This thesis discusses the structure-function studies on triosephosphate isomerase (TIM) from Plasmodium falciparum (Pf), directed towards understanding the roles of highly conserved residues by site derected mutagenesis. Chapter 1 provides an introductory overview to the relevant literature on triosephosphate isomerase. In addition, this Chapter provides an analysis of conserved residues in TIM, and amino acid diversity at specific positions in the structure using a dataset of 503 TIM sequences. Chapter 2 reports the work on the completely conserved residue, C126 in TIM, which is proximal to the active site. Five mutants, C126S, C126A, C126V, C126M and C126T have been characterized. Crystal structures of 3-phosphoglycolate (PGA) bound C126S mutant and the unliganded forms of the C126S and C126A mutants have been determined at a resolution of 1.7 Å to 2.1 Å. Kinetic studies reveal a ~5 fold drop in kcat for the C126S and C126A mutants, while a ~ 10 fold drop is observed for the other three mutants. All the mutants show reduced stability at lower concentration and higher temperature. Chapter 3 presents the kinetic and structural characterization for the E97Q and E97D mutants of Pf TIM. A 4000 fold reduction in kcat is observed for E97Q, 100 fold reduction for the E97D mutant, while a ~ 9000 fold drop in activity for the control mutant, E165A. A large conformational change for the critical K12 side chain is observed in the crystal structure of the E97Q mutant, while it remains unchanged in the E97D structure. The results are interpreted to invoke a direct role for E97 in the catalytic proton transfer cycle, eliminating the need to invoke the formation of the energetically unfavorable imidazolate anion at H95. Chapter 4 reports investigations with position 96 by the biochemical and structural characterization of single mutants, F96Y, F96A and the double mutants, F96S/S73A and F96S/L167V. F96Y showed ~100 fold drop in activity, F96A revealed ~10 fold drop in activity, while F96S/S73A showed 100 fold lower activity than that of the wild type enzyme. Interestingly, the double mutant F96S/L167V proved to be a partial pseudorevertant, showing 10 fold higher activity than the single mutant, F96S. Chapter 5 describes the cloning, and preliminary kinetic and biophysical characterization of the enzyme, Dm TIM. A survey of disease causing mutations in TIM and the relationship of these sites of mutation to the active site and the dimer interface of TIM is presented in this Chapter.

The species Acacia langsdorfii Benth is a tree with scarce geographical distribution not happening report in the literature regarding pharmacotherapeutics properties. The genus Acacia is the largest second in the family Leguminosae and one of the largest is considered in Angiospermas, presenting more of 1.200 species presenting cosmopolitan and typical character of semi-arid hot areas distributed in tropical and temperate hot areas of everyone. The objective of this work is to contribute to the knowledge of chemical composition and biological activity of extracts derived from the leaf and stem of Acacia langsdorfii Benth, hitherto not studied. Several species of Acacia are used traditionally for the treatment of many different diseases. However, of this species, only known to their extracts show immunomodulatory activity and trypanocidal. From the hexane fraction of the stem, was isolated, by chromatographic methods, a steroid and a triterpenoid which were identified as stigmasterol and lupeol, respectively. It was isolated and purified a mixture of triterpenoid of the lupane series (where one is lupeol). The lupeol is the chemical constituent majority. From the Hexane fraction of leaves, was isolated and purified a diterpenoid and triterpenoid, which