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.

Mes travaux de thèse portent sur deux gènes cpbAg1 et cpbAg2 qui codent des enzymes digestives : les carboxypeptidases digestives du moustique Anopheles gambiae, vecteur majeur africain de Plasmodium falciparum, parasite responsable des formes graves du paludisme. La contribution de CPBAg1 dans le développement de P. falciparum chez An. gambiae a été démontrée ainsi qu'un rôle potentiel dans la production des oeufs du moustique (Lavazec et al., 2005 & 2007). Cependant le rôle potentiel de CPBAg2 dans ces deux phénotypes n'avait pas été abordé. Nos travaux ont permis de montrer des propriétés enzymatiques différentes pour chaque carboxypeptidase, avec CPBAg1 qui libère de l'arginine et de la lysine tandis que CPBAg2 est spécifique de l'arginine. La cinétique d'apparition in vivo des protéines montre une production précoce de CPBAg1 et une production constante de CPBAg2 au cours du processus de digestion du repas sanguin. L'analyse fonctionnelle de CPBAg1 et CPBAg2 utilisant l'inactivation génique par ARN interférence a permis de comprendre leurs rôles respectifs. Ainsi, CPBAg1 est impliquée dans le développement de P.falciparum chez An. gambiae, suggérant une plus forte dépendance du parasite pour les ressources en lysine. A l'inverse, CPBAg2 intervient au cours de la vitellogénèse du moustique et son inactivation induit également un retard du développement ovarien. Ainsi, ce dernier semble être plus dépendant de l'arginine, produit principalement par CPBAg2. CPBAg1 et CPBAg2 pourraient de ce fait être utilisées comme les cibles de nouvelles stratégies pour diminuer la transmission du paludisme, en bloquant le parasite chez le moustique tout en diminuant la production d'oeufs.

In response to growing levels of resistance to currently used antimalarials, there is an urgent need to develop drugs that exhibit novel mechanisms to kill Plasmodium parasites. The objective of this study was to examine the antiparasitic activity of newly synthesized compounds based on imidazolium and triazolium rings. According to our structure/activity relationship studies the key components appear to be their positively charged rings and hydrophobic side groups, and bivalent compounds, which incorporate two positively charged rings, show even greater potency than monovalent compounds. Depending on the concentration used, our compounds appear to primarily inhibit intracellular parasite development or invasion into red blood cells. Selected compounds have been tested in vivo using a P. berghei ANKA murine model. Together, our findings demonstrate that small imidazolium- and triazolium-based compounds display both in vitro and in vivo activity through a novel mechanism of action that may involve inhibition of erythrocyte invasion.

In response to growing levels of resistance to currently used antimalarials, there is an urgent need to develop drugs that exhibit novel mechanisms to kill Plasmodium parasites. The objective of this study was to examine the antiparasitic activity of newly synthesized compounds based on imidazolium and triazolium rings. According to our structure/activity relationship studies the key components appear to be their positively charged rings and hydrophobic side groups, and bivalent compounds, which incorporate two positively charged rings, show even greater potency than monovalent compounds. Depending on the concentration used, our compounds appear to primarily inhibit intracellular parasite development or invasion into red blood cells. Selected compounds have been tested in vivo using a P. berghei ANKA murine model. Together, our findings demonstrate that small imidazolium- and triazolium-based compounds display both in vitro and in vivo activity through a novel mechanism of action that may involve inhibition of erythrocyte invasion.

