À l’heure actuelle, le paludisme reste un problème de santé majeur et demeure une des maladies parasitaires les plus menaçantes. Parmi les cinq espèces de malaria infectant l’homme, Plasmodium falciparum est la forme la plus mortelle. Lors de la phase érythrocytaire de son cycle de vie, causant tous les symptômes du paludisme, P.falciparum utilise les phospholipides pour créer les membranes nécessaires au développement de cellules filles. Chez P. falciparum, la phosphatidylcholine est principalement obtenue grâce à la voie de synthèse de novo, dite voie de Kennedy. Dans cette voie de biosynthèse, la seconde étape catalysée par la CTP:phosphocholine cytidylyltransferase [EC 2.7.7.15] est limitante et apparait essentielle pour la survie du parasite murin P. berghei lors de la phase sanguine. Les objectifs de mon travail de thèse ont été de caractériser structuralement cette enzyme et d’identifier des effecteurs, principalement grâce à des approches de « fragment-based drug design » (FBDD). Ainsi, la première structure cristalline du domaine catalytique de l’enzyme (PfCCT) a été déterminée avec une résolution de 2.2 Å. De plus, les structures de trois complexes enzyme-substrat (en présence de CMP, de phosphocholine ou de choline) et d’un complexe enzyme-produit (CDP-Choline) ont été déterminées. Ces structures cristallographiques apportent des informations détaillées sur la poche de liaison de l’enzyme et elles ont révélé des informations sur le mécanisme de la réaction catalytique à l’échelle atomique. La seconde partie de ma thèse présente les méthodes développées pour identifier des inhibiteurs potentiels de la PfCCT. Une approche de FBDD a été utilisée pour identifier et sélectionner de petites molécules (fragments, PM<300 Da) se liant à la PfCCT. Diverses techniques biophysiques (fluorescence-based thermal shift assay, différence de transfert de saturation par RMN, dénaturation chimique isotherme) ont permis la sélection de 23 fragments à partir du criblage d’une bibliothèque (~ 300 molécules). En parallèle, un criblage in silico de plus grandes bibliothèques de fragments (environ 15 000 composés) a permis d’identifier 100 fragments “hits”. Enfin, 5 composés déjà connus pour inhiber la croissance parasitaire (Malaria Box fournit par Medecines for Malaria Venture) ont été sélectionnés pour leur inhibition de l’activité de la PfCCT recombinante. L’ensemble de ces données ouvre la voie pour l’élaboration de futurs composés ciblant la PfCCT et inhibant la biosynthèse de phosphatidylcholine chez P. falciparum. / Malaria remains a major global health problem and the most threatening parasitic disease. Among the 5 malaria species that affect humans, Plasmodium falciparum is the most deadly form. During its life cycle, in erythrocytic stage, which causes all the malaria symptoms, P. falciparum relies on phospholipids to build the membranes necessary for daughter cell development. Approximately 85% of parasite phospholipids consist of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) synthesized by the parasite through the de novo Kennedy pathways. In the pathway of phosphatidylcholine biosynthesis, the second step catalyzed by CTP:phosphocholine cytidylyltransferase [EC 2.7.7.15] is rate limiting and appears essential for the parasite survival at its blood stage. In this PhD thesis I focus on the structural characterization of this enzyme and the identification of effectors mainly by fragment-based drug design approach (FBDD). The first reported crystal structure of the catalytic domain of the enzyme target (PfCCT) has been solved at resolution 2.2 Å. Four other crystal structures of PfCCT in complex with substrates (CMP, phosphocholine and choline) or product (CDP-choline) have been determined. These structural data give detailed images of the binding pocket and reveal the enzyme structures at all catalytic steps that provide crucial information on the catalytic mechanism at atomic level. The second part of the project present the methods developed to identify potential PfCCT inhibitors. A FBDD approach was used in order to identify and select small molecules (fragments, MW< 300 Da) binding to the PfCCT. A combination of biophysical techniques (fluorescence-based thermal shift assay, saturation transfer difference NMR and isothermal chemical denaturation) allowed the selection of 23 fragment hits from the screenings of fragment library (~ 300 molecules). In parallel in silico screening of larger fragment libraries (~15,000 compounds) resulted in 100 selected hits. Finally, 5 compounds already known to inhibit parasite growth (Malaria Box from Medicines for Malaria Venture) were selected for their inhibition of the recombinant PfCCT activity. The results obtained within this thesis brought important knowledge and structural insights on the catalytic mechanism of PfCCT. Taken together, these results pave the way for future structure-based drug design to target PfCCT and to inhibit the essential phosphatidylcholine biosynthesis in P. falciparum.

