Malaria is a major health problem, mainly in developing countries, and causes an estimated 1 million deaths per year. Plasmodium falciparum is the major type of human malaria parasite, and causes the most infections and deaths. Malaria drugs, like any other drugs, suffer from possible side effects and the potential for emergence of resistance. Chloroquine, which was a very effective drug, has been used since about 1945, but its use is severely limited by resistance, even though it has mild side effects, and is otherwise very efficacious. Research has shown that there are chloroquine reversal agents, molecules that can reinstate antimalarial activity of chloroquine and chloroquine-like drugs; many such reversal agents are composed of two aromatic groups linked to a hydrogen bond acceptor several bonds away. By linking a chloroquine-like molecule to a reversal agent-like molecule, it was hoped that a hybrid molecule could be made with both antimalarial and reversal agent properties. In the Peyton Lab, such hybrid "Reversed Chloroquine" molecules have been synthesized and shown to have better antimalarial activity than chloroquine against the P. falciparum chloroquine-sensitive strain D6, as well as the P. falciparum chloroquine-resistant strains Dd2 and 7G8. The work reported in this manuscript involves simplifying the reversal agent head group of the Reversed Chloroquine molecules, to a single aromatic ring instead of the two rings groups described by others; this modification retained, or even enhanced, the antimalarial activity of the parent Reversed Chloroquine molecules. Of note was compound PL154, which had IC50 values of 0.3 nM and 0.5 nM against chloroquine-sensitive D6 and chloroquine-resistant Dd2. Compound PL106 was made to increase water solubility (a requirement for bioavailability) of the simplified Reversed Chloroquine molecules. Molecular modifications inherent to PL106 were not very detrimental to the antimalarial activity, and PL106 was found to be orally available in mice infected with P. yoelli, with an ED50 value of about 5.5 mg/kg/d. Varying the linker length between the quinoline ring and the protonatable nitrogen, or between the head group and the protonatable nitrogen, did not have adverse effects on the antimalarial activities of the simplified Reversed Chloroquine molecules, in accord with the trends observed for the original design of Reversed Chloroquine molecules as found from previous studies in the Peyton Lab. The simplified Reversed Chloroquine molecules even tolerated aliphatic head groups (rather than the original design which specified aromatic rings), showing that major modifications could be made on the Reversed Chloroquine molecules without major loss in activity. A bisquinoline compound, PL192, was made that contained secondary nitrogens at position 4 of the quinoline ring (PL192 is a modification of piperaquine, a known antimalarial drug that contains tertiary nitrogens at position 4 of the quinoline ring); this compound was more potent than piperaquine which had an IC50 value of 0.7 nM against CQS D6 and an IC50 of 1.5 nM against CQR Dd2, PL192 had IC50 values of 0.63 nM against chloroquine sensitive D6 and 0.02 nM against chloroquine resistant Dd2. Finally, the mechanism of action of these simplified "Reversed Chloroquines" was evaluated; it was found that the simplified "Reversed Chloroquines" behaved like chloroquine in inhibiting β-hematin formation and in heme binding. However, the simplified "Reversed Chloroquines" were found to inhibit chloroquine transport for chloroquine resistant P. falciparum chloroquine resistance transporter expressed in Xenopus oocytes to a lesser extant than the classical reversal agent verapamil. From these studies it was noted that the simplified "Reversed Chloroquines" may not behave as well as classical reversal agents would in restoring chloroquine efficacy, but they are very potent, and so could be a major step in developing drug candidates against malaria.