Hest shock protein 70 (Hsp70) class of chaperones is highly conserved and present ubiquitously in all cellular organisms They play important role in folding of nascent polypeptides and translocation of precursor proteins to endoplasmic reticulum, mitochondria and chloroplast Hsp70 assists in assembly of proteins complexes as well as in disassembly e g uncoatmg of clathrin coated vesicles Chaperone function of Hsp70 is modulated by cochaperones of DnaJ class, Hip, Hop etc Hsp70 is a component of multi chaperone complex with Hsp90 and helps in maturation of kinases or transcription factors. Plasmodium falciparum is responsible for most severe form of human malaria Plasmodmm in its intraerythrocytic cycle presents an example of a cell with multiple, complex membrane bound structures both inside the parasite as well as m the infected erythrocyte cytosol Parasite deploys proteins in host erythrocyte cytosol, at erythrocyte plasma membrane or traffics them for secretion outside the infected cell in addition to trafficking of proteins to its own organelles like mitochondria, apicoplast, food vacuole, ER etc It is of interest to malaria biologists to understand these trafficking events and role of chaperones in regulating them This study was aimed at understanding the function(s) of Hsp70 in Plasmodium infected erythrocyte in protein maturation and trafficking events We have attempted to study Hsp70 chaperone present in Plasmodium infected erythrocytes We have largely focused on the cytosohc Hsp70, PfHsp70, in the parasite and systematically analyzed its expression, localization, abundance and complexes in the intraerythrocytic cycle To gain insight into its function, we have identified a subset of PfHsp70 interacting proteins, parasite Hsp90, Hsp70-3, Hsp60 and beta tubulin by coimmunoprecipitation experiments in conjunction with proteomic tools like 2DGE and mass spectrometry Parasite Hsp60 is a mitochondria-targeted protein and we have examined the involvement of PfHsp70 in translocation of Hsp60 precursor protein to parasite mitochondrion PfHsp70 and PfHsp90 were found to be present in a complex Geldanarnycm, a drug that affects Hsp70-Hsp90 complex, was used to investigate the role of PfHsp70 in parasite protein trafficking Since there are no known parasite derived chaperones in erythrocyte cytosol compartment, we have examined the possible "involvement of host Hsp70 in supporting transport and assembly of parasite proteins in erythrocyte cytosol Hsp70 in Plasmodium falciparum intraerythrocytic cycle P. falciparum genome codes for five Hsp70 homologs Two of these, pfHsp70-l and PfBiP are expressed in intraerythrocytic stage and have been localized to nucleocytoplasmic and endoplasmic reticulum fraction of the parasite respectively We have focused this study on PfHsp70 of the parasite We show that PfHsp70 is an abundant protein in the cytosol constituting about 2% of the total soluble pool It gets further induced during stress like heat shock and translocates to nuclear fraction indicating that PfHsp70 may be involved in protective function in the parasite nucleus during stress Nuclear translocation of mammalian Hsp70 during stress has been linked to its phosphorylation at Tyr524 We found PfHsp70 to be phosphorylated by in vivo phosphate labeling m the parasite Analysis of PfHsp70 by 2-dimensional gel electrophoresis on narrow gradient IPG strips indicated that it exists in four forms differing in their isoelectnc points (pi) Phosphatase treatment combined with analysis using a phosphorylation prediction tool,Proteomod (http //www biochem use ernet in/proteomod html) suggested that PfHsp70 is phosphorylated at three residues in the parasite The extent of phosphorylation of PfHsp70 may determine substrate specificity or subcellular localization or both Using 2DGE and mass spectrometry approach, we also identified chaperones like Hsp909 BiP, Hsp60, and protein disulphide isomerase (PDI) m P falciparum proteome In summary, PfHsp70 appears to be a highly abundant, cytosohc chaperone It is inducible by stress and multiply phosphorylated and is likely to participate in multiple processes in the parasite. PfHsp70 complexes and interacting proteins in the parasite To gam insight into the functions of Hsp70, we looked for PfHsp70 interacting proteins in the parasite We used gel filtration chromatography to resolve and enrich PfHsp70 complexes and also employed coimmunoprecipitation approach to identify interacting proteins We found parasite Hsp90, Hsp70-3, Hsp60 and beta-tubulin interact with PfHsp70 Fractionation of parasite lysate indicated that PfHsp70 is present in two major complexes of 200 kDa and 450 kDa We find that PfHsp90 interacts with PfHsp70 and both are present in 450 kDa complex Our analysis indicated that 450-kDa complex is like Hsp70-Hsp90 multichaperone complex described in mammalian cells while 200 kDa complex is likely to be an Hsp70-cochaperone complex Smaller complex appears to be a precursor for multichaperone complex Use of an Hsp90 inhibitor, geldanamycin (GA), to study the function of this multi chaperone showed that GA inhibits parasite growth