Le contrôle du paludisme ne semble aujourd'hui envisageable que par stratégies combinées ciblant différents stades du parasite. Chez le vecteur, certains mécanismes de la réponse immunitaire pourraient être manipulés pour bloquer le développement sporogonique du parasite. Cette thèse examine les effets de la diversité du vecteur et du parasite dans le couple le plus important en termes d'épidémiologie, A. gambiae - P. falciparum. Des polymorphismes de gènes de l'immunité du moustique contrôlant le niveau d'infection ont été identifiés par étude d'association. Certains d'entre eux ont un effet spécifique selon les isolats de parasites, suggérant de potentielles interactions génotype X génotype. Nous avons déterminé un déséquilibre de liaison très bas dans les populations naturelles de vecteurs, validant notre approche par gènes candidats. Les caractéristiques et les forces évolutives faisant d'A. gambiae un vecteur du paludisme majeur sont discutées. Les diverses populations de vecteurs et parasites peuvent interagir de manière spécifique. Pour tester cela, des infections par des isolats de parasites sympatriques et allopatriques ont été comparées, montrant des intensités plus faibles dans les couples sympatriques. Les profils d'expression des gènes montrent cependant peu de régulations spécifiques aux populations, mais plutôt des différences extrêmes selon les isolats de parasites. Ces résultats suggèrent des effets importants de la diversité entre populations et individus. En conclusion, cette thèse souligne l'importance de la prise en compte de la diversité naturelle des vecteurs et parasites dans les recherches futures sur leurs interactions. / To achieve malaria control a variety of approaches must be combined targeting different stages of the parasites life cycle. With better understanding of mosquito immunity, it is hoped that aspects of natural resistance can be manipulated to prevent parasite development. This thesis investigates the effect of both mosquito and parasite diversity on the mosquitoes response to malaria using the most important human malaria system; Anopheles gambiae-Plasmodium falciparum in natural/semi-natural conditions. Mosquito loci are identified that significantly control infection phenotype, some of which act in a parasite isolate specific manner, highlighting their potential involvement in genotype by genotype interactions. Such research is moving towards genomewide studies; however, on finding very low linkage disequilibrium in wild mosquitoes, it favors candidate gene association studies. A. gambiae characteristics that make it such a good malaria vector are discussed and the evolutionary forces driving these traits. Selection behind vector-parasite interactions can differ spatially and temporally causing specificities in sympatric couples. Sympatric and allopatric mosquito infections with malaria are compared, showing that sympatric infections develop lower infection intensities suggesting local adaptation. Mosquito gene expression profiles highlight a small number of genes differentially regulated between sympatric and allopatric infections, however extreme differences in gene regulation are observed within populations, probably driven by the variable nature of malaria parasites. This thesis highlights the importance of taking into account natural diversity in future research.

