by analysis of PlasmoDB is a new opportunity to study the role of genetic variation in this parasite as it needs to adapt to its hosts. This ~ 57 kDa protein contains a C-terminal domain carrying the putative tyrosine recombinase conserved active site residues R-H-K-R-(H/W)-Y, an N-terminus with a predicted alpha-helical bundle and a mixed alpha-beta domain resembling Lambda-Int. Here, we show that the sequence is highly conserved among members of the Plasmodia. It is expressed differentially during distinct life stages as estimated by RT-PCR, namely with a peak in the schizont phase. We then tried to show the involvement of Pf-Int in the parasitic cycle. We were able to create a parasite where the Pf-Int gene was knocked-out. The comparison test showed that Pf-Int has apparently no impact on the intraerythrocytic developmental cycle of the parasite, particularly in the cycle length and the growth rate.At the molecular level, we produced two sets of anti-Pf-Int antibodies using the purified recombinant fragment C-162 (residues 162-490). Comparison of protein extracts from KO and wild parasite by Western blot technique using our antibody has failed to identify the endogenous protein in the wild type parasite.We also tried to determine the subcellular localization of Pf-Int and the role of possible alternate initiation codons by over-expressing different constructs in the parasite Plasmodium falciparum. In order to determine the impact of the N-terminal region (1-190aa) of Pf-Int on its subcellular localization, we also created a chimeric protein using a fusion of Pf-Int(1-190aa) with the GFP. We successfully expressed a variety of the recombinant form of Pf-Int in E. coli. We have first determined its secondary structure content by circular dichroism (CD) and its solution stability by thermal denaturation-CD. An 1-D NMR spectrum was also recorded. The third part of our work has involved the identification of the DNA targets of Pf-Int. Two search strategies conducted in the laboratory using a library of chemically synthesized sequences and a second library made of fragments of genomic DNA of P. falciparum. Both approaches have allowed the identification of two sets of target DNA. Secondly, electrophoretic mobility shift assays (EMSA) were used to show its affinity and specificity for DNA. The recombinant proteins were shown to be functional as they form a covalent complex with DNA. Thus Pf-Int could be a potential agent that binds to and alters DNA, either in a specific or in random fashion. Its conservation and differential expression leads us to conclude that although its role is far from being understood, Pf-Int remains a key target for P. falciparum.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-07T16:03:10Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoNivelConcordancia.pdf: 1258819 bytes, checksum: 6d1ffa3e55c412538882010cdc01808f (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2014-02-13T16:58:07Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoNivelConcordancia.pdf: 1258819 bytes, checksum: 6d1ffa3e55c412538882010cdc01808f (MD5) / Made available in DSpace on 2014-02-13T16:58:07Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_AvaliacaoNivelConcordancia.pdf: 1258819 bytes, checksum: 6d1ffa3e55c412538882010cdc01808f (MD5) Previous issue date: 2007 / O diagnóstico precoce e o tratamento adequado dos casos de malária é a principal estratégia para o controle da doença. Várias alternativas para o diagnóstico microscópico tradicional foram propostas nos últimos anos, os testes imunocromatográficos que capturam antígenos alvos dos parasitos da malária estão sendo propostos, como o teste OptiMAL-IT® que detecta a desidrogenase lática do Plasmodium sp.. O estudo teve como objetivo a avaliação do nível de concordância entre o teste imunocromatográfico (OptiMAL-IT®) e a gota espessa para o diagnóstico da malária no Município de Mazagão – Amapá. Foram analisados 413 indivíduos com sintomatologia de malária, que procuraram o serviço da Unidade Mista de Saúde de Mazagão, com idade entre 01-68 anos. Os resultados do teste OptiMAL-IT® foram comparados com os resultados obtidos (das amostras) através da gota espessa corada pelo Giemsa. Dos 413 pacientes suspeitos de apresentarem malária, 317(76.8%) eram positivos através da GE e 311 (75.3%) eram positivos pelo TDR. Das lâminas de GE positivas, foram encontrados 27.4% de P. falciparum e 72.6% de P. vivax. O teste OptiMAL-IT® detectou 27.7% de P. falciparum e 72.3% de P. vivax. A sensibilidade obtida com o TDR para o P. falciparum foi de 97.7% e para o P. vivax foi de 98.2%, a sensibilidade global do