Ascaris lumbricoides infects at least 10% of the world's population and is a public health issue in most low-to-middle income countries. Survival of this parasite in its host is mediated at least in part by materials exported to the host in secretions. Although very little is known about the composition of these secretions, defining their contents and functions could shed light on the host-parasite interactions that lead to parasite establishment and persistence in the host. Ascaris suum, a parasite of pigs, was used as a model organism because its genome has been sequenced and it is closely related to the human parasite, A. lumbricoides. Excretory/secretory products (ESP), uterine fluid (UF) and perienteric fluid (PE) were collected from adult A. suum. Proteins isolated from these compartments were subjected to LC-MS/MS and analyzed using bioinformatic tools. The ESP fraction included many proteins also present in UF. Proteins found in ESP but not in UF had a considerably different categorical composition than PE or UF, which are similar to each other. We conclude from these data that proteins exported through the secretory apparatus have distinct patterns of biological function and that UF proteins are likely derived from PE. In addition, ESP from A. suum was compared to ESP from Brugia malayi and ESP from Heligmosomoides polygyrus in terms of protein composition. We concluded that A. suum secretome is conserved through both phylogeny and predilection site. / Ascaris lumbricoides infecte au moins 10% de la population mondiale et est une problématique de santé publique dans les pays en voie de développement. La survie de ce parasite dans son hôte est médiée d'une part par des substances exportées à son hôte par voies de secrétions. Bien que peu d'informations soient connues sur la composition de ces substances, définir leur contenu ainsi que leurs fonctions pourraient aider à clarifier la relation entre le parasite et son hôte. Ascaris suum est un parasite du porc utilisé comme organisme-modèle en raison de son génome séquencé et de sa similarité morphologique avec le parasite de l'homme, A. lumbricoides. Les produits de secrétions/excrétions (PSE), le fluide perientérique (FPE) et le fluide utérin (FU) ont été obtenus des femelles adultes d'A. suum. Les protéines contenues dans ces fluides ont été isolées et soumises à LC-MS/MS et ont ensuite été soumises à des analyses bioinformatiques. Une fraction de PSE inclut plusieurs protéines qui se trouvent aussi dans FU. Les protéines trouvées dans les PSE, mais absentes du FU, avaient une composition catégorique différente comparée aux FPE et au FU, lesquels montraient une composition similaire. Nous concluons par ces résultats que les protéines exportées par l'appareil de sécrétion ont des motifs distincts en termes de fonctions biologiques et que les protéines du FU sont dérivées du FPE. De plus, le PSE d' A. suum a été comparé au PSE de Brugia malayi et au PSE de Heligmosomoides polygyrus. Nous avons conclu que le secretome d' A. suum est conservé à la fois par phylogénie et l'emplacement de l'infection dans l'hôte.

Parasites of the genus Leishmania cause a broad array of diseases, collectively termed leishmaniasis. These diseases range in morbidity; the cutaneous form is typically self healing, while the mucocutaneous and visceral manifestations require chemotherapeutic intervention to avoid lethality. At present there is no vaccine, and current methods of chemotherapeutic intervention have severe drawbacks, together creating a dire need for new options to combat these destructive diseases. An organelle within the parasite, the glycosome, has been identified as an attractive drug target. The glycosome compartmentalizes several enzymes from important biosynthetic and metabolic pathways, which has been shown to be necessary for the viability of the parasite. Although the organelle is structurally and evolutionarily related to peroxisomes of higher eukaryotes, the import machinery of the organelles differs significantly. The majority of proteins entering the glycosome contain a C-terminal PTS1 tri-peptide sequence, which is readily recognized and bound by the