Résumé : La biologie évolutive permet de comprendre et de retracer l'origine des espèces ou des populations, de comprendre leurs dispersions dans différentes zones et d'analyser les différentiations résultant de ces évolutions. L'invasion biologique et les espèces envahissantes en général sont de bons modèles pour étudier et comprendre l'adaptation à de nouveaux environnements. Plasmodium falciparum, un protozoaire parasite agent du paludisme, a envahit de nouvelles populations hôtes et de nouvelles espèces de vecteurs à plusieurs reprises. Notre objectif était d'étudier (i) l'introduction, l'origine et la distribution de P. falciparum dans des environnements radicalement différents : dans une nouvelle aire géographique tout d'abord (en Amérique du Sud) puis dans une nouvelle espèce hôte (chez les primates) et (ii) de déterminer les gènes potentiellement impliqués dans l'adaptation à ces nouveaux environnements. Ces questions ont été abordées à travers différentes approches réunissant des analyses de génétique des populations, de phylogéographie ainsi que des analyses phylogénétiques. Les résultats obtenus démontrent pour la première fois que, P. falciparum a été introduit par l'homme au moins à deux reprises en Amérique du Sud à partir de l'Afrique. Cette thèse a aussi permis de démontrer pour la première fois que ce parasite circule naturellement chez les primates non-humains. L'analyse des patrons de sélection sur des gènes candidats jouant un rôle dans l'invasion des hématies par le parasite a été réalisée afin de déterminer si des évolutions adaptatives particulières avaient opérées sur ces gènes dans ces nouveaux environnements. L'ensemble de nos résultats démontrent que P. falciparum peut être considéré comme une espèce envahissante et que ce parasite n'est en fait pas spécifique à l'homme. L'ensemble de notre travail nous a permis d'avancer dans la connaissance de ce modèle biologique en termes de stratégie d'émergence ou de réémergence dans différents environnements. Nos résultats soulignent les changements qui ont opéré dans la distribution géographique et l'émergence du spectre d'hôte utilisé par P. falciparum au cours de son histoire évolutive passée et présente ce qui peut laisser craindre d'autres évolutions à l'avenir.Mots clés : Invasion biologique, espèce invasive, parasite, origine, adaptation, sélection, maladies émergente, Plasmodium falciparum, Amérique du Sud, génétique des populations, phylogéographie, marqueurs moléculaires, singes. / Abstract: The evolutionary biology allows to understand and to trace the origin of species or populations, to understand their dispersions in different areas and analyse the resulting differentiation of these developments. The biological invasion and invasive species, in general, are good models to study and understand the adaptation to new environments. Plasmodium falciparum, a protozoan parasite, agent of the malaria, invades a new host and new vector species at several times. The objective of this thesis was to analyse (i) introduction, origin and distribution of P. falciparum in radically different environments; first, a new geographical area (South America); second, a new host species (in primates); and (ii) identify genes potentially involved in adaptation to new environments. I addressed these questions using different approaches, including population genetics, phylogeographic analyses, and also phylogenetic analyses. The results demonstrate for the first time, P. falciparum has been introduced by humans at least twice in South America from Africa. This thesis has also demonstrated for the first time that this parasite circulates naturally in nonhuman primates. The analysis of the patterns of the selection on candidate genes play a role in the invasion of erythrocytes by the parasite was performed to determine if adaptive evolutions were occur on these specific genes in these new environments. Overall, our results demonstrate that P. falciparum can be considered an invasive species and that parasite is not specific to humans. All of our work allowed us to advance in the knowledge of the biological model in terms of strategy emergence or reemergence in different environments. Our results highlight the changes that have taken place in the geographical distribution and the emergence of host range used by P. falciparum during its evolutionary history, past and present which may raise concerns of other developments in the future. Keywords : Biological invasion, invasive species, parasite, origin, adaptation, selection, emerging infectious diseases, Plasmodium falciparum, South America, population genetics, phylogeography, molecular markers, apes.

RNAs não codificantes (ncRNAs) têm ganho crescente prestígio nos últimos anos devido a recentes e contínuas descobertas revelando sua diversidade e importância. Porém, a identificação dessas moléculas ainda é um problema em aberto. Em particular, Plasmodium falciparum é um desafio para a pesquisa de ncRNAs, onde poucos foram identificados até o momento. P. falciparum é o parasita que causa uma malária humana letal. A descoberta de novos ncRNAs neste organismo pode auxiliar no desenvolvimento de novos tratamentos. Este trabalho faz um estudo sobre técnicas computacionais para a predição de ncRNAs e, utilizando como objeto de estudo P. falciparum, propõe uma metodologia de predição que seja aplicável inclusive a genomas com viés composicional. A ênfase deste estudo foi a predição de ncRNAs família-específicos, utilizando o componente RNA da telomerase como objeto de estudo. Este é um importante RNA que, devido à sua alta taxa de mutação, é de difícil identificação. Este RNA ainda não foi identificado em P. falciparum. No entanto, evidências biológicas indicam que este RNA é presente, funcional e deve ser essencial ao parasita, caracterizando-se como um alvo de drogas. Além disso, foi realizado um trabalho preliminar sobre a predição de ncRNAs em geral em P. falciparum utilizando uma abordagem comparativa. / Noncoding RNAs (ncRNAs) have been receiving increasing prestige in the last years due to recent and continuous discoveries revealing their diversity and importance. However, the identification of these molecules is still an open problem. In particular, Plasmodium falciparum is a challenge for the ncRNA research, in which few ncRNAs have been identified. P. falciparum is the parasite that causes a lethal human malaria. The discovery of new ncRNAs in this organism may help in the development of new treatments. This work does a research of computational techniques for the ncRNA prediction and, by using P. falciparum as target, proposes a prediction methodology which is also applicable to compositionally biased genomes. The emphasis of this study was the prediction of family-specific ncRNAs, by using the telomerase RNA component as target. This is an important RNA that has a high mutation rate, being difficult to predict. This RNA has not been identified in P. falciparum, yet. However, biological evidences indicate this RNA is present, functional and might be essential for the parasite, being a drug target. In addition, this work presents preliminary results about the prediction of general ncRNAs in P. falciparum by using a comparative approach.