were identified as ent-atisan-7α,16α-diol and lupeol, respectively. The lupeol is the chemical constituent majority. In addition, from the ethyl acetate fraction of leaves was isolated and purified by column chromatography, two flavan-3-ols, one flavanone and two flavones which were identified by spectroscopic techniques such as catechin, epicatechin, naringenin, 4'-hydroxy-5,6,7-trimethoxyflavone and morin-3-O-rutinoside, respectively. The morin-3-Orutinoside is the chemical constituent majority of this fraction. The structures were identified using spectroscopic techniques of IR, MS, 1D and 2D NMR (H1, C13, DEPT 90 °, 135 °, DEPT, COSY, HSQC, HMBC and NOESY), and determinations of the melting point, rotation optical and comparisons with literature data. Substances ent-atisan-7α,16α-diol, morin-3-O-rutinoside, 4'-hydroxy-5 ,6,7-trimethoxyflavone were first reported in the genus. The hexane fractions derived from both the stem and leafs showed a high rate (> 90%) inhibits activity the proliferation of Plasmodium falciparum. The fractions derived from both the stem and leafs showed a high rate (> 80%) inhibits activity the linfoproliferation. / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A espécie Acacia langsdorfii Benth é uma árvore com distribuição geográfica escassa não ocorrendo registro na literatura quanto às suas propriedades fármaco-terapêuticas. O gênero Acacia é o segundo maior na família Leguminosae e é considerado um dos maiores nas Angiospermas, apresentando mais de 1.200 espécies apresentando caráter cosmopolita e típico de regiões quentes semi-áridas distribuídas em regiões quentes tropicais e temperadas de todo o mundo. O objetivo deste trabalho é contribuir para o conhecimento da composição química e da atividade biológica dos extratos oriundos da folha e do caule da espécie Acacia langsdorfii Benth, até então não estudada. Diversas espécies de Acacia são utilizadas tradicionalmente para o tratamento das mais diferentes patologias. Porém, Não há registro na literatura quanto às propriedades fármaco-terapêuticas desta espécie, apenas sabe-se que seus extratos apresentaram atividade imunomoduladora e tripanocida. Da fração de hexano do caule foram isolados, por métodos cromatográficos, um esteróide e um triterpenóide, os quais foram identificados como sendo estigmasterol e lupeol, respectivamente. Também foi isolado e purificado uma mistura contendo triterpenóides da série lupano (onde um é o lupeol). Da fração hexânica das folhas foi isolado e purificado um diterpenóide e um triterpenóide, os quais foram identificados como sendo ent-atisan-7α,16α-diol e lupeol, respectivamente. Além disso, da fração acetato de etila das folhas foi isolado e purificado por cromatografia em coluna dois flavan-3-óis, uma flavanona e duas flavonas as quais foram identificadas por técnicas espectroscópicas como sendo catequina, epicatequina, naringenina, 4’-hidroxi-5,6,7-trimetoxiflavona e morina 3-O-rutinosídeo respectivamente. A morina 3-O-rutinosídeo é o constituinte químico majoritário desta fração. As estruturas foram identificadas com o uso de técnicas espectroscópicas de IV, RMN 1D e 2D (H1, C13, DEPT 90°, DEPT 135°, COSY, HSQC, HMBC e NOESY) e espectrométrica (EM), além de determinações do ponto de fusão, rotação óptica e comparações com dados da literatura. As substâncias ent-atisan-7α,16α-diol, morina 3-O-rutinosídeo, 4’-hidroxi-5,6,7-trimetoxiflavona foram relatadas pela primeira vez no gênero. As frações oriundas do hexano, tanto do caule como em folhas, apresenta um alto índice (> 90%) de atividade inibidora da proliferação do Plasmodium falciparum. As frações obtidas da partição do extrato em etanol das folhas e caule da A. langsdorfii apresentaram uma alta inibição da linfoproliferação (>80%).