soluble cytosolic receptor LPEX5. The receptor binds PTS1 cargo in the cytosol, shuttling it to the glycosomal membrane where the protein interacts with LPEX14, a peripherally membrane bound protein. The interaction with LPEX14 at the glycosomal membrane, facilitated by several other biogenesis proteins, initiates the formation of a transient import pore. In this thesis research project the role of Leishmania donovani PEX5 (LdPEX5) in formation of this crucial import pore was analyzed. Using biophysical techniques, it was found that interaction of the receptor with a PTS1 did not cause major changes in secondary structure, although did provoke a conformational change in the protein, preceding and possibly facilitating its interactions with LdPEX14 at a glycosomal membrane. Using large unilamellar vesicles mimicking the glycosomal lipid composition, the domain of LdPEX5 necessary to interact with LdPEX14 was then narrowed to 268-302. Furthermore, using serial carbonate-urea extractions, the domain identified to be necessary for interaction with LdPEX14 at a glycosomal mimetic was also found to insert into the liposomal membrane, implying that the insertion of LdPEX14 into the glycosomal membrane could be drawing LdPEX5 into the membrane as part of pore formation. In conclusion, this study has implicated LdPEX5 in having a central role in formation of the transient glycosomal import pore. / Les parasites du genre Leishmania provoquent un large éventail de maladies, appelées collectivement leishmanioses. Ces maladies varient en termes de morbidité ; la forme cutanée se conclut généralement par une auto-guérison, alors que les manifestations cutanéo-muqueuses et viscérales nécessitent une intervention chimiothérapeutique pour éviter le décès. À l'heure actuelle, il n'existe aucun vaccin, et les méthodes actuelles d'intervention chimiothérapeutique présentent de graves conséquences. Il existe aujourd'hui un besoin urgent de trouver de nouvelles options pour lutter contre ces maladies destructrices. Un organite dans le parasite, le glycosome, a été identifié comme une cible thérapeutique intéressante. Le glycosome compartimente plusieurs enzymes de biosynthèses et voies métaboliques importantes; il a été prouvé que cet organite est nécessaire pour assurer la viabilité du parasite. Bien que l'organite soit structurellement et évolutivement lié aux peroxysomes des eucaryotes supérieurs, le mécanisme d'importation des organites diffère sensiblement. La majorité des protéines entrant dans le glycosome contient une séquence tri-peptide PTS1 C-terminal, qui est facilement reconnue et liée par le récepteur cytosolique soluble LPEX5. Le récepteur se lie au cargo PTS1 dans le cytosol, le conduisant vers la membrane glycosomale où la protéine interagit avec LPEX14, une protéine liée à la membrane périphérique. L'interaction avec LPEX14 au niveau de la membrane glycosomale, facilitée par plusieurs autres protéines de biogenèse, initie la formation d'un pore d'importation transitoire. Dans ce projet de thèse, le rôle de PEX5 dans la formation de ce pore d'importations essentiel a été analysé chez Leishmania donovani. En utilisant des techniques biophysiques, il a été constaté que l'interaction du récepteur avec un PTS1 n'a pas causé de changements majeurs dans la structure secondaire, bien qu'elle ait provoqué un changement de conformation de la protéine, précédant et éventuellement facilitant ses interactions avec LdPEX14 à une membrane glycosomale. Grâce à l'utilisation de grandes vésicules unilamellaires mimant la composition lipidique glycosomale, le domaine de LdPEX5 nécessaire pour interagir avec LdPEX14 fut ramené à 268-302. En outre, en utilisant des extractions carbonate-urée en série, il a été prouvé que le domaine identifié comme étant nécessaire pour l'interaction avec LdPEX14 au mimétique glycosomal, s'insère dans la membrane liposomale. De ce fait, l'insertion de LdPEX14 dans la membrane glycosomale pourrait tirer LdPEX5 dans la membrane dans le contexte de la formation de pores. En conclusion, cette étude a démontré que LdPEX5 possède un rôle central dans la formation du pore d'importation glycosomale transitoire.