É proposto que vias de sinalização controlem a sobrevivência e adaptação do Plasmodium, nos diferentes hospedeiros. No presente trabalho buscamos por diferentes abordagens estudar a via de sinalização de melatonina em P. falciparum. Para isso, avaliamos os níveis de RNA mensageiro de genes do sistema-ubiquitina proteossomo (UPS) bem como o perfil de ubiquitinação resultante do tratamento de parasitas com melatonina. Mostramos que a proteína quinase 7 de P. falciparum (PfPK7) atua na modulação dos genes do UPS em resposta a melatonina. Avaliamos também se o parasita é responsivo ao ácido indol-3-acético (AIA). Sabendo-se da importância de receptores de membrana na regulação de diversas funções celulares incluindo a percepção do meio externo, buscamos caracterizar um receptor serpentino putativo identificado previamente pelo grupo. Pudemos concluir que a via de sinalização por melatonina em P. falciparum envolve a participação da PfPK7, uma vez que em parasitas nocautes para pfpk7 são irresponsivos à melatonina quando comparados ao parental. / It is proposed that signaling pathways can control the parasite survival and adaptation into the hosts. In the present work we inquire about to study the melatonin signaling pathway trhough different metodologies. For this purpose we have analized post-translational modification of melatonin signaling, through ubiquitin-proteasome system (UPS) mRNA levels as well as the profile of ubiquitination resulted of melatonin treatment when compared with control. Moreover, we have found here that the P. falciparum protein kinase 7 (PfPK7) plays a major role in ubiquitin-proteasome system mRNA modulation in response to melatonin since parasites knockout to pfpk7 gene do not upregulate the UPS genes in response to melatonin. As for melatonin we have evaluated if P. falciparum parasites were responsive to indoleacetic acid. Last but not least, we made an effort to characterize a putative serpentine receptor previously identified by our group. We conclude that melatonin signaling pathway involves PK7 participation since pfpk- parasites are irresponsives to melatonin.

A detecção microscópica de gametócitos pode subestimar sua prevalência e induzir uma avaliação errônea do potencial de transmissão da malária em áreas endêmicas, mantendo desconhecida a proporção de indivíduos infectados que são potenciais transmissores da doença. Este trabalho teve como objetivo a padronização de métodos moleculares para detecção e quantificação de gametócitos de P. falciparum e P. vivax. Descrevemos um método de qRT-PCR, que tem como alvo transcritos do gene pvs25 e pfs25. Detectamos transcritos de pvs25 em 96,4% das amostras sanguíneas provenientes de indivíduos infectados por P. vivax. qRT-PCR foi mais sensível do que a RT-PCR convencional, que tem como alvo o mesmo gene. Descobrimos que a maioria (61,9%) dos portadores de gametócitos são assintomáticos ou tem parasitemias subpatentes e não teriam sido detectados pelas estratégias de controle de rotina da malária. Porém, esses portadores de gametócitos não identificados, geralmente possuem baixas densidades de gametócitos e contribuem com até 4% da carga global de gametócitos na comunidade. / The microscopic detection of gametocytes may underestimate their prevalence, leading to an inaccurate assessment of the potential for malaria transmission in receptive areas and a poor estimate of the proportion of infected individuals who are infectious. We aimed to standardize molecular methods for detection and quantification of gametocytes of Plasmodium falciparum and Plasmodium vivax. Here, we describe a qRT-PCR that targets transcripts of the mature gametocyte-specific pvs25 gene. We found mature gametocytes in 53 of 55 (96.4%) P. vivax infections diagnosed during an ongoing cohort study in a farming settlement located in the southern of Amazonas state. SYBR green qRT-PCR was more sensitive than a conventional RT-PCR that targets the same gene. Most (61.9%) gametocyte carriers were either asymptomatic or had subpatent parasitemias, and would have been missed by routine malaria control strategies. However, potentially undiagnosed gametocyte carriers usually had low-density infections and contributed up to 4% to the overall gametocyte burden in the community.