Le paludisme demeure un problème de santé majeur en Afrique subsaharienne. Le nombre limité d'antipaludiques, l’apparition de résistances et l’absence d’un vaccin efficace, font de la lutte anti-vectorielle (LAV) la principale stratégie préventive de cette maladie. Les méthodes actuelles de LAV visant à limiter ou à interrompre le développement du parasite chez le moustique vecteur, il est donc nécessaire d’améliorer notre compréhension des interactions entre le vecteur Anopheles, son environnement et le parasite Plasmodium. A Madagascar, Anopheles gambiae s.l. et Anopheles funestus sont les vecteurs majeurs de Plasmodium falciparum et de Plasmodium vivax. Anopheles mascarensis, espèce endémique, peut également être un vecteur important. Dans ce contexte, l’objectif premier de ma thèse a été d’approfondir les connaissances sur An. mascarensis à travers une revue. Les données collectées plaident davantage qu’An. mascarensis est un complexe d'espèces et permettent de poser les bases pour une analyse moléculaire ciblée. En parallèle, j’ai contribué à la mise en place de la première plateforme expérimentale de Madagascar pour infecter des anophèles par P. falciparum et P. vivax, afin d’évaluer leur compétence vectorielle. Enfin, en associant entomologie et immuno-parasitologie, nous avons analysé la contribution des vecteurs à la transmission du paludisme dans deux villages adjacents. L’ensemble des travaux réalisés durant de ma thèse contribue à une meilleure connaissance de la diversité de la transmission du paludisme à Madagascar. De plus, la mise en place de la plateforme expérimentale d’infection permettra l’analyse de la compétence des populations d’anophèles vecteurs. / Malaria remains a major health concern in sub-Saharan Africa. The limited number of antimalarial drugs, the emergence of resistances and the lack of an effective vaccine, make vector control the main preventive strategy for this disease. Current methods of vector control aim at limiting or interrupting parasite development in the vector mosquito. It is therefore necessary to improve our understanding on interactions between the Anopheles vector, its environment and the parasite Plasmodium. In Madagascar, Anopheles gambiae s.l. and Anopheles funestus are the major vectors of Plasmodium falciparum and Plasmodium vivax. Anopheles mascarensis, an endemic species, may also be an important vector. In this context, the main objective of my PhD was to deepen the knowledge on An. mascarensis through a review. The data collected indicate that An. mascarensis is a complex of sibling species. I could thus provide the foundation for targeted molecular analysis. In parallel, in order to evaluate their vector competence, I contributed in a major way to the establishment of the first experimental platform of Madagascar to infect anopheline mosquitoes by P. falciparum and P. vivax. Finally, combining entomology and immuno-parasitology, we analysed the contribution of vectors to malaria transmission in two neighbouring villages. All the work done during my PhD contributes to a better knowledge of the diversity of malaria transmission in Madagascar, especially on the effective contribution of the different vector species. In addition, the establishment of the experimental platform for infections will further allow the analysis of the competence of vector Anopheles populations.

Plasmodium falciparum is a protozoan parasite responsible for causing the most severe form of malaria in humans. This species is responsible for over 90% of malaria mortalities which occur predominantly in Africa. An increase in drug resistant parasites in recent years is threatening the progress made against malaria and thus new antimalarial drugs and vaccines are needed to combat this disease. During the intraerythrocytic phase, merozoites egress from mature schizonts to invade new uninfected erythrocytes. Glycophosphatidylinositol (GPI) -anchored proteins cover most of the exterior surface of the merozoite prior to invasion, while other GPI-anchored proteins are released onto the merozoite surface through apical organelle secretions. These proteins are involved in interactions with erythrocytes and are thought to be vital to erythrocyte invasion. GPI-anchored proteins have also been implicated as a cause of pathogenic symptoms and activation of immune components. These proteins are then released or cleaved to enable merozoite entry into the erythrocyte. Several enzymes are thought to be involved in their cleavage including the serine proteases subtilisin-like