The lethal form of human malaria is caused by the most prominent human protozoan parasite, Plasmodium falciparum, responsible for an estimated ~1 million deaths annually. Malaria treatment and, prevention heavily rely on antimalarial drugs. The synthetic substitute of quinine, chloroquine, was the most effective treatment for this disease. The rise of chloroquine resistant malaria in endemic areas has suppressed control efforts. Chloroquine resistance has been associated with mutations in a transmembrane protein on the digestive vacuole of the parasite, designated PfCRT. Moreover, the normal function(s) and natural substrate(s) of PfCRT remain a matter of speculation as direct evidence for the normal functions or substrates are yet to be determined. The objectives of this thesis are to develop and characterize two antisera to the N- and C termini of PfCRT as tools for the identification of PfCRT interacting proteins in P. falciparum. Although the molecular mass of PfCRT was estimated as 48.67 kDa, immunobiochemical characterizations using different antiserums raised against the N- and C-cytosolic domains of this protein throughout the published literature yielded varying molecular masses of PfCRT on SDS PAGE. In this thesis, we report the generation and biochemical characterization of two antisera raised against the N- and C-cytosolic domains of PfCRT. Our results show PfCRT-C antiserum to detect non-phosphorylated epitopes or sequences in PfCRT. Moreover, our high-resolution epitope mapping results confirm the specificity of PfCRT-C antiserum to a non-phosphorylated form of PfCRT and show that phosphorylation at two sites in PfCRT sequence, Ser411 and Thr416, prevent its binding to the full-length and phosphorylated protein. In addition, we have identified a novel truncated form of PfCRT that is both unphosphorylated, as it is recognized by PfCRT-C antiserum, and likely to represent a differentially spliced product of PfCRT, that is expressed in vivo on the parasite digestive vacuole. Furthermore, our findings confirm the molecular mass of the full-length and phosphorylated PfCRT as a 52 kDa protein band on SDS PAGE. Using PfCRT antisera together with three different methods of protein interactions, we provide the first evidence of proteins interacting with PfCRT. Identification of these proteins which ranges from ~20 kDa to 200 kDa is under active investigation. / La forme létale du paludisme humain est causée par le plus important parasite protozoaire humain, Plasmodium falciparum, responsable d'environ ~1 million de mort annuellement. Le traitement et la prévention du paludisme dépend des médicaments antipaludiques. Le substitut synthétique de la quinine, la chloroquine, était le traitement le plus efficace pour cette maladie. La montée du paludisme résistant à la chloroquine dans les pays endémiques a supprimé les efforts de contrôle. La résistance à la chloroquine a été associée aux mutations dans la protéine transmembranaire présente sur la vacuole digestive du parasite, désigné PfCRT. De plus, la ou les fonctions normales et substrats naturels de la protéine PfCRT restent une affaire de spéculation étant donné qu'une évidence directe des fonctions normales et des substrats de cette protéine sont encore à déterminer. Les objectives de cette thèse sont de développer et de caractériser deux antisérums de l'extrémité N- et C-terminale de PfCRT comme outils d'identification des protéines interagissant avec la protéine PfCRT de P. falciparum. Bien que la masse moléculaire de la protéine PfCRT était estimée à 48.67 kDa, des caractérisations immunobiochimiques utilisant différents antisérums dirigés contre les domaines cytosoliques N- et C-terminal de cette protéine et publiés à travers la littérature ont abouti à des masses moléculaires variable de la protéine PfCRT sur le SDS PAGE. Dans cette thèse, nous reportons la génération et la caractérisation biochimique de deux antisérums dirigés contre les domaines cytosoliques N- et C de PfCRT. Nos résultats montrent que l'antisérum C de PfCRT détecte les épitopes non-phosphorylés ou des séquences dans la protéine PfCRT. En outre, nos résultats de la cartographie à haute résolution de l'épitope confirment la spécificité de l'antisérum C de PfCRT à la forme non-phosphorylé de PfCRT et montrent que la phosphorylation à deux sites au niveau de la séquence de la protéine, Ser411 et Thr416, qui empêche sa fixation à la protéine complète et phosphorylée. De plus, nous avons identifié une nouvelle forme tronquée de PfCRT qui est à la fois phosphorylée puisqu'elle est reconnue par l'antisérum C de PfCRT, et susceptible de représenter un produit différemment épissé de PfCRT, qui est exprimé in vivo sur la vacuole digestive du parasite. En outre, nos résultats confirment la masse moléculaire de la protéine complète et phosphorylée de PfCRT à 52 kDa sur un SDS PAGE. En utilisant les antisérums de PfCRT combiné à trois méthodes différentes d'interactions protéiques, nous fournissons la première preuve de protéines interagissant avec la protéine PfCRT. L'identification de ces protéines dont la masse varie entre ~20 kDa et 200 kDa est sous enquête active.

