abstract: The complex life cycle and widespread range of infection of Plasmodium parasites, the causal agent of malaria in humans, makes them the perfect organism for the study of various evolutionary mechanisms. In particular, multigene families are considered one of the main sources for genome adaptability and innovation. Within Plasmodium, numerous species- and clade-specific multigene families have major functions in the development and maintenance of infection. Nonetheless, while the evolutionary mechanisms predominant on many species- and clade-specific multigene families have been previously studied, there are far less studies dedicated to analyzing genus common multigene families (GCMFs). I studied the patterns of natural selection and recombination in 90 GCMFs with diverse numbers of gene gain/loss events. I found that the majority of GCMFs are formed by duplications events that predate speciation of mammal Plasmodium species, with many paralogs being neutrally maintained thereafter. In general, multigene families involved in immune evasion and host cell invasion commonly showed signs of positive selection and species-specific gain/loss events; particularly, on Plasmodium species is the simian and rodent clades. A particular multigene family: the merozoite surface protein-7 (msp7) family, is found in all Plasmodium species and has functions related to the erythrocyte invasion. Within Plasmodium vivax, differences in the number of paralogs in this multigene family has been previously explained, at least in part, as potential adaptations to the human host. To investigate this I studied msp7 orthologs in closely related non-human primate parasites where homology was evident. I also estimated paralogs’ evolutionary history and genetic polymorphism. The emerging patterns where compared with those of Plasmodium falciparum. I found that the evolution of the msp7 multigene family is consistent with a Birth-and-Death model where duplications, pseudogenization and gene lost events are common. In order to study additional aspects in the evolution of Plasmodium, I evaluated the trends of long term and short term evolution and the putative effects of vertebrate- host’s immune pressure of gametocytes across various Plasmodium species. Gametocytes, represent the only sexual stage within the Plasmodium life cycle, and are also the transition stages from the vertebrate to the mosquito vector. I found that, while male and female gametocytes showed different levels of immunogenicity, signs of positive selection were not entirely related to the location and presence of immune epitope regions. Overall, these studies further highlight the complex evolutionary patterns observed in Plasmodium. / Dissertation/Thesis / Doctoral Dissertation Biology 2016

Malaria, babesiosis, trypanosomosis, and leishmaniasis are some of the most life-threatening parasites, but the range of drugs to treat them is limited. An effective, safe, and low-cost drug with a large activity spectrum is urgently needed. For this purpose, an aryl amino alcohol derivative called Alsinol was resynthesized, screened in silico, and tested against Plasmodium, Babesia, Trypanosoma, and Leishmania. In silico Alsinol follows the Lipinski and Ghose rules. In vitro it had schizontocidal activity against Plasmodium falciparum and was able to inhibit gametocytogenesis; it was particularly active against late gametocytes. In malaria-infected mice, it showed a dose-dependent activity similar to chloroquine. It demonstrated a similar level of activity to reference compounds against Babesia divergens, and against promastigotes, and amastigotes stages of Leishmania in vitro. It inhibited the in vitro growth of two African animal strains of Trypanosoma but was ineffective in vivo in our experimental conditions. It showed moderate toxicity in J774A1 and Vero cell models. The study demonstrated that Alsinol has a large spectrum of activity and is potentially affordable to produce. Nevertheless, challenges remain in the process of scaling up synthesis, creating a suitable clinical formulation, and determining the safety margin in preclinical models. / Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS) / Revisión por pares

Adaptive phenotypic plasticity, the ability of a genotype to give rise to different phenotypes in different environments, evolves to allow organisms to fine-tune their life-history traits according to the varying conditions they encounter during their lives. Reproductive investment - the manner in which organisms divide their resources between survival and reproduction - is well studied in evolutionary ecology because it is a key determinant of fitness. However, whilst plasticity in reproductive effort is well understood for free-living multicellular taxa (such as insects, birds, and mammals), the application of evolutionary theory for plasticity and life history strategies to unicellular parasites and pathogens is lacking. In this thesis, I use empirical and theoretical approaches to uncover how differential resource allocation to non-replicating, sexual stages (gametocytes) versus asexually replicating stages can be harnessed by the rodent malaria parasite Plasmodium chabaudi to maximise its fitness across the often very variable conditions it encounters during infections. Differential allocation between those stages is equivalent to the fundamental life-history trade-off between survival and reproduction because gametocytes are responsible for between-host transmission (i.e. reproduction of the infection) whereas asexual parasites mediate host exploitation and within-host survival. A suite of within-host models reveal that malaria parasites could gain considerable fitness benefits in the face of low levels of drug treatment if they reduce their investment into gametocyte production ("reproductive restraint"), thereby assuring the continuity of the infection and capitalising on opportunities for future transmission. In contrast, high levels of drug treatment typically select parasites to commit all of their resources to gametocyte production ("terminal investment"), to escape a host that does not offer much opportunity for future transmission. My experiments reveal that P. chabaudi increases both its reproductive investment and its asexual replication rate in anaemic hosts (i.e. host that have a low density of red blood cells), suggesting that parasites profit from host anaemia and can afford high investment in gametocytes ("affluent investment"). I also uncover plasticity in a number of traits that underpin asexual replication rate, including invasion preference for different ages of red blood cells, but it is plasticity in the number of progeny (merozoites) per infected cell that is the main contributor to asexual replication rate. My experiments also reveal genetic variance in plasticity of the life-history traits investigated, which has profound implications for their evolution. Furthermore, plastic modification of these traits is associated with minimal costs or constraints, so that parasites can rapidly match life-history traits appropriately to the within-host environment. Severe anaemia is one of the deadliest symptoms of malaria, so observing that virulence and infectiousness increases in anaemic hosts has also fundamental clinical implications. Finally, the empirical and theoretical observations of affluent investment, reproductive restraint and terminal investment match theoretical predictions of how organisms should behave in varying environments, confirming P. chabaudi as a useful model system to test life-history theory.