Background Promotion of job opportunities and tourism in African countries has led to an increase in expatriates in malaria endemic areas. A paucity of data exist on characteristics and numbers of expatriates and long-term travellers being evacuated from sub-Saharan Africa for suspected malaria infections diagnosed while still in Africa. Methods A retrospective flight record review of a South African fixed-wing air-ambulance provider from June 2006 through July 2009 was performed. Adult expatriates and long-term travellers with suspected malaria being evacuated from sub-Saharan African countries to Johannesburg, South Africa were included. Results Suspected malaria was the single most common diagnosis for dispatching airambulances with 81 (11.9%) of the 679 flights. Accuracy of the initial diagnosis, based on confirmation of malaria at the receiving facility was 78.4% for blood smears, 92.3% for rapid detection tests and 42.8% for clinical signs alone. P. falciparum (alone, or in combination with other Plasmodium species) was the most frequently isolated species at both the referring (100%) and receiving (88.2%) facilities in cases where the species was documented. The suspected malaria patients were predominantly male 69 (84.1%), with a mean age of 42.1 ±12.8 years, and were in sub-Saharan Africa for occupational reasons 65 (79.3%). Angola, the Democratic Republic of Congo and Mozambique were the countries of origin in 48 (58.5%) of the suspected malaria flights. Compliance on appropriate malaria chemoprophylaxis was documented in two (2.4%) suspected malaria patients. Intubation as a marker of severity was required for 15 (18.3%) patients, and one (1.2%) patient died inflight. No statistically significant difference (p=0.50) was shown for intubation requirements when comparing patients who had utilised malaria chemoprophylaxis with the patients who had not utilised chemoprophylaxis. Conclusions Patients presented in advanced stages of severe/complicated malaria with concurrent poor chemoprophylaxis utilisation and compliance. Appropriate chemoprophylaxis did not decrease the severity of presentation (based on intubation requirements) and did not guarantee complete malaria protection.

The strides made in malaria eradication efforts have been aided by a combination of vector control and chemoprevention. However, Plasmodium resistance to first-line artemisinin-based combination therapies (ACTs), and mosquito resistance to insecticides threatens the progress made. Innovative vector control measures, vaccines and antimalarial drugs with novel modes of action are key to disease eradication. High-throughput phenotypic screening of chemical libraries tested directly against all the stages of the Plasmodium lifecycle have been the mainstay of antimalarial drug discovery efforts and have identified compounds that are effective in parasite clearance. Unfortunately, these screens are handicapped in that they are unable to specify the actual compound targets in the Plasmodium parasites. As a result, many candidate hits have had to be re-screened in specific assays to determine putative mechanisms of antiplasmodial action. Predictably, this has elevated target-specific screens as the next frontier in drug discovery. This shift has been aided by a number of factors, including the cost effectiveness of these screens and the fact that target-specific screens do not always require specialized access to parasites. When combined with knowledge of the target’s structure, where known, target-specific screens have the potential to give lead compounds with impeccable potency and selectivity. This approach has already been successfully put to use, for example, in the identification of P. falciparum p-type ATPase 4 (PfATP4) and P. falciparum phosphatidylinositol 4-kinase (PfPI(4)K) inhibitors. The new challenge now is the identification of quality targets. Here, computational biology ‘omics’ tools have proved to be an invaluable resource. Two of the more commonly used of these tools are genomics and metabolomics. In-vitro evolution assays followed by whole genome sequencing analysis is a popular genomics approach and helps unveil novel target genes. Plasmodium parasites are exposed to sublethal doses of a compound until an upward shift in the half-maximal inhibitory concentration (IC50), indicative of resistant parasites, is observed in the culture. Sequenced genomes of the resistant parasite clones are compared to those of the drug-naive parent to reveal genetic changes, which include both single nucleotide polymorphisms (SNPs) and copy number variations (CNVs). While these genomic changes may point to genes encoding actual drug targets, they often reveal mediators of drug resistance or tolerance. Follow-up assays like SNP validation