<p>Malaria remains the most important parasitic disease worldwide. It is estimated that over 500 million infections and more that 2.7 million deaths arising from malaria occur each year. Most (90%) of the infections occur in Africa with the most affected groups being children of less than five years of age and women. this dire situation is exacerbated by the emrggence of drug resistant strains of Plasmodium falciparum. The work reported in this thesis focuses on improving the purification of PfHPRT by investigating the characteristics of anion exchange DE-52 chromatography (the first stage of purification), developing an HPLC gel filtration method for examining the quaternary structure of the protein and possible end stage purification, and initialcrystalization trials. a homology model of the open, unligaded PfHPRT is constructed using the atoomic structures of human, T.ccruz and STryphimurium HPRT as templates.</p>

Les espèces de Plasmodium induisent une réponse immunitaire spécifique, qui stimulent la libération de cytokines et entraînent des réponses protectrices ou pathologique. Dans les modèles murins, ses réponses dépendent des combinaisons de la souche de souris et de l’espèce Plasmodium utilisés. Les souris C57BL/6 infectées, avec un nombre relativement faible de Plasmodium chabaudi AS, expérimentent une infection qui n’est pas fatale et une résistance à l'infection associée avec une réponse inflammatoire forte qui implique l'interleukine-12 et l'activation précoce des cellules T CD4+ avec une production élevée de IFN-γ et TNF-α. Cette réponse pro inflammatoire est par la suite contrôlée par l'IL-10 qui mène au contrôle et à la résolution de l'infection. Cependant, les souris qui succombent à l’infection développent la parasitémie fulminante et une infection mortelle. La cause des différences de résolution n'est pas bien comprise, bien que les principaux composants de protection et les réponses immunitaires pathologiques sont bien connus. Cette étude utilise le modèle de Plasmodium chabaudi dans le souris C57BL/6, pour: 1) comparer la résolution de l'infection et de la pathologie entre le P. chabaudi AS qui est relativement non virulentes et la plus virulentes de P. chabaudi CB; 2) déterminer si les différences dans la réplication des parasites, charge parasitaire ou les réponses immunitaire de l'hôte contribuent a des différences pathologiques; 3) ) chercher à déterminer ce qui mène les différences dans la réponse immunitaire de l'hôte. P. chabaudi CB provoque une infection plus sévère chez les souris C57BL/6 par rapport à P. chabaudi AS, avec une mortalité de ±40% endéans 12 jours et une moyenne des pics de 50% par rapport à 30% observé dans des souris infectées avec P. chabaudi AS. Aucune différence n’a été observé entre la charge corporelle des parasites durant les infections de P. chabaudi AS comparée a celle de P. chabaudi CB. Un taux élevé de cellules NK a été constaté dans les rate des souris infectées par P. chabaudi AS au niveau maximum de parasitémie, tandis que le taux des cellules NK dans les rates des souris infectées par P. chabaudi CB est resté constant tout au long de l'infection. Par conséquent, la poussée du taux des cellules NK contribue a l’infection non virulente des clones P. chabaudi AS. Ceci est du à la capacité cytolytique des cellules NK ainsi qu’à la production des IFN- γ. Le nombre de lymphocytes T CD4+ présents dans les rates des souris C57BL/6 infectées par P. chabaudi CB était inférieur a celui des lymphocytes T CD4+ présent dans les rates des souris infectées par P. chabaudi AS ; 1 fois moins de cellules T CD4+l'IFN-γ+ et 2 fois moins de cellules l'IL-10 +T CD4+ ont été observés. Une analyse plus approfondie a illustré que les cellules l'IL-10+ IFN-γ+ T CD4+ sont plus fréquentes chez les souris infectées par