The protozoan parasite Entamoeba histolytica is the etiological agent of human amebiasis. Trophozoites colonize the colonic mucus layer and may invade the epithelium subsequent to overcoming the mucus barrier. MUC2 is the major gel-forming mucin secreted by goblet cells in the colon and serves to maintain epithelial barrier function as well as acting as a major host defense against invading pathogens. The polymerization of MUC2 monomers via the N- and C- terminal cysteine rich D-domains is essential for mucus gel formation and confers protection to the underlying mucosa. Amoebae secrete cysteine proteinases, glycosidases and an unidentified mucus secretagogue, which may play a role in overcoming the protective mucus barrier. We hypothesize that E. histolytica cysteine proteinases as well as glycosidases are involved in mucus degradation and weakening of the mucus barrier by disrupting mucin polymerization. Amoebae secreted cysteine proteinases were shown to degrade the cysteine rich regions of MUC2 involved in polymerization and abrogate its protective function. More importantly, the major E. histolytica surface proteinase, cysteine proteinase 5 (EhCP5) was shown to specifically degrade [35S]cysteine labeled colonic mucin as effectively as secreted components. Moreover, trophozoites genetically engineered to express low levels of CP activity were incapable of traversing a mucus barrier and destroying the underlying epithelium, indicating a strong dependence between amebic invasiveness and cysteine protease activity. In addition, we have demonstrated that EhCPs specifically target the MUC2 C-terminus resulting in destabilization of the mucin polymeric network. Parasite glycosidase activity was also shown to contribute to mucin oligosaccharide degradation. Taken together, these results indicate that E. histolytica can substantially weaken the colonic mucus barrier via proteolytic degradation and glycosidase activity to compromise the gel and

Schistosomes are among the most complex parasites affecting humans. They are dioecious parasites with an indirect life cycle that requires two hosts, a snail intermediate host and a human definitive host. The complexity of the life cycle is made possible, in part by the parasite's nervous system, which coordinates behavior and all major physiological activities of the worm. Neuronal signaling in schistosomes is mediated by a variety of neurotransmitters and associated receptors. Among these transmitters are dopamine and the neuroactive amino acid, glutamate. Both have been implicated in the control of neuromuscular function and movement but their mode of action is largely unknown. Here we provide the first molecular evidence for the existence of dopamine and glutamate receptors in Schistosoma mansoni. Two glutamate receptors were identified: One (SmGluR) is distantly related to heptahelical (metabotropic) glutamate receptors from other species and the other (SmGBP) is an unusual, truncated receptor that resembles the extracellular binding domain of glutamate receptors but lacks the remaining heptahelical transmembrane sequence. When expressed heterologously in vitro, SmGluR and SmGBP were both responsive to glutamate but did not recognize many classical (mammalian) glutamate agonists and antagonists, suggesting these are novel receptors with different pharmacological properties. Immunolocalization studies revealed that the two glutamate receptors have different tissue distributions. SmGluR is strongly expressed in neurons of the central and peripheral nervous system, including the peripheral innervation of the body wall muscles and the acetabulum, and it is also present in the female reproductive tract. In contrast, SmGBP is male-specific and was found only in the tegument, particularly the tubercles. The third receptor described in this thesis, SmD2, is structurally related to dopaminergic type 2 receptors and it is selectively activated by dopamine in vitro. SmD2 is localized entirely in the body wall musculature of both male and female worms and larvae. Together these results suggest that SmD2 and SmGluR could play important roles in the control of schistosome movement, SmD2 by targeting the musculature directly, whereas SmGluR works indirectly