proteases (SUB) 1 and 2, and phosphatidylinositol-phospholipase C (PIPLC); GPI-anchored proteins are also generally sensitive to phospholipase A2 (PLA2). Cleaved proteins are released into the host blood system, while uncleaved proteins are carried into the erythrocyte during invasion. Merozoites have a limited period in which they retain invasive capacity. A previous lack of available techniques that are specifically adapted to merozoite analysis has resulted in an incomplete understanding of invasion and GPI-anchored protein involvement in invasion. This study aimed to determine how GPI-anchored proteins on the merozoite surface are altered in the invasive phase, and explore the possibility of using merozoite GPI-anchored proteins as potential drug targets to block erythrocyte invasion. Optimised methods of in vitro parasite culturing which produce highly synchronised merozoites was essential to this study. Parasite culturing techniques were optimised by utilising low haematocrit cultures with frequent culture splitting and optimised synchronisation. The “Malarwheel” is a tool that was developed for this research to provide a means for scheduling sorbitol treatments and MACs isolations. This tool and optimised culturing methods enabled large volumes of highly synchronised invasive merozoites to be harvested. Four compounds (vanadate, edelfosine, dioctyl sodium sulfosuccinate (DSS), and gentamicin) suspected to interfere with GPIanchored cleavage or processes were screened on intraerythrocytic stages and merozoites. Antimalarial and anti-invasive properties of these compounds were screened by modified malaria SYBR Green I-based fluorescence (MSF) assay and merozoite invasion assays (MIA) respectively. DSS and gentamicin showed limited potential as antimalarials or as anti-invasive agents. Vanadate and edelfosine both showed antimalarial and anti-invasive activity, while edelfosine was the most potent anti-invasive agent at physiological concentrations. The merozoite GPI-anchored proteome was analysed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) followed by complete gel lane analyses conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on soluble and pelleted merozoite proteins in samples from either invasive or non-invasive merozoites. Thirteen known or predicted GPI-anchored proteins were identified in samples. Several changes were identified in merozoite GPI-anchored proteins between the invasive phase and after its completion, and minor differences were observed following treatment with edelfosine. Edelfosine showed partial inhibition of erythrocyte invasion, however, the primary cause of inhibition cannot be directly related to interferences with GPI-anchored proteins. These results suggest that GPIanchored proteins are controlled by various complex processes, and are cleaved or processed by diverse mechanisms during the invasive phase. These mechanisms may be controlled by multiple signals which effect proteins or groups of proteins in specific ways. These signals may be influenced by “checkpoints” during invasion processes including the time period after egress from schizonts, and possibly the recognition of erythrocyte targets. These methods and results provide a foundation for future research to enable culturing of P. falciparum parasites specifically for merozoite research, and to identify merozoite proteins active during the invasive phase. These results confirm and challenge previous ideas reported in literature on the GPI-anchored processes of merozoites and further characterise less studied GPIanchored proteins. The results suggest that the processes controlling GPI-anchored proteins may be more complex than previously thought. These results form a basis to further identify and characterise GPI-anchored proteins in the aim to develop antimalarial medications and vaccines that target merozoites and their GPI-anchored processes. / Dissertation (MSc)--University of Pretoria, 2017. / Pharmacology / MSc / Unrestricted