P. chabaudi AS. L'absence des cellules d'IL-10+ IFN-γ+ T CD4+ chez les souris infectées par P. chabaudi CB contribue à la pathologie, parce que ces cellules limitent les réactions inflammatoires mortelles et améliorent la perte de poids, l'hypothermie et l'anémie, en régulant l'immunopathologie. Les différences observées dans les réponses immunitaires ne sont pas contrôlées par l'interaction des parasites spécifiques tel que glycophosphatidylinositol avec TLR2, mais plutôt par l'IFN-α/β. Bien qu’ IFN-α/β n’ont pas été détecté dans le plasma, des niveaux plus élevés d'IFN-α/β d'ARNm transcrits ont été trouvés dans la rate de C57BL/6 infectées par P. chabaudi CB au cinquième jour d’infection. Le manque d’IFN-α/βR limite la pathologie et l’infection P. chabaudi CB virulentes sans affecter les infections P. chabaudi AS non virulentes. / Plasmodium species induces a specific immune response, stimulating the release of cytokines, resulting in either protective or pathological responses. In mouse models, this is depended on mouse strain or parasite combination used. C57BL/6 infected with relatively low numbers of Plasmodium chabaudi AS pRBC experience a non-lethal infection and resistance is associated with robust inflammatory responses that involves IL-12, early activation of CD4+Th1 cells with production of high levels of IFN-γ and TNF-α, this pro-inflammatory response is subsequently controlled by IL-10 leading to control and resolution of infection. However, mice that succumb to infection develop fulminant parasitaemia and increase mortality. The cause of the differences in infection outcome is not well understood, although the principal components of protective and pathological immune responses are well known. Using the model of Plasmodium chabaudi in C57BL/6, this study addresses the following; 1) compares the course of infection and pathology between the relatively avirulent P. chabaudi AS and the more virulent P. chabaudi CB; 2) investigates whether differences in parasite replication, parasite load or host immune responses contribute to differences in pathology; 3) what mediates the differences in host immune response? P. chabaudi CB causes a more severe infection in C57BL/6 compared to P. chabaudi AS, with ±40% mortality within 12 days and mean peak parasitaemia of 50% compared to 30% in P. chabaudi AS infected mice. There was no difference in total body parasite load between the P. chabaudi AS and CB infections. A High number of NK cells was found in the spleen of P. chabaudi AS infected mice at peak parasitaemia, whereas NK cells numbers in spleen of P. chabaudi CB infected mice remained constant throughout infection. Hence, the elevated NK cells contribute to parasite clearance in the avirulent P. chabaudi AS clone, because of its IFN-γ production and cytolytic activity. P. chabaudi CB infected C57BL/6 were found to have reduced number of CD4+T cells in the spleen, with 1-fold decrease in IFN-γ+CD4+T cells and 2-fold decrease in IL-10+ CD4+T cells when compared to CD4+T cells from P. chabaudi AS infected mice. Further analysis showed that IL-10+IFN-γ+CD4+T cells were more prevalent in P. chabaudi AS infected mice. The absence of IL-10+IFN-γ+CD4+T cells in P. chabaudi CB infected C57BL/6 contributes to pathology, because these cells limit lethal inflammatory responses and ameliorate weight loss, hypothermia and anemia, by regulating immunopathology. The differences observed in immune responses are not mediated by interaction of parasite specific GPIs with TLR2, but rather by IFN-α/β. Although IFN-α/β were not detected in plasma, higher levels of IFN-α/β mRNA transcripts were found in the spleen of P. chabaudi CB infected C57BL/6 on day 5. IFN-α/βR deficiency limits virulent P. chabaudi CB infections with no effect on avirulent P. chabaudi AS infections.