TDR foi de 98.1% e a especificidade global e para ambas as espécies foi de 100%. Foram encontrados valores preditivos positivos e negativos de 100% e 94.1%, respectivamente. O teste OptiMAL-IT®, teve uma alta concordância com a GE, foi específico e eficiente, podendo ser usado no diagnóstico de malária nas situações onde a microscopia não está disponível. / The precocious diagnosis and the opportune treatment of the cases of malaria is one of the main strategies for the control of the disease. Several alternatives for the traditional microscopic diagnosis were proposed in the last years, the Immunochromatographic tests that capture white antigens of the parasites of the malaria they are being proposed, as the test OptiMAL-IT® that captures the lactic desidrogenase of the Plasmodium sp.. The study had as objective the evaluation of the level of agreement between the Immunochromatographic test (OptiMAL-IT®) and the thick drop for the diagnosis of the malaria in the City of Mazagão – Amapá, Brazil. 413 individuals were analyzed with malaria sintomatology that had looked for the service of the unit of health service of the city, with age among 01-68 years. The results of the OptiMAL-IT® test were compared with the obtained results, of the same samples, through the thick drop red-faced by the Giemsa. Of the 413 patients suspicious to present malaria, 317(76.8%) were positive through GE and 311 (75.3%) were positive for TDR OptiMAL-IT®. Of the positive blades of GE, had been found 27.4% of P. falciparum and 72.6% of P. vivax . The OptiMAL-IT® test detected 27.7% of P. falciparum and 72.3% of P. vivax. The sensibility obtained with TDR for P. falciparum was of 97.7% and for P. vivax was of 98.2%, the global sensibility of TDR was of 98.1% and the global specificity for both the species was of 100%. They were found preditivos values positive and negative of 100% and 94.1%, respectively. The OptiMAL-IT® test had a high agreement with thick drop, it is specific and efficient. It can be used in the diagnosis of malaria in the situations where microscopy is not available.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-02-12T12:04:47Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_MonitoramentoConcentracaoPlasmatica.pdf: 1602384 bytes, checksum: d99ae72ff74a26891b27d49d62237a6a (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-04-22T14:14:11Z (GMT) No. of bitstreams: 2 Dissertacao_MonitoramentoConcentracaoPlasmatica.pdf: 1602384 bytes, checksum: d99ae72ff74a26891b27d49d62237a6a (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) / Made available in DSpace on 2014-04-22T14:14:11Z (GMT). No. of bitstreams: 2 Dissertacao_MonitoramentoConcentracaoPlasmatica.pdf: 1602384 bytes, checksum: d99ae72ff74a26891b27d49d62237a6a (MD5) license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Previous issue date: 2006 / Governo do Estado do Amapá / A correlação entre resposta parasitológica e a concentração plasmática de quinina + doxiciclina e de mefloquina foi monitorada por 28 dias em trinta pacientes com malária por Plasmodium falciparum no Estado do Amapá. No grupo (A) 12 pacientes receberam o esquema terapêutico oral de quinina (3 dias) + doxiciclina (5 dias), e no grupo (B) 18 pacientes receberam mefloquina oral dose única (1º dia). No grupo (A) quatro pacientes (33,3%) apresentaram plasmódios resistentes do tipo RI, e (66,7%) foram sensíveis (S). A média da concentração plasmática de quinina em D1, D3 e D5 foi de 2,575μg/mL, 2,3334μg/mL e 0,532μg/mL para os (S) e de 2,4667μg/mL, 2,1784μg/mL e 0,450μg/mL para os (RI), não houve diferença estatisticamente significativa entre essas concentrações. No grupo (B), 100% dos pacientes apresentaram plasmódios sensíveis. A média da concentração plasmática da mefloquina foi: D0(0 μg/mL), D1 (0,709μg/mL), D3 (0,543 μg/mL), D5 (0,361 μg/mL), D7 (0,187 μg/mL), D14 (0,125 μg/mL), D21 (0,046 μg/mL) e D28 (0 μg/mL). A correlação entre resposta parasitológica e a concentração plasmática de quinina +doxiciclina foi de 94,23% para os (S) e de 76,70% para os (RI). Houve absorção adequada desses fármacos tanto para os (S), quanto, para os (RI), portanto , não foi a má absorção a causa da recrudescência. Os resultados sugerem que houve resistência do P. falciparum ao esquema administrado. Para os pacientes que receberam mefloquina a correlação foi de 91,82%. Houve absorção e manutenção da concentração desse fármaco durante o período de tempo necessário para completa eliminação dos parasitas. / The correlation between the plasma concentration of quinine and doxycycline and of mefloquine with the parasitological answer was monitored by 28 