MSc (Biochemistry) / Department of Biochemistry / Malaria is a disease that claims about half a million lives annually, mainly children. There are 5 Plasmodium species that cause malaria; namely, P. falciparum, P. ovale, P. malariae, P. knowlesi and P. vivax. P. falciparum is the most virulent of them all. The parasite upregulates some heat shock proteins (Hsps) in response to stress it encounters during its life cycle. These Hsps play a major role in proteostasis. The drug resistance of P. falciparum to traditionally used remedies has led to a need for the development of novel drugs. Hsps have been implicated as antimalarial drug targets. Hsps act as molecular chaperones and some make complexes, which are important in facilitating protein folding. As an example, heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) form a functional complex through an adaptor protein, Hsp70-Hsp90 organizing protein (Hop). P. falciparum expresses six Hsp70s that are localized in different subcellular compartments. Amongst them, P. falciparum Hsp70-x (PfHsp70-x), is exported to the erythrocyte where it is implicated in host cell remodeling. PfHsp70-x possesses an ATPase domain, substrate binding domain and a C-terminal subdomain. PfHsp70-x possesses an EEVN motif on its C-terminus which is implicated in interactions with co-chaperones amongst them, Hop. Although some of the chaperone functions of PfHsp70-x have been reported, its interaction with human chaperones has not been investigated. The availability of PfHsp70-x in the infected erythrocyte cytosol presents a possibility that this protein may functionally cooperate with human Hsp90 via human Hop (human Hop). This hypothesis that PfHsp70-x interacts with human chaperones is strengthened by the absence of Hsp90 and Hop of parasite origin in the infected erythrocytes. The main aim of this study was to explore the chaperone activity of PfHsp70-x and its functional co-operation with human Hop. Recombinant PfHsp70-x (full length and EEVN deletion mutant) proteins were expressed in E. coli XL1 Blue cells and purified using nickel affinity chromatography. PfHsp70-x was found to be structurally comprised of mostly alpha helices and demonstrated heat stability based on circular dichroism (CD) spectrometry studies. It was established that the EEVN motif may be important for the ATPase activity of PfHsp70-x. However, it was established that the EEVN motif was not important in regulating the holdase chaperone (protein aggregation suppression) function of PfHsp70-x. Furthermore, PfHsp70-x and its mutant preferentially bound to asparagine-rich peptides. Parasite proteins have high asparagine repeat regions as compared to human proteins. In addition, preference for asparagine-rich proteins iii could signify that PfHsp70-x is biased towards binding proteins of parasitic origin. Surface plasmon resonance (SPR) analysis suggested that PfHsp70-x interacts with human Hop with relatively higher affinity compared to its EEVN minus derivative. In conclusion, the removal of the EEVN motif of PfHsp70-x does not affect the chaperone function of PfHsp70-x. However, the EEVN motif is essential for the interaction of PfHsp70-x with human Hop.

MSc (Biochemistry) / Department of Biochemistry / Plasmodium falciparum is a unicellular protozoan parasite that causes malaria in humans. The parasite is passed to humans through mosquito bites and migrates to the liver before it infects host erythrocytes. It is at the erythrocytic stage of development that the parasite causes malaria pathology. Malaria is characterized by the modification of host erythrocytes making them cytoadherent. This is as a result of formation of protein complexes (knobs) on the surface of the erythrocyte. The knobs that develop on the surface of the erythrocyte are constituted by proteins of host origin as well as some proteins that the parasite ‘exports’ to the host cell surface. Nearly 550 parasite proteins are thought to be exported to the infected erythrocyte. Amongst the exported proteins is P. falciparum heat shock protein 70-x (PfHsp70-x). Hsp70 proteins are known to maintain protein homeostasis. Thus, the export of PfHsp70-x may be important for maintaining protein homeostasis in the host cell. PfHsp70-x is not essential for parasite survival although is implicated in the development of parasite virulence. This is possibly through its role in facilitating the trafficking of parasite proteins to the erythrocyte as well as supporting the formation of protein complexes that constitute the knobs that develop on the surface of the infected erythrocyte. The main objective of the current study was to investigate protein interaction partners of PfHsp70-x. It is generally believed that PfHsp70-x interacts with various proteins of human and parasite origin. Potential candidate interactors include its protein substrates, Hsp70 co-chaperones such as Hsp40 members, and human Hsp70-Hsp90 organizing protein (hHop). The establishment of the PfHsp70-x interactome would highlight the possible role of PfHsp70-x in the development of malaria pathogenicity. Based on bioinformatics