via the innervation of the musculature. The results also identify novel functions for glutamate receptors in the control of the acetabulum (SmGluR), egg production in females (SmGluR) and the tubercles of the male tegument (SmGBP). The widespread distribution of these receptors, combined with their unusual structures and pharmacological profiles, make them promising targets for discovery of new anti-schistosomal drugs. / Schistosoma est une des espèces les plus complexes de parasites humains. Il est dioïque et requiert un cycle de vie indirect : un hôte intermédiaire, l'escargot, et un hôte définitif, l'homme. Le système nerveux du parasite contrôle le comportement et toutes les activités physiologiques importantes et est en grande partie responsable de la complexité du cycle de vie du parasite. La signalisation neuronale des schistosomes est médiée par une variété de neurotransmetteurs et récepteurs correspondants, dont la dopamine et le glutamate. Ces transmetteurs régissent en partie les fonctions neuromusculaires et le mouvement, mais leur mode d'action demeure inconnu. Cette étude divulgue l'existence de récepteurs de la dopamine et du glutamate chez Schistosoma mansoni. Deux récepteurs de la dopamine ont été identifiés : SmGluR est indirectement associé au domaine heptahélice (métabotropique) des récepteurs du glutamate chez d'autres espèces, et SmGBP est un récepteur tronqué qui ressemble au domaine de liaison extracellulaire des récepteurs du glutamate dont la séquence transmembranaire heptahélice est absente. SmGluR et SmGBP sont tout deux sensibles au glutamate lorsqu'exprimés in vitro de façon hétérologue, mais ne reconnaissent pas plusieurs agonistes et antagonistes classiques (mammaliens) du glutamate. Cela suggère que ce sont de nouveaux récepteurs ayant des propriétés pharmacologiques différentes. Ces deux récepteurs du glutamate ont une expression tissulaire différente : SmGluR est fortement exprimé dans les neurones du système nerveux central et périphérique, et l'innervation périphérique des muscles de la paroi corporelle et de l'acétabulum, ainsi que dans le système reproductif de la femelle. Par contre, SmGBP est spécifique aux mâles et ne s'exprime que dans le tégument, particulièrement les tubercules. Le troisième récepteur identifié, SmD2, est structurellement lié aux récepteurs dopaminergiques de type 2, et sélectivement activé in vitro par la dopamine. SmD2 est uniquement retrouvé dans la musculature de la paroi corporelle chez les vers adultes et les larves des deux sexes. SmD2 et SmGluR semblerait donc important quant à la mobilité des schistosomes. SmD2 ciblerait la musculature directement, et SmGluR agirait indirectement sur l'innervation musculaire. Ces résultats définissent aussi de nouvelles fonctions des récepteurs du glutamate : le contrôle de l'acétabulum (SmGluR), la production des œufs chez les femelles (SmGluR) et des tubercules du tégument chez les mâles (SmGBP). La grande distribution de ces récepteurs combinée à leurs structures et profils pharmacologiques inhabituels en font des cibles prometteuses quant au développement de nouvelles thérapies contre la bilharziose.

The objective of this study was to further characterize the inhibitory serum protein crisis form factor, or CFF, and identify candidate proteins responsible for CFF activity. Four models for serum CFF induction were tested: C57BL/6 mice infected with P. chabaudi adami AS, C57BL/6 mice inoculated with BCG + LPS, and BALB/c mice infected with P. chabaudi adami DS or DK. C57BL/6 mice infected with P. chabaudi adami AS produced sera with the most pronounced level of inhibitory activity and were used for CFF analysis. Heat inactivation did not affect CFF activity, indicating the observed effect was not due to complement proteins, function was lost after heating serum to 100°C. Gel filtration determined that CFF may be in the range of 20 kDa – 80 kDa. Serum depletion by IgY retained CFF activity in the low abundance protein fraction (LAP), which was analyzed by MALDI and LC-QToF mass spectrometry and compared to the LAP from naïve mice. A total of 68 proteins were identified as either up-regulated or unique to the CFF serum, and qualitative analysis revealed one potential CFF candidate: neutrophil gelatinase-associated lipocalin. In conclusion, we have established a model for inducing serum CFF, characterized some of its physical properties, and through proteomic analysis identified a potential CFF candidate. / L'objectif de cette étude visait d'une part à approfondir la caractérisation du facteur de la forme de crise (CFF; « crisis form factor »), un facteur protéique inhibiteur présent dans le sérum, et d'autre part à identifier des protéines candidates responsable de l'activité de CFF. Quatre modèles murins d'induction sérique du CFF ont été testés: des souris C57BL/6 infectées par la souche de P. chabaudi adami AS, des souris C57BL/6 inoculées avec BCG + LPS, et des souris BALB/c infectées respectivement avec les souches de P. chabaudi adami DS ou DK. L'activité inhibitrice la plus prononcée a été observé à partir du sérum prélevé chez les souris C57BL/6 infectées par la souche de P. chabaudi adami AS. Le sérum issu de ce modèle a donc été utilisé pour la suite des analyses de CFF. Nous avons déterminé que l'activité de CFF ne provient pas des protéines du complément et que CFF est inactivé lorsque le sérum est bouilli à 100oC. Le fractionnement du sérum par chromatographie d'exclusion nous a permis de déterminer que CFF est éluée dans les fractions protéiques allant de 20 kDa à 80 kDa. Le sérum a ensuite été soumis à une colonne d'affinité IgY afin d'y dépléter les protéines majoritaires. Suite à cette déplétion, nous avons déterminé que l'activité de CFF est préservée uniquement dans la fraction contenant les protéines sériques de faible abondance (LAP; « low abundance protein »). Nous avons donc analysé la fraction LAP du sérum induit pour CFF par spectrométrie de masse de type MALDI et LC-QToF, puis comparé son profil protéique à celui de la fraction LAP de sérum issu de souris naïves. Ces profils protéiques révèlent qu'un total de 68 protéines sont soit surexprimées, soit exclusives au sérum démontrant une activité CFF. De plus, l'analyse qualitative nous a permis d'identifier une protéine candidate potentiel pour CFF : la gélatinase de neutrophile associée à la lipocaline (NGLA; « neutrophil gelatinase-associated lipocalin »). En concluant, nous avons établi un modèle d'induction sérique de CFF, avons caractérisé certaines de ses propriétés physiques et avons identifié, par l'entremise de la protéomique, NGLA en tant que candidate potentielle de CFF.

This research investigated the concurrent effects of a gastrointestinal nematode infection Heligmosomoides bakeri, pregnancy, and protein deficiency (PD) during late pregnancy throughout lactation on energetic, morphologic and immunological responses in CD1 mice and their offspring. Our novel findings can be summarized broadly into three key themes: (i) energetics and resting metabolic rate, (ii) bone metabolism and (iii) immune development. This work highlights the largely independent ways in which the mouse responds to competing demands of pregnancy, infection, and PD. Pregnancy increased RMR while PD lowered RMR; a trickle infection was not associated with any change. During pregnancy, these additional energetic demands were met by increased food intake and fat utilization. During infection mice lowered their body temperature. Finally, the reduction in RMR during PD was associated with higher serum corticosterone and leptin concentrations. A second novel finding was that both infection and PD impacted on maternal and neonatal bone development. Infection lowered maternal femur bone area which was associated with elevated serum IFN-γ in heavily infected pregnant mice and reduced foetal crown-rump length consistent with higher amniotic fluid IL-1β. Lower bone mineralization in PD dams was associated with elevated serum corticosterone and leptin whereas it was associated with elevated serum IL-1β and IL-6 during infection. The elevated serum IL-1β, lower leptin and IGF-1 in pups of PD and infected dams were consistent with the shorter crown-rump length. Finally, we explored for the first time the impact of maternal PD and infection on neonatal immune development. Both maternal infection and PD reduced lymphoid organ mass in pups whereas the percentage of T cells and T:B cell ratio in the spleen was increased only