through gene editing are necessary to distinguish between actual targets, resistance mechanisms and random background mutations. Expectedly, genetic changes in uncharacterized Plasmodium genes are the bottle-necks in the identification of novel druggable targets. Even so, this genomics method has uncovered or reconfirmed novel antimalarial drug targets, including the proteasome, aminophospholipid-transporting P-type ATPase (PfAT-Pase2) and cGMP-dependent protein kinase (PfPKG). Metabolomic profiling and transcriptomics narrows down a compound’s mode of action. Here, parasites are treated with a compound of interest and the metabolites extracted and analyzed using liquid chromatography-mass spectrometry (LC-MS). The metabolomics fingerprint or metaprint is then compared to that of untreated parasites. While this method rarely provides the exact drug target, it narrows down the compound’s mode of action, which is valuable for target validation and characterization. The issue of non-specific or non-viable phenotype metabolite signals is easily filtered out by treating parasites with various drug concentrations and/or over a period of time. Other areas that limit the effectiveness of this tool and need to be addressed include the analysis of compounds that do not act through metabolic pathway disruption and potential host contamination. Nonetheless, metabolomics are a key player in drug discovery and have successfully been used in the study of pantothenamides (MMV689258) where the observed CoA analog buildup helped identify their mechanism of action in sequestering coenzyme A to block acetyl-CoA anabolism. Presented herein is a culmination of my graduate research in antimalarial drug discovery. Three independent projects are presented, and they all have either been published or are currently under reviewership. Chapter 1 is an introduction to malaria, a disease that has and continues to claim hundreds of thousands of lives, especially in my home continent of Africa. In chapter 2, I detail the experimental procedures used to generate the data presented in chapters 3-5. Chapter 3 is a detailed susceptibility profiling and metabolomic fingerprinting of Plasmodium falciparum asexual blood stages (ABS) to clinical and experimental antimalarials. This work, published in Cell Chemical Biology (2020), presents to the malaria research community a medium-throughput assay that can be utilized to identify new antimalarial lead compounds and novel assayable targets. Chapter 4 presents a detailed analysis of a novel ATP-binding cassette (ABC) transporter that confers pleiotropic antimalarial drug resistance in P. falciparum and that was first identified through in vitro evolution assays. This work is currently under review in Cell Chemical Biology. Chapter 5 presents work on an promising new preclinical compound, MMV688533, that provides single-dose cure and that was discovered using an innovative orthology-based screen by the Sanofi drug discovery team. In this chapter, I also present in detail the assays performed to better understand this compound’s mode of antiplasmodial action and the potential drivers of parasite resistance. This work has been accepted, pending minor textual revisions, in Science Translational Medicine. Finally in chapter 6, I summarize chapters 3-5 and share future follow-up work needed to strengthen and contextualize some of the experimental findings presented here.

En 2020, la malaria a provoqué 241 millions de cas d'infections et 627 000 morts. La faible efficacité du vaccin disponible et la résistance aux traitements rendent indispensable l'identification de cibles thérapeutiques. Plasmodium falciparum (Pf) envahit les érythrocytes pour s'y répliquer. Pour cela, de protéines contenues dans des organites d'invasion, tels que les micronèmes et les rhoptries rassemblés à un complexe apical, sont sécrétées. Le trafic des protéines entre l'appareil de Golgi et les organites d'invasion est médié par la PfSortiline. Dans les organismes modèles, la sortiline est recyclée entre les organites cibles et l'appareil de Golgi via le complexe protéique rétromère composé des protéines de tri vacuolaire (Vps) Vps26-29-35. Ce complexe est recruté via les complexes VpsC, composé de Vps11-16-18-33, CORVET composé de Vps3-8 et HOPS, composé de Vps39-41. Chez Pf, l'ensemble des composants des complexes VpsC et rétromère sont conservés. Seulement PfVps3 du complexe CORVET est retrouvée et le complexe HOPS est absent. L'hypothèse du projet est que ces protéines conservées sont impliquées dans la biogenèse des organites du complexe apical via le recyclage de la PfSortiline vers l'appareil de Golgi. Pour vérifier cela, des souches de parasites exprimant les protéines de fusion PfVps3-11-16-18-29 étiquetées à un domaine GFP ont