analysis, PfHsp70-x was predicted to interact with some exported P. falciparum Hsp40s, hHop and human Hsp90 (hHsp90). Recombinant forms of PfHsp70-x (full length and a truncated form that lacks the C-terminal EEVN motif implicated in co-chaperone binding) were expressed in E. coli BL21 Star (DE3) cells. Recombinant hHop and hHsp70 were expressed in E. coli JM109 (DE3) cells. The proteins were successfully purified using nickel affinity chromatography. Co-affinity chromatography using recombinant PfHsp70-x and immuno-affinity chromatography using PfHsp70-x specific antibody did not confirm the direct interaction of PfHsp70-x with human Hop. However, the direct interaction of hHop and PfHsp70-x has previously been validated in vitro and the current bioinformatics data support ii the existence of such a complex. PfHsp70-x was not stable in the cell lysate that was prepared and this could explain why its interaction with hHop could not be ascertained. However, taken together the evidence from a previous independent study, and the predicted interaction of PfHsp70-x with human chaperones suggests cooperation of chaperone systems which possibly facilitates the folding and function of parasite proteins that are exported to the infected erythrocyte. / NRF

MSc (Biochemistry) / Department of Biochemistry / Sustaining proteostasis is essential for the survival of the cell and altered protein regulation leads to many cellular pathologies. Heat shock proteins (Hsps) are involved in the regulation of the protein quality control. Hsps are a group of molecular chaperones that are upregulated in response to cell stress and some are produced constitutively. The Hsp70 family also known as DnaK in Escherichia coli (E. coli) is the most well-known group of molecular chaperones. Structurally, Hsp70s consist of a nucleotide binding domain (NBD) and a substrate binding domain (SBD) conjugated by a linker sub-domain. ATP binding and hydrolysis is central to the Hsp70 functional cycle. Hsp70s play a role in cytoprotection especially during heat stress in E. coli. Hsp70s from different organisms are thought to exhibit specialized cellular functions. As such E. coli Hsp70 (DnaK) is a molecular chaperone that is central to proteostasis in E. coli. On the other hand, Plasmodium falciparum Hsp70s are structurally amenable to facilitate folding of P. falciparum substrates. The heterologous production of P. falciparum proteins in E. coli towards drug discovery has been a challenge. There is need to develop tools that enhance heterologous expression and proper folding of P. falciparum proteins in an E. coli expression system. To this end, a chimeric Hsp70, KPf consisting of E. coli DnaK NBD and P. falciparum Hsp70-1 (PfHsp70-1) SBD was previously designed. KPf was shown to confer cytoprotection to E. coli DnaK deficient cells that were subjected to heat stress. In this study it was proposed that KPf has an advantage over E. coli DnaK and PfHsp70-1 in its function as a protein folding chaperone. Therefore, the main aim of this study was to characterize the chaperone function of KPf relative to the function of wild type E. coli and P. falciparum Hsp70s. The recombinant forms of KPf, DnaK and PfHsp70-1 proteins were successfully expressed and purified using nickel affinity chromatography. Circular Dichroism (CD) structural study demonstrated that KPf and PfHsp70-1 are predominantly α-helical and are also heat stable. Tertiary structure studies of PfHsp70-1 and KPf using tryptophan fluorescence revealed that both confirmations of recombinant proteins are perturbed by the presence of ATP more than ADP. Interestingly, the substrate binding capabilities of these proteins were comparable both in the absence or presence of nucleotides ATP/ADP. KPf is an independent chaperone, that exhibit nucleotide binding and hydrolysis. The current study has established unique structure-function features of KPf that distinguishes it from its “parental” forms, DnaK and PfHsp70-1. / NRF