by maternal PD. These changes were associated with elevated corticosterone and IL-6 concentration in milk, and lower pup serum leptin and IGF-1 i / L'impact sur les réactions immunologiques, énergétiques et morphologiques a été étudié suite à une combinaison de facteurs tels une infection par Heligmosomoides bakeri, un nématode du système gastro-intestinal, la grossesse et la déficience protéique (DP) durant les derniers mois de grossesse et pendant la lactation chez les souris CD1 et leur descendance. Nos découvertes peuvent se résumer selon trois thèmes clés: (i) énergétique et métabolisme de base (MB), (ii) métabolisme osseux et (iii) développement immunitaire. Ce travail met en évidence les diverses voies indépendantes utilisées par la souris pour répondre à trois évènements: la grossesse, l'infection et la DP. La grossesse a augmenté le MB tandis que la DP l'a diminué. Une infection peu sévère n'a été associée à aucun changement. Durant la grossesse, les besoins énergétiques supplémentaires ont été comblés en augmentant l'apport nutritionnel et l'utilisation des graisses. Pendant la période d'infection, la température corporelle des souris a diminué. Enfin, la réduction du MB lors de la DP a corrélé avec une plus grande concentration de corticostérone et de leptine. Notre seconde découverte montre que l'infection et la DP ont un impact sur le développement osseux maternel et néonatal. L'infection a diminué l'os du fémur et a entraîné une forte concentration d'IFN-γ dans le sérum des souris gestantes fortement infectées. Elle a aussi diminué la distance vertex-coccyx tout en augmentant IL-1β dans le fluide amniotique. Une plus faible minéralisation osseuse chez les souris souffrant de DP coïncidait avec une forte concentration de corticostérone et de leptine dans le sérum bien qu'elle coïncidait avec une forte concentration d'IL-1β et d'IL-6 lors de l'infection. Une concentration élevée de IL-1β mais plus faible de leptine et de IGF-1 chez les souriceaux de mères infectées et souffrant de DP était compatible avec des distances$

<p>Passerine birds that primarily use grassland habitats are rarely the focus of a parasite study. With many rapidly declining bird populations that breed at even faster decreasing grassland habitat, it is important to know the potential risks to the birds posed by blood parasites. During the breeding seasons of 2009-2011, 150 samples from 148 individual birds (fourteen species) were collected from five grassland sites in northwest Minnesota, USA and surveyed for blood parasites using microscopy and molecular methods. Eighty-five (56.67%) of the 150 samples were infected with at least one of three haemosporidian genera: Haemoproteus, Plasmodium and Leucocytozoon. Seventy (46.67%) of the 150 samples were infected with either Haemoproteus or Plasmodium (fourteen infections were Haemoproteus, forty were Plasmodium and sixteen were undetermined due to dual infections or lack of sequences) and 41 samples (27.33%) were infected with Leucocytozoon, for a total of 111 infections. Plasmodium infections in two juvenile bobolinks provide evidence of active transmission within the study area. Haemoproteus/Plasmodium prevalence was significantly higher in May and June than in later collection months (July-Sept.) and dual infections were significantly higher in June compared with other sampling months. Of the three bird species that were sampled most, clay-colored sparrows (Spizella pallida) had significantly more Haemoproteus infections than savannah sparrows (Passerculus sandwichensis) and bobolinks (Dolichonyx oryzivorus). Only bobolinks were classified based on sex and/or age and adult males had significantly more Leucocytozoon and dual infections than adult females or juveniles. Parasite prevalence did not differ significantly between study sites or years. Phylogenetic reconstructions based on Maximum Likelihood and Bayesian analyses produced three major clades, corresponding to the three haemosporidian genera. Bird host species were well mixed within the trees, indicating infective vectors fed on bird species opportunistically rather than selectively and also shows that the Haemosporidia are generalists, being able to infect a wide range of the sampled bird species.