Etliche Membran Transport Proteine (MTP) sind essentiell in den Plasmodium Blutstadien, und geraten zunehmend in den Fokus der Wirkstoffentwicklung. Die physiologischen Rollen der Transporter sind jedoch oft ungeklärt. In dieser Arbeit wurden mittels experimenteller Genetik funktionelle Charakteristika der MTPs untersucht. Am Maus Parasiten Plasmodium berghei und der Plasmodium falciparum Blutstadien-Kultur wurden vier MTPs ausgewählt: ein konservierter Folat Transporter (FT2), sowie eine P. falciparum-spezifisches P-Typ ATPase und zwei essentielle MTPs (CRT und ATP4). Diese Auswahl verkörpert ein breites Spektrum an MTP Kandidaten und reflektieren zudem das Potenzial und die Grenzen funktioneller Analysen von Plasmodium MTPs mittels reverser Genetik. Für den Folat Transporter 2 (FT2) wurde eine Kombination von transgenen Strategien auf P. berghei angewandt. Durch ein endogenes tag von FT2 wurde die Lokalisierung im Apicoplast, sowie dessen Expression über fast den kompletten Zyklus hinweg gezeigt. Nach der Deletion von FT2, wiesen die Parasiten einen Defekt während der Sporulation auf. Demzufolge bilden sich nur nicht infektiöse Sporozoiten, was letztendlich zur Unterbrechung des Lebenszyklus der Parasiten führt. Eine Aminophospholipid P-Typ ATPase, wurde mittels CRISPR/Cas9 in P. falciparum genetisch deletiert und die Mutante analysiert. Im Gegensatz zu den meisten vitalen P-Typ ATPasen erweist sich das Gen in den asexuellen Blutstadien als entbehrlich. Des Weiteren bilden die MTPs ATP4 und CRT einen einflussreichen Faktor bei Malaria-Therapien. Eine umfassende Analyse von räumlichen und zeitlichen Expressionsmustern von transgenen Parasiten mit mCherry-getaggten Proteinen zeigt ein Expression der beiden MTPs über die Blutstadien hinaus, was auf zusätzliche Funktionen in den jeweiligen Stadien verweist. Diese Studie trägt, basierend auf Lokalisation, Expression und funktioneller Deletion, zur funktionellen Entschlüsselung der vier untersuchten MTPs bei. / Many membrane transport proteins (MTP) are essential for Plasmodium infection and gain importance as candidate drug targets in malaria therapy, whereas the physiological functions often remain enigmatic. In this thesis, we applied experimental genetics to determine key characteristics of four Plasmodium MTPs. We employed the murine malaria model parasite Plasmodium berghei and in vitro blood cultures of Plasmodium falciparum. We selected one conserved MTP called FT2, which was previously shown to transport folate, a P-type ATPase that is specific for P. falciparum as well as two essential MTPs, CRT and ATP4. These targets exemplify the range of druggable candidates and illustrate the potential and limitations of reverse genetics to decipher their physiological roles. A combination of transgenic and knockout strategies was applied to the P. berghei folate transporter 2 (FT2). We show that endogenously tagged FT2 localises to the apicoplast membranes, and is broadly expressed throughout the parasite’s life cycle. Analysis of FT2-deficient parasites revealed a severe sporulation defect in the vector; the vast majority of ft2– oocysts form large intracellular vesicles which displace the cytoplasm. Very few sporozoites are generated and these are non-infectious to the mammalian host, resulting in a complete arrest of Plasmodium transmission. A candidate aminophospholipid P-type ATPase, was assessed by a CRISPR/Cas9-mediated gene disruption. Compared to many vital P-type ATPases this gene is dispensable for asexual blood replication. Two MTPs, ATP4 and CRT are prime targets for antimalarial therapies. A comprehensive spatio-temporal expression analysis of transgenic parasites expressing mCherry-tagged proteins revealed expression beyond blood infection, indicative of functions in additional parasite stages. The findings of this study contribute towards a better understanding of the roles of the four MTPs based on localisation, expression and functional deletion.