Maturation of four phosphoproteins interacting with PfHsp70 was affected by GA implicating them in regulation of parasite growth GA appeared to mediate its effects by inhibiting H§p^0 phosphorylation Amongst the other three interacting proteins, PfHsp70-3 is amoveJ/Hsp70 homolog that was found at the protein level for the first time in this study PfHsp60 is mitochondria-targeted protein in the parasite and it is likely that cytoshc PfHsp70 helps in translocation of PfHsp60 to mitochondria from cytosol Tubuhn is a cytoskeletal protein and its interaction with PfHsp70 suggests possible role of PfHsp70 in cytoskeleton organization during invasion, growth or cell division In all, we find that Hsp70 in the parasite exist in a multi chaperone complex with Hsp90 which might be responsible for maturation of signaling molecules important for growth The smaller complex of PfHsp70 is a precursor of multi chaperone complex and is likely to be an Hsp70- co chaperone complex Role of Hsp70 in protein translocation and trafficking Cytosolic Hsp70 aids in translocation of precursor proteins from cytosol to mitochondria (or chloroplast) We found a mitochondnal chaperone, PfHsp60, interact with PfHsp70 and we examined the possibility that PfHsp60 translocation is assisted by cytosolic PfHsp70 We found that PfHsp60 had a cleavable, N-thermal targeting sequence Examination of PfHsp60 forms present in mitochondnal and cytosolic fraction of the parasite showed that mitochondnal form was more acidic in pi than cytosolic form as expected after targeting sequence cleavage Cytosolic PfHsp60 interacted with both PfHsp70 and PfHsp90 Interestingly, while mitochondnal PfHsp60 appeared to be in a chaperonm like complex, as expected, cytosolic form was present in smaller ohgomeric complex of about 450 kDa This suggested that PfHsp60 precursor form could be bound to multichperone complex All these experiments together strongly indicated that PfHsp60 precursor interacts with cytosolic Hsp70 and Hsp90 before former's translocation into mitochondria This interaction might be required to keep the precursor in the transport competent state P falciparum lives inside a vacuole in the infected cells but it deploys a number of proteins to host cell cytosol and to the plasma membrane To examine the involvement of multichaperone complex in trafficking, we studied the effect of GA on targeting of two parasite proteins, knob associated histidme-rich protein (KAHRP) and glycogen synthase kinase (GSK) KAHRP is indispensable for the formation of cytoadherence complexes called knobs at erythrocyte plasma membrane We found that KAHRP transport to erythrocyte plasma membrane was blocked in GA-treated parasites and it appeared all over the infected cell Further analysis showed that GA caused block in KAHRP transport at some step beyond its exit from parasite ER The targeting of GSK to membranous inclusions in the infected RBC cytosol was not severely affected m the GA-treated parasites suggesting that GSK transport may not be regulated by multi chaperone complex It also indicated that parasite may be using different pathways for trafficking of proteins to the host compartment In summary, PfHsp70 and PfHsp90 interact with PfHsp60 precursor in the cytosol They probably help keep the precursor in transport competent form before arrival at the translocase complex of mitochondria The multi chaperone complex may also be important for trafficking of at least one parasite protein, KAHRP, to the host cell compartment Analysis of erythrocyte Hsp70 in Plasmodium falciparum infected cells The remodeled plasma membrane of parasite-infected erythrocytes is important for the cytoadherence property of the infected cells Knobs, supramolecular complexes on the infected cell surface, formed by parasite proteins, PfEMPl, KAHRP, and PfEMP3 are responsible for cytoadherence of infected cells to vascular endothehum or placenta KAHRP transport is BFA-sensitive inside the parasite while PfEMP proteins undergo vesicle mediated trafficking in the erythrocyte cytosol The involvement of molecular chaperones has been implicated in the trafficking and assembly of knob components in the erythrocyte cytosol There is no evidence for the presence of bona fide parasite derived chaperones in the host compartment The chaperones of the erythrocyte origin, Hsp70, Hsp90, Hip and Hop were readily detected in the host cytosol, on the other hand By analyzing localization, abundance and biochemical characteristics of the host chaperones of erythrocyte origin, we examined if host chaperones are being utilized by the parasite for its functions Localization experiment showed that while PfHsp70, PfHsp90 and PfBiP were present in the parasite compartment, host-Hsp70 was present in erythrocyte cytosol fraction Host~Hsp70 was about 60% as abundant as PfHsp70 and was potentially capable of facilitating chaperone function in the erythrocyte cytosol Though host-Hsp70 was soluble in unmfected cells, it was present in membrane bound, triton-insoluble complexes, containing KAHRP, in infected cells Since knobs are triton-insoluble complexes at the erythrocyte plasma membrane, we