Malaria control programs in the past decade have been successful in decreasing the global burden of Plasmodium falciparum, the more virulent of the Plasmodium species causing malaria in humans. However, as the public health community gears towards elimination and eradication of malaria, there is need for an attention shift towards research of neglected Plasmodium species. A central event in malaria pathogenesis is the invasion of host red blood cells (RBCs) mediated by specific interactions between parasite ligands and RBC receptors. These interactions, also called invasion pathways, can be major determinants of host tropism. Restriction in invasion due to tropism is attributed to limiting disease severity, as it limits the proliferation of the parasite in vivo. In this dissertation, we determine the host cell tropism of Plasmodium knowlesi and Plasmodium vivax, two understudied Plasmodium species. The zoonotic P. knowlesi is now the major cause of malaria in parts of South East Asia, and P. vivax is the most widespread species worldwide causing substantial morbidity. After a review of the current literature on biological and clinical features of these species and their invasion pathways in Chapter 1, we determined the tropism of P. knowlesi in human RBCs in Chapter 2. We used density-based enrichment methods to test the invasion of P. knowlesi into human RBCs of varying age. Incorporating mathematical modeling and experimental adaptation, we demonstrated that an expansion of host cell niche is required for the parasite to reach densities observed clinically. We also obtained parasite lines adapted to efficient proliferation in human RBCs. In Chapter 3, we investigated the molecular basis of increased invasion of human RBCs through whole genome sequencing analysis of newly human-adapted lines, as well as historical strains we adapted to in vitro culture. We showed that different genetic changes in P. knowlesi can lead to the upregulation of PkDBPα-DARC pathway, and have provided evidence of major divergence in invasion ligands in recent field isolates. Finally, in Chapter 4, we studied the variation in human RBC preference of patient isolates for P. knowlesi and P. vivax. We confirmed that recent human P knowlesi isolates also vary in the niche of susceptible RBCs, with a subset exhibiting a lack of restriction in invading human RBCs. We also evaluated ex vivo P. vivax patient isolates for their host RBC preference. We determined that P. vivax field isolates differ in their level of reticulocyte preference. We further found an association between increased reticulocyte preference and schizont maturation. This body of work aims to contribute to our overall understanding of human RBC tropism of Plasmodium species of public health importance and the implication this has for parasite adaptation to humans. / Biological Sciences in Public Health

Approximately one third of the world’s population is at risk of contracting malaria. The World Health Organization estimates there were over 200 million news cases of malaria in 2015, resulting in nearly 500,000 deaths from this preventable disease. The majority of fatalities occur in Sub-Saharan Africa, where Plasmodium falciparum malaria causes severe disease in children under the age of five and pregnant women. In the last decade, increased anti-malaria interventions have resulted in substantial decreases in cases and fatalities. However, the recent emergence of artemisinin drug resistance in Southeast Asia threatens these gains, and the loss of another first-line antimalarial therapy would be a devastating setback. The first goal of this work was to identify genetic markers of artemisinin drug resistance. Identifying the genetic determinants and molecular mechanisms of artemisinin resistance is crucial for understanding the emergence of this phenomenon and tracking the spread of these drug resistant parasites. Over the course of four years, we used an in vitro drug resistance selection approach to generate three independent artemisinin-resistant lines. Here we characterize those lines, and present Pfcoronin, a kelch13-like protein, as a novel candidate marker for artemisinin resistance. This study identifies additional non-kelch13 molecular markers of artemisinin resistance, increases our understanding of how this resistance is acquired, and sheds light on the molecular mechanisms of artemisinin resistance in the parasite. In contrast to in vitro selection, natural selection of parasites occurs during natural infection. Investigation of specific genes under selection in the parasite will increase our understanding of biological processes that provide a fitness advantage, and potentially identify novel pathways for therapeutic development. Here, we focused on the acyl Co-A synthetase (ACS) gene family, previously shown to be under recent positive selection in P. falciparum. The signatures of recent positive selection identified in natural parasite populations suggest that particular ACS alleles may confer a selective advantage. Using molecular genetics approaches, we show distinct expression and localization patterns for individual ACS isoforms, and identify a growth defect in the ACS5 knockout line. Follow up studies characterize the fatty acid and metabolic profiles of individual ACS knockout lines, and point to a role for ACS5 in central carbon metabolism in P. falciparum. Our investigation of the ACS gene family and their role in P. falciparum growth and metabolism led us to hypothesize a link between ACS activity and central carbon metabolism. In the final chapter, we explore the basic fatty acid and glucose requirements for P. falciparum growth in vitro, and present a metabolic profile for these starved parasites. Under starvation conditions, we were able to demonstrate fatty acid oxidation activity in the parasite. This is an unexpected finding, as this pathway was not previously annotated in the genome. Taken together, these two projects tell a story of the selective pressures acting on P. falciparum parasites. Investigating in vitro selected artemisinin-resistant lines provides important insights into genetic markers and acquisition of resistance. Molecular and biochemical characterization of a gene family under natural selection in P. falciparum increases our understanding of important metabolic pathways that support parasite growth. / Biological Sciences in Public Health