Student Number : 9605361W - PhD thesis - School of Pathology - Faculty of Science / Malaria is one of the most debilitating parasitic infections to have afflicted humanity and remains an expanding health risk for many countries. This is attributed largely to the complexity of the parasite’s life cycle and refined ability to evade host immunity. During development within the erythrocyte, Plasmodium falciparum induces a wide array of changes to the ultrastructure, function and antigenic properties of the host membrane. Numerous proteins encoded by the parasite associate with the erythrocyte skeleton and appear to be essential for P. falciparum survival. The elucidation of new protein-protein interactions has therefore formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of phage display technology was used in this research. P. falciparum phage display libraries were created and biopanned against human erythrocyte skeletal protein 4.1 (4.1R). DNA sequencing and bioinformatic investigations uncovered a number of parasite proteins with binding specificity toward 4.1R. They included five hypothetical proteins, two invasion proteins, namely erythrocyte binding antigen-175 (EBA-175) and EBA-181, two predicted protein kinases and a putative aminopeptidase. A common binding motif displaying homology to muscle myosin and neurofilament sequences was also identified in four of the ten proteins. The interaction between EBA-181 and 4.1R was characterised further by mapping the domain in 4.1R responsible for binding to the parasite protein. Recombinant proteins were used in blot-overlay and pull-down experiments, which revealed specific interaction between the highly conserved 10kDa domain and the 4.1R binding region in EBA-181. Binding was concentration dependent, as well as saturable and was abolished by heat denaturation of 4.1R. Functions of the 4.1R-specific parasite proteins remain to be determined, however, they are potentially involved in parasite growth and survival during intra-erythrocytic development. Furthermore, these proteins may also participate in the entry and/or exit of parasites from the human erythrocyte. The interaction of EBA-181 with the 10kDa domain of 4.1R provides new insight into the molecular mechanisms utilised by P. falciparum during erythrocyte entry. It also highlights the multifunctional role of malaria invasion proteins, which may contribute to the success of the pathogenic stage of the parasite’s life cycle.

A Thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg for the Degree of Doctor of Philosophy / This thesis describes the characterisation of southern African isolates of Plasmodium falciparum according to isoenzyme type, antigen variants and drug sensitivity. Nineteen southern African isolates and a Gambian reference isolate were cultured in vitro in gassed tissue culture flasks. Polyacrylamide gel electrophoresis with use of 5 enzymes revealed little variation amongst the isolates and the frequencies of enzyme forms were similar to those of isolates from other parts of the world. Antigenic diversity was demonstrated using a panel of 9 monoclonal antibodies in the indirect fluorescent antibody test. The antigenic composition of 70% of isolates was markedly different to any obtained in other geographical areas. Both characterisation techniques revealed a mixture of parasite types in some isolates. However I the characteristics of most of these heterogeneous isolates were not stable with time in culture. Most isolates proved to be resistant to chloroquine in a 48-hour growth inhibition test. A wide range in Pyrimethamine susceptibilities was detected although most isolates exhibited a low level of resistance to this drug. In the radioisotope uptake assay, the halofantrine IC50 values obtained were comparable with those of West African isolates but were higher than the TC50s of south-East Asian isolates. All of the isolates resistant to chloroquine in the 48-hour test were resistant in the radioisotope assay. However, 1 isolate shown to be sensitive to chloroquine in the first test was found to be resistant in the 3Hhypoxanthine incorporation method. This was assumed to be the result of selection of resistant clones. Southern African isolates were shown to be fully susceptible to mefloquine, but had reduced susceptibility to quinine and sulphadoxine/ pyrimethaInine. Most isolates were sensitive to amodiaquine. A field study of 31 KwaZulu isolates of P. falciparum obtained in 1987 and 1988 showed 29 to be resistant to chloroquine in the 24-hour microtechnique, with the majority being highly resistant. Chloroquine resistance was confirmed in this area in a field study carried out in 1989 but in 1990 all 4 isolates tested were sensitive to the drug. In 1989, 12 out of 13 KwaZulu isolates were resistant to amodiaquine and most isolates showed reduced susceptibility to quinine and sulphadoxine/ pyrimethamine. / Andrew Chakane 2018