isolated erythrocyte ghost (plasma membrane) fraction and could detect both Hsp70 and KAHRP Hsp70 association with erythrocyte plasma membrane was specific as it could be crosshnked to KAHRP in ghost fraction of infected cells Host-hsp70 was present in purified cytoskeleton fraction containing knobs from infected cells along with cochaperone Hop All these evidences suggest that parasite may be exploiting host-Hsp70 in erythrocyte cytosol compartment Summary This study gives insight into some functions performed by PfHsp70 in mtraerythrocytic cycle of malarial parasite PfHsp70 is an abundant cytosohc chaperone in the parasite It gets induced during stress and translocates to the nucleus It is also phosphorylated at three sites Analysis of Pfhsp70 complexes shows that it is present in bimodal complexes (450 kDa and 200 kDa), which are in equilibrium PfHsp70 and PfHsp90 interact and are part of 450 kDa multichaperone complex This multichaperone complex appears to regulate trafficking of one parasite protein to host cytosol compartment In addition, PfHsp70 and PfHsp90 are also bound to mitochondria-targeted PfHsp60 precursor in the cytosol probably keeping them m a transport competent state In addition to PfHsp90 and PfHsp60, PfHsp70 interacts with a novel Hsp70 homolog of the parasite, PfHsp70-3, and cytoskeletal protein, beta-tubuhn Examination of chaperones available in erythrocyte cytosol, showed that parasite chaperones were absent while host chaperone (Hsp70) was present and exhibited altered properties during parasite infection It was associated with membrane-bound, triton-insoluble complexes on the infected cell plasma membrane suggesting that host-Hsp70 might be involved in trafficking and/or assembly of parasite proteins In all, PfHsp70, as part of multichaperone complex, appears to be regulating translocation and trafficking of parasite proteins to organellar locations or outside the parasite Host-Hsp70, in erythrocyte cytosol, might also be engaged in specific chaperone function upon infection

El objetivo de esta investigación retrospectiva, fue determinar la asociación existente entre mutaciones puntuales en los genes dihidrofolato reductasa (Pfdhfr) y dihidropteroato sintasa (Pfdhps) de Plasmodium falciparum y la respuesta clínica en pacientes con diagnóstico de malaria no complicada causada por P. falciparum, admitidos en un estudio de eficacia in vivo de sulfadoxina-pirimetamina (SP) llevado a cabo en dos áreas de la región Loreto en 1999. Se tomaron muestras de sangre de 86 pacientes antes de administrarles SP, las que se analizaron usando PCR-anclado específico de alelo para estudiar a los codones S108N/T, N51I, C59R, I164L y C50R del gen Pfdhf y los codones A436G, A437G, K540E, A581G y A613S/T del gen Pfdhps, encontrándose que las infecciones causadas por parásitos con 3 mutaciones en Pfdhfr (108Asn/51Ile/164Leu) y 2 (581Gli/437Gli) ó 3 mutaciones en Pfdhps (581Gli/437Gli/540Glu) denominados el quíntuple mutante y el séxtuple mutante, respectivamente, se encontraban asociadas con la falla del tratamiento con SP. Además, se estableció que cuanto más alto era el número de mutaciones tanto en Pfdhfr como en Pfdhps, más alto era el riesgo de fallar al tratamiento con SP, según resultado del análisis de regresión logística empleado para asociar a estas dos variables. Este estudio contribuye en brindar evidencias científicas de la asociación existente entre las variables en la región Loreto contribuyendo a su validación y su futuro uso para estudios de vigilancia de fármaco resistencia a SP no solo en el Perú sino en general para los países de América del Sur que comparten territorio de la selva Amazónica. / The objective of this retrospective study was to determine the asociation between point mutations in dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) Plasmodium falciparum genes, and clinical outcome of patients with non complicated P.falciparum malaria diagnosis, admitted to a in vivo sulphadoxine-pirymethamine (SP) drug efficacy study conducted in 1999, in two areas of Loreto region. We used allelic specific-nested PCR to analize 86 blood samples collected before SP treatment, and study mutations at codons S108N/T, N51I, C59R, I164L and C50R in Pfdhfr and codons A436G, A437G, K540E, A581G y A613S/T in Pfdhps genes, and found that Infections caused by parasites harbouring 3 mutations in Pfdhfr (108Asn/51Ile/164Leu) and either 2 or 3 mutations in Pfdhps (581Gly/437Gly and 540Glu) called the quintuple and sextuple mutants, respectively, were associated to failure with SP treatment. Logistic regression analysis was used to look for association between the variables, helping to establish the higher the number of mutations in both Pfdhfr and Pfdhps genes, the higher the risk of treatment failures when using SP.The contribution of this study is to provide scientific evidences of the association between both variables in Loreto region supporting its validation and future application in surveillance studies for SP drug resistance, that can be conducted not only in Peru but also in South American countries that share the Amazon basin territory.