Introducción: La malaria es una enfermedad metaxénica producida por Plasmodium spp. Las especies que causan más daño en salud pública son P. vivax y P. falciparum. Malaria severa se refiere a un cuadro de malaria en presencia de alteraciones de signos físicos y laboratoriales definidos por la OMS. Aunque la malaria severa se ha relacionado tradicionalmente con malaria por P. falciparum, también está asociada a malaria por P. vivax. La prevalencia de malaria severa por P. vivax no ha sido bien estudiada. Por ello, el objetivo de este estudio fue identificar algunos factores epidemiológicos y clínicos asociados a malaria severa en los pacientes con malaria por Plasmodium vivax y Plasmodium falciparum, en la población de la provincia de Maynas entre los años 2014-2019. Métodos: Estudio observacional analítico de tipo transversal. Se revisaron todas las historias clínicas de pacientes con diagnóstico confirmado de malaria por P. vivax o P. falciparum del hospital Regional de Loreto en el periodo 2014-2019. Se determinó malaria severa por los criterios de la OMS y se estratificaron los casos por especie. Luego, se realizaron análisis univariados, bivariados, y multivariados; en las que los factores asociados a malaria severa eran las variables independientes, y tener malaria severa por una u otra especie eran las variables dependientes. Para realizar el análisis multivariado se usó el modelo lineal generalizado de la familia Poisson para obtener razones de prevalencia crudas y ajustadas (RP). Resultados: Se revisaron 590 historias clínicas de pacientes con malaria del Hospital Regional de Loreto. De estas, 456 historias tenían información sobre algún criterio para evaluar malaria severa. Se encontró malaria severa en 55 (16,2%) pacientes con malaria por P. vivax, y en 22 (19%) pacientes con malaria por P. falciparum. En los análisis multivariados, no se encontraron asociación entre malaria severa ni por P. vivax ni por P. falciparum, con la edad, sexo, episodios previos de malaria, ocupación ni número de leucocitos en sangre. Conclusiones: En el Hospital Regional de Loreto, la frecuencia de malaria de malaria severa por P. vivax y P. falciparum fue de 16,2% y 19,0%, respectivamente. Los criterios de malaria severa más prevalentes fueron anemia severa (9,3%) e injuria renal aguda (14,5%). No se encontró asociación entre malaria severa ni por P. vivax ni por P. falciparum, y la edad, sexo, episodios previos de malaria, ocupación ni número de leucocitos en sangre. / Introduction: Malaria is a vector-borne disease caused by Plasmodium spp. The most important species and that cause more damage in Public Health are P. vivax and P. falciparum. Severe malaria refers to malaria episodes with altered physical and laboratory signs defined by the WHO. Although severe malaria has traditionally been associated with P. falciparum, it is also associated with P. vivax malaria. The prevalence of severe P. vivax malaria has not been well studied. Therefore, the objective of this study was to identify some epidemiological and clinical factors associated with severe malaria in patients with malaria caused by Plasmodium vivax and Plasmodium falciparum, in the population of the province of Maynas between the years 2014-2019. Methods: Cross-sectional and analytical observational study. All the medical records of patients with a confirmed diagnosis of P. vivax or P. falciparum malaria from the “Hospital Regional de Loreto” in the period 2014-2019 were reviewed. Severe malaria was determined by the 2015 WHO criteria and cases were stratified by species. Then, univariate, bivariate, and multivariate analyzes were performed; in which factors associated with severe malaria were the independent variables, and having severe malaria due to one or another species were the dependent variables. To perform the multivariate analysis, the generalized linear model of the Poisson family was used to obtain crude and adjusted prevalence ratios (PR). Results: We reviewed 590 medical charts of patients with malaria from the “Hospital Regional de Loreto”. Out of them, 456 charts had some criteria to evaluate severe malaria. Severe malaria was found in 55 (16.2%) patients with P. vivax malaria, and in 22 (19%) patients with P. falciparum malaria. In the multivariate analyzes, no association was found between severe malaria neither by P. vivax nor by P. falciparum, and age, sex, previous malaria episodes, occupation or leukocytes count. Conclusions: The prevalence of severe malaria due to P. vivax and P. falciparum was 16.2% and 19,0%, respectively. No association was found between severe malaria neither by P. vivax nor by P. falciparum, and age, sex, previous malaria episodes, occupation or leukocytes count. / Tesis