A thesis submitted to the Faculty of Health Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2014 / Malaria is the most serious tropical infectious disease in humans, caused by parasites of the Plasmodium genus and transmitted by anopheline mosquitoes. The interaction between the parasites and vectors has become a focus for malaria research as it may present an alternative disease control method by enhancing anti-plasmodial factors within the mosquito to impede parasite development and transmission. Anopheles gambiae is the best studied African malaria vector and is often used with the murine malaria parasite, Plasmodium berghei, for investigating parasite-vector interactions in the laboratory setting. Anopheles funestus has not been studied and its interactions with Plasmodium were unknown, until now. Although the Vector Control Reference Laboratory routinely maintains An. funestus and a number of An. gambiae colonies, none have been infected with Plasmodium since their establishment. This study aimed to use P. berghei to determine the vectorial capacity of these colonies and to examine the involvement of the 2La paracentric chromosomal inversion and antimicrobial peptides during Plasmodium infection in An. gambiae and An. funestus, respectively. Most of the An. gambiae complex colonies were susceptible to P. berghei, but the range of feeding and infection rates varied considerably. The infection rates for some of the older colonies were lower than previously documented. Anopheles funestus colonies were all viable vectors and there was an inverse correlation between the insecticide resistance profile and parasite susceptibility. Increased detoxification enzyme activities may have been contributing to a greater degree of parasite elimination. In An. gambiae, molecular karyotyping of the 2La inversion using PCR was validated against traditional cytogenetic techniques. The PCR was shown to be a reliable substitute for identifying the inversion. Using molecular karyotyping on 2La polymorphic colonies infected with P. berghei, it was found that infected females were more likely to carry the 2La inversion, indicating possible correlation between the inversion and susceptibility to parasites. In An. funestus, the expression of antimicrobial pepetide genes during P. berghei infection was examined using real-time PCR. Although all three genes showed increased activity at certain points of the infection, none displayed significant anti-plasmodial properties. However, in the less parasite susceptible strain, expression of two genes was higher towards the end of the infection, which was not observed in the other strains. It is possible that the co-expression of both peptides has led to a decrease in parasite load in late infection, but given the multi-factorial nature of the parasite-vector interaction, further investigation is required.

Malaria is a tropical disease affecting millions of people worldwide. Plasmodium falciparum is the causative agent of the most severe form of malaria, and therefore insights into the molecular mechanisms by which it functions are critical. The intraerythrocytic stage of the life cycle is responsible for the clinical manifestations of the disease. Numerous proteins are required for the invasion and remodelling of host erythrocytes, and need to be transported to the highly specialized organelles from which they are secreted (invasion proteins), or to the erythrocyte cytoplasm or membrane (exported proteins). It is postulated that newly synthesized proteins are transported from the Golgi network to their target destinations by specific interactions of target sequences of the proteins with the medium subunit (μ) of an adaptor protein (AP1) complex. Bioinformatic analysis of the putative P. falciparum AP1μ subunit, encoded by Pf13_0062, revealed a cargo-binding domain. Three regions, one of which encompassed the putative binding domain, while the other two interrupted this domain, were cloned into the pGEX-4T-2 expression vector. These recombinant proteins were expressed in E. coli with a GST tag, purified and immobilized on glutathione magnetic beads and used to