days in thirty patients with malaria by Plasmodium falciparum in the State of the Amapá. In the group (A) twelve patient received the oral scheme of quinine (3 days) + doxycycline (5 days), and in the group (B) eighteen patients received mefloquine oral unique dose (1º day). In the group (A) four sick (33,3%) presented Plasmodium resistant of the type RI, and (66,7%) were sensible (S). On average of the plasma concentration of the quinine in D1, D3 and D5 was of 2,5750μg/mL, 2,3334μg/mL and 0,532μg/mL for the (S) and of 2,4667μg/mL, 2,1784μg/mL and 0,450μg/mL for the (RI), had not difference statistical significant between those concentrations. In the group (B), patients 100% presented sensible Plasmodium. The average of the concentration plasma of the mefloquine was: D0 (0μg/mL), D1 (0,709μg/mL), D3 (0,543 μg/mL), D5 (0,361 μg/mL), D7 (0,187 μg/mL), D14 (0,125 μg/mL), D21 (0,046 μg/mL) and D28 (0μg/mL). The correlation between parasitological answer and the plasma concentration of quinine + doxicyclyne was of 94,23% for the (S) and of 76,70% for the (RI), the quinine and doxycycline adequately was absorption so much for the (S), as well as, for the (RI), therefore, was not to bad absorption of the drug the cause of the recrudescence. The results suggest that there was resistance of the P. falciparum to the administered treatment. For the patients that received mefloquine the correlation was of 91,82%, having the absorption and maintenance of the drug concentration during the period of necessary time for complete elimination of the parasites.

Cerebral malaria is characterised by an accumulation of infected erythrocytes in the microvasculature of the brain. Plasmodium falciparum infected erythrocytes have been shown to bind to a Human Brain Endothelial Cell line (HBEC-5i) in vitro. This provides a model for the investigation of interactions between P. falcuparum and human brain endothelium. Currently neither the parasite adhesion ligands on infected erythrocytes, nor the host endothelial cell receptors necessary for this interaction have been identified. In this work, the identity of the host receptor on brain endothelial cells was addressed by binding assays of selected and unselected parasites on a wide range of malaria-associated host molecules. The identity of the parasite ligand was investigated by microarray analysis of parasites after selection for cytoadherence to HBEC-5i. The hypothesis being tested was that the gene encoding the parasite cytoadherence ligand would show significant upregulation in selected compared to unselected paarasites. The P. falciparum laboratory strains 3D7, HB3 and IT/FCR3 were selected for binding to HBEC-5i using a panning assay. Compared to unselected parasites, HBEC-5i selected parasites showed a distinct phenotype with reduced platelet-mediated clumping. There was no significant increase in binding of parasites to any of the known endothelial cytoadherence receptors for P. falciparum after selection on HBEC-5i. Binding inhibition assays with various antibodies and soluble receptors did not greatly block the adhesion of parasites to HBEC-5i except for heparin. Altogether, the receptor(s) mediating the interation with HBEC-5i remains unknown. In order to carry out transcriptional analysis of selected and unselected paarasites form all three parasite strains, it was necessary to update the existing microarray chip which is based on the 3D7 genome. This is because each parasite train has a unique repertoire of variant surface antigens (VSAs) including var, rif and stevor genes. Therefore, to fully analysis HB3 and IT genomes. Unique oligonnucleotide probes were then designed for each new sequence and the 3D7-based microarray chip was updated. Transcriptional analysis was then carried out on selected and unselected parasites of all strains. Microarray data clearly indicated that the most highly upregulated genes after selection were group A or group A-like var genes (HB3var3, 3D7_PFDOO2Oc, ITvar7 and ITvar19), showing 11 to over 100 fold upregulation in selected parasites. The rif gene adjacent to the upregulated var gene was also highly expressed. To a lesser extent some exported proteins like RESA-1, PfEMP3 or PHIST family members also showed increased transcription in HBEC-selected parasites (2-3 fold upregulation). Reverse transcriptase-PCR confirmed the upregulation of group A var genes in selected parasites, suggessted that the group A PfEMP1 variants are major candidate ligands for parasite binding to HBEC-5i. These findings are consistent with previous work showing an association between Group A var genes and cerebral malaria.