L'apparition récurrente de Plasmodium falciparum résistant aux antipaludiques est un obstacle majeur au contrôle du paludisme. Introduite en 2000, une nouvelle association très bien tolérée, l'atovaquone-proguanil est rapidement devenue le traitement de choix des accès palustres simples dans certains hôpitaux français. Ce travail de recherche avait pour objectif d'approfondir les connaissances sur la résistance à cette association. Nous n'avons détecté aucune résistance naturelle à l'atovaquone-proguanil en Afrique de l'Ouest et dans l'Océan Indien parmi 477 isolats. La majorité des rechutes précoces sont liées à une malabsorption des principes actifs alors que les échecs tardifs sont liés à la présence de parasites hautement résistants in vitro présentant, au moment de la rechute, une mutation au niveau du codon 268 du cytochrome b (Y268S ou Y268C) sans augmentation du nombre de copies de ce gène, évalué par PCR en temps réel à 16 ± 9 copies par parasite. Le séquençage du génome mitochondrial et l'analyse de marqueurs microsatellites chromosomiques des parasites isolés avant et après la rechute parasitaire montrent que la mutation associée à cette résistance est apparue indépendamment chez chacun des six patients en échec étudiés. L'atovaquone-proguanil est efficace pour le traitement des voyageurs avec moins de 0,1% de résistance. Le risque actuel de dispersion des résistances est négligeable puisqu'elles émergent chez des patients traités hors de zone de transmission. Par contre, si cette association devait être déployée en zone d'endémie, il serait indispensable de la combiner avec d'autres molécules.

A new era of malaria eradication programs relies on increased knowledge of the parasite through sequencing of the Plasmodium genome. Programs call for re-orientation at specific epidemiological markers as regions move from control towards pre- and total elimination. However, relatively little is known about the effects of intervention strategies on the parasite population or if the epidemiological cues correspond to effects on the parasite population. We hypothesized that genomic tools could be used to track population changes in Plasmodium falciparum to detect significant shifts as eradication programs apply interventions. Making use of new whole-genome sequencing data as well as GWAS and other studies, we used SNPs as biological markers for regions associated with drug resistance as well as a set of neutral SNPs to identify individual parasites. By utilizing tools developed as proxy for full genomic sequencing of the human pathogen Plasmodium falciparum, we characterized and tracked parasite populations to test for changes over time and between populations. When applied to markers under selection - those associated with reduced antimalarial drug sensitivity - we were able to track migration of resistance-associated mutations in the population and identify new mutations with potential implications for resistance. Using a population genetic analysis toolbox to study changes in neutral allele frequencies in samples from the field, we found significant population changes over time that included restricted effective population size, reduced complexity of infections, and evidence for both clonal and epidemic propagation of parasites.