Duplications and deletions are a major source of genomic variation. Duplications, specifically, have a significant impact on gene genesis and dosage, and the malaria parasite P. falciparum has developed resistance to a growing number of anti-malarial drugs via gene duplication. It also contains highly duplicated families of antigenically variable allelic genes. While specific genes and families have been studied, a comprehensive analysis of duplications and deletions within the reference genome and population has not been performed. We analyzed the extent of segmental duplications (SD) in the reference genome for P. falciparum, primarily by a whole genome self alignment. We discovered that while 5% of the genome identified as SD, the distribution within the genome was partition clustered, with the vast majority localized to the subtelomeres. Within the SDs, we found an overrepresentation of genes encoding antigenically diverse proteins exposed to the extracellular membrane, specifically the var, rifin, and stevor gene families. To examine variation of duplications and deletions within the parasite populations, we designed a novel computational methodology to identify copy number variants (CNVs) from high throughput sequencing, using a read depth based approach refined with discordant read pairs. After validating the program against in vitro lab cultures, we analyzed isolates from Senegal for initial tests into clinical isolates. We then expanded our search to a global sample of 610 strains from Africa and South East Asia, identifying 68 CNV regions. Geographically, genic CNV were found on average in less than 10% of the population, indicating that CNV are rare. However, CNVs at high frequency were almost exclusively duplications associated with known drug resistant CNVs. We also identified the novel biallelic duplication of the crt gene – containing both the chloroquine resistant and sensitive allele. The synthesis of our SD and CNV analysis indicates a CNV conservative P. falciparum genome except where drug and human immune pressure select for gene duplication.

Each year countless lives are affected and about half a million people die from malaria, a disease caused by parasites originating from the Plasmodium family. The most virulent species of the parasite is Plasmodium falciparum (P. falciparum).   Calmodulin (CaM) is a small, 148 amino acid long, highly preserved and essential protein in all eukaryotic cells. Previous studies have determined that CaM is important for the reproduction and invasion of P. falciparum in host cells. The primary structure of human CaM (CaMhum) and CaM from P. falciparum (CaMpf) differ in merely 16 positions, making differences in their structures and ligand affinity interesting to study. Especially since possible inhibitors of CaMpf in favor of CaMhum, in extension, could give rise to new malaria treatments.   Some antagonists, functioning as inhibitors of CaM, have already been analysed in previous studies. However, there are also compounds that have not yet been studied in regards to being possible antagonists of CaM. This study regards three known antagonists; trifluoperazine (TFP), calmidazolium (CMZ) and artemisinin (ART) and also three recently created fentanyl derivatives; 3-OH-4-OMe-cyclopropylfentanyl (ligand 1), 4-OH-3OMe-4F-isobutyrylfentanyl (ligand 2) and 3-OH-4-OMe-isobutyrylfentanyl (ligand 3).   Bioinformatic methods, such as modelling and docking, were used to compare the structures of CaMhum and CaMpf as well as observe the interaction of the six ligands to CaM from both species. In addition to the differences in primary structure, distinguished with ClustalW, disparities in tertiary structure were observed. Structure analysis of CaMhum and CaMpf in PyMOL disclosed a more open conformation as well as a larger, more defined, hydrophobic cleft in CaMhum compared to CaMpf. Simulated binding of the six ligands to CaM from both species, using Autodock 4.2, indicated that TFP and ART bind with higher affinity to CaMhum which is expected. Ligand 2 and ligand 3 also bound with higher affinity and facilitated stronger binding to CaMhum, which is reasonable since their docking is based on how TFP binds to CaM. However, ligand 1 as well as CMZ both bound to CaMpf with higher affinity. Despite promising results for ligand 1 and CMZ, no decisive conclusion can be made solely based on bioinformatic studies.    To gain a better understanding on the protein-ligand interactions of the six ligands to CaMhum and CaMpf, further studies using e.g. circular dichroism and fluorescence would be advantageous. Based on the results from this study, future studies on the binding of CMZ and ligand 1 to CaM as well as ligands with similar characteristics would be especially valuable. This is because they, based on the results from this study, possibly are better inhibitors of CaMpf than CaMhum and thereby could function as possible antimalarial drugs.