biopan P. falciparum phage display libraries to identify interacting proteins. No binding was observed with the truncated domains, but several specific interactions were identified with the binding domain. One of these peptides was 13 amino acids long and contained a Yxx motif, indicating that PfAP1, like its homologues in higher eukaryotes, binds specifically to this motif in cargo proteins. Other sequences identified included a RRNIFLFINRKKE peptide; exported protein PHISTa; and conserved protein PFL0675c. In the C-terminal region of PFL0675c an armadillo repeat structure was predicted, just downstream of the binding domain identified by biopanning. This region of PFL0675c was therefore cloned into the pET-15b expression vector and expressed as a recombinant His-tagged protein. Slot overlays and far western blotting confirmed the specificity of the interaction with PfAP1. Since PFL0675c does not display the characteristics typical of AP1 cargo, it is postulated to be an accessory protein to the complex. Localization studies performed by transfection V of P. falciparum parasites with pARL2AP1GFP showed that in vivo, PfAP1 localized to distinct foci around the nucleus. Co-localization studies confirmed that PfAP1 localizes to the cis-Golgi in P. falciparum. PfAP1 may therefore be involved in trafficking proteins from the Golgi network to specific subcellular compartments within the parasite. This is the first study identifying interacting partners of PfAP1, and demonstrating its localization in P. falciparum 3D7 parasites.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johannesburg, November 2012. / Malaria continues to be a devastating disease. Plasmodium falciparum is the most lethal human malaria parasite, responsible for the majority of the hundreds of millions of cases of malaria and approximately 665,000 thousand deaths in 2010. Understanding the biology of the parasite is vital in identifying potential drug targets necessary to develop novel treatments to combat the disease. During every 48-hour asexual intra-erythrocytic replication cycle, a single parasite can produce up to 32 progeny. This extensive proliferation implies that parasites require substantial amounts of lipid precursors. Glycerol kinase (GK) is a highly conserved enzyme that functions at the interface of lipid synthesis and carbohydrate metabolism. GK catalyzes the ATP-dependent phosphorylation of glycerol to glycerol-3-phosphate, a major phospholipid precursor. In this study, the full length 1,506bp P. falciparum glycerol kinase (PfGK) gene was cloned and expressed as a glutathione S-transferase (GST) fusion protein in E. coli. The recombinant PfGK (rPfGK) enzyme was predominantly expressed as an insoluble aggregate, however, ~3μg soluble rPfGK was purified from an 800ml induced culture. SDS-PAGE analysis showed that the protein migrated at ~73kDa and its enzyme activity was verified using an ADP-coupled spectrophotometric assay. The kinetic parameters for rPfGK were Km = 15.7μM for glycerol and Km = 15.9μM for ATP. To evaluate the role of the enzyme in asexual blood-stage development, PfGK was disrupted using double crossover homologous DNA recombination to generate a glycerol kinase knockout parasite line (3D7ΔPfGK). Southern hybridization and PfGK mRNA expression analysis verified that the gene had been disrupted. 3D7ΔPfGK growth rates were evaluated using thiazole orange, a DNA staining dye, coupled to flow cytometry analysis for improved sensitivity. Highly synchronized ring stage parasites were monitored over one 48-hour developmental cycle and results showed that 3D7ΔPfGK growth was significantly reduced to 56.5 ± 1.8% when compared to wild type parasites. This reduced proliferation of 3D7ΔPfGK knockout parasites suggests that PfGK is required for optimal proliferation during the blood stages but is not essential for viability and therefore, not a potential drug target. However, PfGK mRNA expression is markedly elevated in gametocytes and sporozoites. This suggests that PfGK may play a significant role in the mosquito- and liver-stage parasites, with implications for a potential transmission-blocking target. Thus, using a novel bioinformatics method, Evolutionary Patterning, in combination with structural modelling, three potential drug target sites that were different to the human GK orthologue and less likely to develop resistance to compounds were identified. Further studies in the mosquito stages will provide insight into the role of PfGK in the lifecycle of P. falciparum parasites.

A Thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg in the fulfillment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2017. / Malaria continues to be a global health problem. Despite a marked reduction in mortality over the last 15 years, these hard-fought gains are threatened by growing resistance of the Plasmodium falciparum malaria parasite to artemisinin, the frontline drug used in treatment of the disease. Clinical symptoms of malaria are caused by the intra-erythrocytic phase of the parasite life cycle. Entry into the erythrocyte is accomplished by several specialised invasion proteins, which are stored in unique apical secretory organelles known as micronemes and rhoptries. Very little is known about the trafficking signals and transport mechanisms of invasion proteins to these organelles. Three micronemal proteins, Apical Membrane Antigen-1 (PfAMA-1), Subtilisin-like protease 2 (PfSUB2) and Erythrocyte Binding Antigen 181 (PfEBA181) were investigated with the aim of identifying domains responsible for targeting the micronemes. Selected domains were amplified and mini-genes were created by overlap extension PCR. A pARLmCherry plasmid containing a Pfama-1 stage-specific promoter that is only active during the schizont stage of parasite development when micronemes are formed, was used to create mCherry-tagged constructs. P. falciparum parasites were transfected by electroporation of the plasmid constructs. Transgenic parasites were selected by drug pressure and the expression of red fluorescent mCherry-tagged chimaeric proteins was visualised in live parasites. Co-localisation studies were performed with a microneme marker to assess if the transgenic mini-proteins reached their destination. Interestingly, all three proteins required different domains to target the micronemes: PfEBA181 required an extended region of a conserved cysteine-rich domain, PfAMA-1 required the prodomain, and PfSUB2 required the transmembrane domain. Since no common targeting signal was identified, the possibility of a protein escorter was explored. The PfAMA-1 prodomain was expressed as a recombinant histidine-tagged protein and immobilised onto Nickel-coated beads, which were exposed to a P. falciparum phage display library for four rounds of biopanning. Two novel binding partners were identified: a putative Chaperone Binding Protein and a putative Formin 2. The identification of the molecular trafficking determinants of three invasion proteins, as well as a potential protein escorter for microneme targeting, represent novel findings that extend our knowledge of a fundamental biological process in the malaria parasite. This pathway may be exploited for drug development and new malaria treatment strategies. / MT2017

Os mosquitos pertencentes à família Culicidae (Diptera) desempenham importante papel como vetores de patógenos causadores de doenças que acometem o homem em várias partes do mundo (MARCONDES, 2001), causando um grande impacto socioeconômico e de saúde pública nas áreas em que ocorrem. Aspectos biológicos e comportamentais dos mosquitos também contribuem decisivamente para sua atuação como transmissor de patógenos que atingem as populações humanas. O repasto sanguíneo é um processo fisiológico fundamental no ciclo de vida destas espécies. Por outro lado, a alimentação sanguínea infectada causa mudanças no metabolismo das fêmeas recémingurgitadas, desencadeando a expressão diferencial de genes envolvidos em respostas imunológicas, como também de genes envolvidos em outros processos fisiológicos. Como um dos interesses de nosso grupo é estudar as alterações sofridas pelo inseto vetor após a infecção por patógeno, nesse trabalho o ponto de partida para esse estudo foi a escolha dos genes DV431431, DV340891 e DV411647 que nas análises in silico apresentaram expressão diferencial mediante a infecção por vírus da Dengue ou Plasmodium gallinaceum. Após experimentos de RT-PCR em tempo real, foi mostrado que apenas o gene DV411647 apresentou expressão elevada 48 horas após a alimentação sanguínea infectada tanto por P. gallinaceum como por vírus dengue. Na busca por uma função predita desse gene em mosquitos, nós utilizamos a ferramenta Editseq através do software DNAstar para traduzir o peptídeo que apresentou maior número de aminoácidos, e confrontamos através de Blastp com o banco de dados GenBank, obtivemos uma provável desidrogenase/redutase de cadeia curta (AAK55494). Experimentos de Western blot evidenciaram que esta proteína esta presente no intestino médio das fêmeas 72 horas após a alimentação com sangue infectado por vírus dengue tipo 2. / Mosquitoes belonging to the family Culicidae (Diptera) play an important role as vectors of disease-causing pathogens that affect humans in various parts of the world (Marcondes, 2001), causing a great socioeconomic impact and public health areas in which they occur. Biological and behavioral aspects of mosquito also contribute decisively to its role as a transmitter of pathogens that affect human populations. The blood meal is a fundamental physiological process in the life cycle of these species. On the other hand, the infected blood meal causes changes in the metabolism of newly engorged females, triggering the differential expression of genes involved in immune responses, as well as genes involved in other physiological processes. As one of the interests of our group is to study the changes undergone by the insect vector after infection with the pathogen, in this work the starting point for this study was the selection of genes DV431431, DV340891 and DV411647 that the in silico analysis showed differential expression by Dengue virus infection of Plasmodium gallinaceum.Após or RT-PCR experiments in real time, it was shown that only the gene DV411647 had high expression 48 hours after the blood meal infected by both P. gallinaceum as for dengue virus. In the search for a predicted function of this gene in mosquitoes, we use the tool Editseq by DNAstar software to translate the peptide with the highest number of amino acids, and confronted by blastp with the GenBank database, we obtained a probable dehydrogenase / reductase short chain (AAK55494). Western blot experiments showed that this protein is present in the midgut of females 72 hours after feeding with blood infected with dengue virus type 2.