Ce travail avait pour objectif : 1) évaluer le portage de gamétocytes et leur génotype avant et après le traitement d’une part, et d’étudier leur infectivité ; 2) exprimer le domaine catalytique (PfMCA1-cd-Sc) de la métacaspase de Plasmodium falciparum (PfMCA1) chez la levure et 3) tester in vitro l’activité antiplasmodiale de nouvelles molécules synthétiques dérivées des pyrano et ferro-quinoléines sur des clones de laboratoire 3D7 et Dd2. Pour cela, le test in vivo de 28 jours de l’OMS, les marqueurs moléculaires de résistance et le « direct feeding » ont été utilisés pour le premier objectif. La culture des levures, l’expression des protéines de la métacaspase 1 de Plasmodium falciparum, le western blot, le test de prolifération et de survie, et les marqueurs de mort cellulaire ont servi pour le second objectif et enfin, la culture parasitaire et tests in vitro par la méthode de fluorimétrie au Sybr Green I ont permis l’évaluation de l’activité antiplasmodiale de nouvelles molécules. Nous avons démontré que les gamétocytes post-traitement étaient porteurs de mutations ponctuelles et plus infectants dans le groupe chloroquine ; que l’expression hétérologue du domaine catalytique de la métacaspase de Plasmodium falciparum (PfMCA1) dans la levure Saccharomyces cerevisiae entraînait une mort clonale de type apoptotique et un retard de croissance dépendant de l’activité VAD-Protéase et enfin, que les substitues aromatiques à base de pyrimidine ou de benzylméthylamine ferrocène révèlent une activité satisfaisante par rapport à la méthoxyéthylidene sur les clones 3D7 et Dd2. / Plasmodium species use programmed cell death for the survival of their offspring as some prokaryotic parasites. This study was designed to - assess the gametocytes carrier and their genotypes before/ after treatment and studying their infectivity; - express the catalytic domain (PfMCA1-cd-Sc) of Plasmodium falciparum metacaspase (PfMCA1) in yeast; - Test the “in vitro” anti-plasmodial activity of pyrano and ferro- quinolines derived new synthetic molecules on 3D7 and Dd2 Chloroquine laboratory clones. The 28-day “in vivo” WHO test, molecular markers of resistance and direct feeding; yeast culture, protein expression of P. falciparum metacaspase 1, Western blot, proliferation and survival test, and cell death markers were used to achieve the first two objectives while parasite culture and in vitro tests by the method of fluorimetry in SYBR Green I was used to evaluate the anti-plasmodial activity of new molecules. Results show that post-treatment gametocytes were carriers of point mutations and the most infective in the Chloroquine group. The heterologous expression of PfMCA1 catalytic domain in Saccharomyces cerevisiae resulted in apoptotic clonal death and growth retardation activity-dependent Protease-VAD, showing the involvement of PfMCA1 in the process of cell death. The aromatic substitutes with pyrimidine or benzyldimethylamine ferrocene residues showed satisfactory activity against the methoxyethylidene on 3D7 and Dd2. The data suggest that the structural optimization of these compounds based on pyrimidine and ferrocene is more interesting from the standpoint anti-plasmodial activity for candidate molecules in the near future.

The in vitro production of gametocytes and oocysts of the apicomplexan parasite genus Eimeria is still a challenge in coccidiosis research. Until today, an in vitro development of gametocytes or oocysts had only been shown in some Eimeria species. For several mammalian Eimeria species, partial developments could be achieved in different cell types, but a development up to gametocytes or oocysts is still lacking. This study compares several permanent cell lines with primary fetal cells of the black rat (Rattus norvegicus) concerning the qualitative in vitro development of the rat parasite Eimeria nieschulzi. With the help of transgenic parasites, the developmental progress was documented. The selected Eimeria nieschulzi strain constitutively expresses the yellow fluorescent protein and a macrogamont specific upregulated red tandem dimer tomato. In the majority of all investigated host cells the development stopped at the second merozoite stage. In a mixed culture of cells derived from inner fetal organs the development of schizont generations I-IV, macrogamonts, and oocysts were observed in crypt-like organoid structures. Microgamonts and microgametes could not be observed and oocysts did not sporulate under air supply. By immunohistology, we could confirm that wild-type E. nieschulzi stages can be found in the crypts of the small intestine. The results of this study may be helpful for characterization of native host cells and for development of an in vitro cultivation system for Eimeria species.

The in vitro production of gametocytes and oocysts of the apicomplexan parasite genus Eimeria is still a challenge in coccidiosis research. Until today, an in vitro development of gametocytes or oocysts had only been shown in some Eimeria species. For several mammalian Eimeria species, partial developments could be achieved in different cell types, but a development up to gametocytes or oocysts is still lacking. This study compares several permanent cell lines with primary fetal cells of the black rat (Rattus norvegicus) concerning the qualitative in vitro development of the rat parasite Eimeria nieschulzi. With the help of transgenic parasites, the developmental progress was documented. The selected Eimeria nieschulzi strain constitutively expresses the yellow fluorescent protein and a macrogamont specific upregulated red tandem dimer tomato. In the majority of all investigated host cells the development stopped at the second merozoite stage. In a mixed culture of cells derived from inner fetal organs the development of schizont generations I-IV, macrogamonts, and oocysts were observed in crypt-like organoid structures. Microgamonts and microgametes could not be observed and oocysts did not sporulate under air supply. By immunohistology, we could confirm that wild-type E. nieschulzi stages can be found in the crypts of the small intestine. The results of this study may be helpful for characterization of native host cells and for development of an in vitro cultivation system for Eimeria species.