Protozoa and bacteria can easily cause disease in humans, specifically E. coli, Plasmodium falciparum, and Cryptosporidium parvum. These three pathogens are associated with large public health concerns that span the globe. The variety of locations in which these can be found is extremely high. Cryptosporidium spp. are extremely resilient when in oocyst form, P. falciparum is in the Anopheles spp. mosquito, while E. coli can be found on anything from food and water, to the skin and gut. The diverse range of locations these can be found in means that a portable sensor for their detection is necessary. In detecting Cryptosporidium, microscopy is the preferred method of identification currently. This requires a trained lab technician as well as calibrated and expensive optical equipment. Technician error can lead to false negative or positive diagnoses as well as sample destruction. A method to remove this technician interaction is thus necessary. This method must allow for objective results that are not open to interpretation. Particle immunoagglutination assays with Mie scatter allow for such an approach using inexpensive components. Particle immunoagglutination relies on the principles of antibody-antigen interaction and antibody conjugated latex particles. Using highly carboxylated latex particles, it is possible to attach IgG antibodies that are specific to a target antigen. Mie scatter is governed by particle size rather than wavelength as other forms of scatter. These two combined allow for an increase in light scatter based on particle size. This is correlated in a linear manner as long as the number of antibody-conjugated particles is higher than the number of antigens. Microfluidics is an ever growing field in the field of lab-on-a-chip that works very well with particle immunoagglutination. In this paper, a method to rapidly identify the presence of Cryptosporidium using microfluidics and particle immunoagglutination is discussed and analyzed. This method allows for a low detection limit of 1-10 oocysts/sample and an assay time of approximately 10 minutes. Results are displayed on a computer screen as the value of light scatter intensity and, when compared to a standard curve, is an objective way to identify the concentration and presence of oocysts in a diverse range of samples. These samples include PBS, pool water, and sump water. This system also works with P. falciparum, which causes malaria in rural and urban poor regions of the world. With the low income and remote nature of these locations, a portable microfluidic device is necessary. Smartphones allow for a portable microfluidic device that can detect P. falciparum antigens in 10% whole blood. This system is capable of detecting as little as 1 pg/mL antigen. The microfluidic chip is inexpensive and disposable, allowing for a portable and inexpensive system. Using a single smartphone, a lab technique requiring a spectrometer, light source, and laptop can be made portable and less expensive, while maintaining sensitivity and specificity. In order to identify biological agents, there are commonly 3 methods for doing so: PCR, culturing, and ELISA. Culturing can take more than 24 hours, but results in a high signal to initial target ratio, while ELISA has poor sensitivity due to a 1:1 signal to target ratio, though is much quicker than culturing at usually 3 hours or less. PCR manages to solve both these problems by exponentially increasing the number of copies of target genetic material in a relatively short time frame of 1-3 hours. PCR relies on 4 basic components: target genetic material, primers to set a start and end location for duplication, polymerase to add base pairs to the strand beginning at the primers, and heat. PCR has worked very well during the past 31 years. It has worked so well that it is often the gold standard. However, there are flaws built into today's systems. These largely come in the form of inefficient heat transfer via conduction and large sample volumes due to unnecessary additions of nuclease free water (NFW). Both of these can be easily overcome by droplet PCR. Droplet PCR relies on small sample volumes of between 8 and 12 μL and convection in oil rather than conduction through plastic. In this study, it was found that droplet PCR could be performed on genomic E. coli DNA in as little as 15 minutes for 30 cycles. Sensitivity was also analyzed and found to be 2.62 pg DNA/μL or about 5 x 10² cfu/sample. PCR has a theoretical lower limit of 1 copy of genetic material and this is only 2 orders of magnitude above that. The system was also tested for portability and resistance to shock and vibration. It was found that the surface heated, thermocouple guided system is more shock and vibration resistant than standard wire guided, hanging droplet PCR systems. It was also found that the use of coconut oil allows for the system to be transported without fear of the contents spilling out and contaminating other samples. This is because of coconut oil's high melting temperature.

Here we report the identification of 15 potential MSP-like proteins from the P. falciparum 3D7 genome using a bioinformatics-based approach. One candidate, renamed URF1, was further characterized by cloning into the Gateway system. We were able to demonstrate expression of URF1 during the blood stage, especially the trophozoite, early and late schizont phases, by immunofluorescence on infected RBC using antisera raised in mice with an URF1 DNA vaccine. URF1 expression in the merozoite stage could not be confirmed in this study. Future co-localization and immunosorbent electron microscopy (EM) experiments would help us determine the exact localization of URF1 on the parasite before officially categorizing URF1 as a merozoite surface protein. As a whole, this research project demonstrates the success of using bioinformatics in identifying potential new MSP-like proteins found in the malaria genome. Further characterization and sequence analysis of the other 15 candidates may reveal other novel antigens expressed during the erythrocytic stage, especially in the merozoite stage. Such antigens may prove to be good vaccine candidates.