Os mosquitos pertencentes à família Culicidae (Diptera) desempenham importante papel como vetores de patógenos causadores de doenças que acometem o homem em várias partes do mundo (MARCONDES, 2001), causando um grande impacto socioeconômico e de saúde pública nas áreas em que ocorrem. Aspectos biológicos e comportamentais dos mosquitos também contribuem decisivamente para sua atuação como transmissor de patógenos que atingem as populações humanas. O repasto sanguíneo é um processo fisiológico fundamental no ciclo de vida destas espécies. Por outro lado, a alimentação sanguínea infectada causa mudanças no metabolismo das fêmeas recémingurgitadas, desencadeando a expressão diferencial de genes envolvidos em respostas imunológicas, como também de genes envolvidos em outros processos fisiológicos. Como um dos interesses de nosso grupo é estudar as alterações sofridas pelo inseto vetor após a infecção por patógeno, nesse trabalho o ponto de partida para esse estudo foi a escolha dos genes DV431431, DV340891 e DV411647 que nas análises in silico apresentaram expressão diferencial mediante a infecção por vírus da Dengue ou Plasmodium gallinaceum. Após experimentos de RT-PCR em tempo real, foi mostrado que apenas o gene DV411647 apresentou expressão elevada 48 horas após a alimentação sanguínea infectada tanto por P. gallinaceum como por vírus dengue. Na busca por uma função predita desse gene em mosquitos, nós utilizamos a ferramenta Editseq através do software DNAstar para traduzir o peptídeo que apresentou maior número de aminoácidos, e confrontamos através de Blastp com o banco de dados GenBank, obtivemos uma provável desidrogenase/redutase de cadeia curta (AAK55494). Experimentos de Western blot evidenciaram que esta proteína esta presente no intestino médio das fêmeas 72 horas após a alimentação com sangue infectado por vírus dengue tipo 2. / Mosquitoes belonging to the family Culicidae (Diptera) play an important role as vectors of disease-causing pathogens that affect humans in various parts of the world (Marcondes, 2001), causing a great socioeconomic impact and public health areas in which they occur. Biological and behavioral aspects of mosquito also contribute decisively to its role as a transmitter of pathogens that affect human populations. The blood meal is a fundamental physiological process in the life cycle of these species. On the other hand, the infected blood meal causes changes in the metabolism of newly engorged females, triggering the differential expression of genes involved in immune responses, as well as genes involved in other physiological processes. As one of the interests of our group is to study the changes undergone by the insect vector after infection with the pathogen, in this work the starting point for this study was the selection of genes DV431431, DV340891 and DV411647 that the in silico analysis showed differential expression by Dengue virus infection of Plasmodium gallinaceum.Após or RT-PCR experiments in real time, it was shown that only the gene DV411647 had high expression 48 hours after the blood meal infected by both P. gallinaceum as for dengue virus. In the search for a predicted function of this gene in mosquitoes, we use the tool Editseq by DNAstar software to translate the peptide with the highest number of amino acids, and confronted by blastp with the GenBank database, we obtained a probable dehydrogenase / reductase short chain (AAK55494). Western blot experiments showed that this protein is present in the midgut of females 72 hours after feeding with blood infected with dengue virus type 2.