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.

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

Ein Kennzeichen der Plasmodium-Infektion ist die Induktion von strukturellen Veränderungen der Membranen der Wirtszelle. Die Parasiten bilden sich Membranstrukturen im Zytoplasma der Wirtszelle aus. Die Maurersche Fleckung ist einer der am besten charakterisierten Struktur von Plasmodium falciparum, die für den Transport von Virulenzfaktoren an die Oberfläche der infizierten Erythrozytenmembran wichtig ist. Plasmodium berghei, eine Nagetier-infizierende Spezies, bildet ähnliche Strukturen aus, die als intra-erythrozytäre P. berghei induzierte Strukturen (IBIS) bezeichnet werden. In beiden Strukturen können Proteine gefunden werden, die für die Sequestrierung von infizierten roten Blutkörperchen innerhalb des Wirts verantwortlich sind. Zwei P. berghei Proteine, IPIS2 und IPIS3, sind im Leber- und Blutstadium der Infektion präsent. Im Blutstadium werden sie in die IBIS-Kompartimente exportiert. Das speziesübergreifende Vorkommen von IPIS2 und IPIS3 deutet darauf hin, dass sie konservierte Funktionen haben könnten, die für das intrazelluläre Wachstum von Plasmodium wichtig sind. Diese Arbeit beschreibt Rollen für beide Proteine an der Wirts-Parasit Schnittstelle während der Infektion. Um einen Einblick in die induzierten Veränderungen zu erhalten, wurde die Infektion von P. berghei in Abwesenheit von IPIS2 beziehungsweise IPIS3 charakterisiert. Darüber hinaus interessierte uns auch, wie sich die Orthologe von IPIS2- und IPIS3 in den Blutstadien von P. knowlesi verhalten. Darüber hinaus zeigt diese Arbeit, dass die Deletion von entweder IPIS2 oder IPIS3 zu leicht reduzierten Wachstumsraten der Parasiten im Blut infizierter Tiere führte und die Anzahl der zirkulierenden Schizonten im peripheren Blut erhöhte, während das Leberstadium unbeeinflusst blieb. Zusammengenommen deuten diese Daten darauf hin, dass IPIS2 und IPIS3 für den Wirtszell-Umbau erforderlich sind, der für die effiziente Sequestrierung von infizierten Erythrozyten aus dem Blutkreislauf verantwortlich ist. / One characteristic of Plasmodium infection is the induction of structural changes in the membranes of the host cell. The parasites form membrane structures in the cytoplasm of the host cell. Maurer's clefts are one of the best characterised structures of Plasmodium falciparum, which are important for the transport of virulence factors to the surface of the infected erythrocyte membrane. Plasmodium berghei, a rodent-infecting species, forms similar structures called intra-erythrocyte P. berghei induced structures (IBIS). In both structures, proteins can be found that are responsible for sequestering infected red blood cells within the host. Two P. berghei proteins, IPIS2 and IPIS3, are present in the liver and blood stages of infection. In the blood stage, they are exported to the IBIS compartments. The cross-species presence of IPIS2 and IPIS3 suggests that they may have conserved functions important for Plasmodium intracellular growth. This work describes roles for both proteins at the host-parasite interface during infection. To gain insight into the induced changes, infection of P. berghei was characterized in the absence of IPIS2 and IPIS3, respectively. Furthermore, we were also interested in how the orthologues of IPIS2- and IPIS3 behave in the blood stages of P. knowlesi. Furthermore, this work shows that deletion of either IPIS2 or IPIS3 resulted in slightly reduced parasite growth rates in the blood of infected animals and increased the number of circulating schizonts in the peripheral blood, while the liver stage was unaffected. Taken together, these data suggest that IPIS2 and IPIS3 are required for host cell remodeling, which is responsible for the efficient sequestration of infected erythrocytes from the bloodstream.