été construites. Des techniques de Western Blot et de microscopie à fluorescence ont montré que ces protéines de fusion sont exprimées lors du cycle érythrocytaire. Il semble que PfVps29-GFP localise à des structures semblables aux endosomes et partiellement aux micronèmes. Les protéines PfVps16-18-GFP semblent localiser aux micronèmes et partiellement aux rhoptries et à l'appareil de Golgi. Finalement, des souches dans lesquelles les protéines PfVps3-16-29-GFP peuvent être délocalisées de façon conditionnelle ont été construites. Il a été montré que PfVps16-GFP semble essentielle à la survie de Pf. Ce projet participe à la caractérisation de nouvelles pistes thérapeutiques antipaludiques. / Malaria was responsible for 627,000 deaths and 241 million infections in 2020 alone. Drug resistance, and the poor efficacy of the only available vaccine, are strong arguments supporting the need to identify therapeutic targets. The malaria parasite Plasmodium falciparum (Pf) invades erythrocytes and multiplies inside them. To do so, it secretes invasion proteins located inside organelles, like micronemes and rhoptries, which are localised at an apical complex. Protein trafficking from the Golgi apparatus to these organelles is dependent on PfSortilin. In model organisms, this protein is recycled between the target organelles and the Golgi Apparatus by a protein complex called retromer. This complex is composed of Vacuolar Sorting Proteins (Vps)-26-29-35. The retromer complex is recruited by complexes, composed of Vps11-16-18-33, CORVET, composed of Vps3-8, and HOPS, composed of Vps39-41. In Pf, all components of the retromer and the VpsC complexes are conserved. However, only PfVps3 of the CORVET complex is conserved and the HOPS complex is absent. We hypothesized that conserved proteins play a key role in apical complex biogenesis by recycling PfSortilin to the Golgi apparatus. To verify the hypothesis, parasite strains coding the fusion proteins PfVps3-11-16-18-29 tagged with a GFP were generated. Western Blot and fluorescence microscopy showed that those proteins are expressed during the erythrocyte life cycle. PfVps29-GFP seemed to localize at endosome-like structures and partially at micronemes. PfVps16-18 seemed to localise at micronemes too and partially at rhoptries and at the Golgi apparatus. Finally, strains where PfVps3-16-29 could be functionally mislocalized have been generated. This technique showed that PfVps16-GFP were essential for Pf survival. Our work could lead to the characterization of new antimalarial drug targets.

The emergence and spread of drug resistance in Plasmodium falciparum has consistently been a major barrier to the control and eradication of malaria. Resistance to the affordable and fast-acting former first-line drug chloroquine (CQ) was first observed in the 1950s near the Thai-Cambodian border and in South America. Resistance later spread from Asia to highly endemic regions in Africa, with reports of up to 6-fold increases in regional malaria mortality rates. The replacement drug, sulfadoxine-pyrimethamine (SP), encountered resistance within one year of clinical use. Artemisinin-(ART) based combination therapies (ACTs), which consist of a fast-acting ART derivative and a slower-acting partner drug, became the global first-line standard in 2000 and, along with mosquito vector control measures, helped decrease mortality rates by 60%. Unfortunately, parasites resistant to ART derivatives arose in Southeast Asia. This compromised the effectiveness of the ACT partner drug piperaquine (PPQ) and resistance to this drug was first reported in 2015, around a decade after the introduction of dihydroartemisinin-PPQ. By 2019, PPQ resistance, driven primarily by a series of mutations in the P. falciparum chloroquine resistance transporter (PfCRT), was widespread in Southeast Asia, resulting in >50% failure upon treatment with dihydroartemisinin-PPQ. Malaria mortality rates have surged recently, causing an estimated 619,000 deaths in 2021. Sub-Saharan Africa is most heavily affected by this disease where 78.9% of deaths are of young children. To this date, PPQ remains effective in Africa. It is a major concern that PPQ resistance will arise on this continent, however, given the importance of PPQ in current efforts to expand the range of antimalarial interventions and reverse the current rise of malaria cases in Africa. Understanding PPQ resistance mechanisms and their effect on parasite biology is critical to creating effective treatments and minimizing the impact of drug-resistant P. falciparum malaria. This thesis aims to investigate the earliest reports of PPQ resistance, to define the PPQ susceptibility and parasite fitness of contemporary SE Asian parasite strains, and to predict future dominant strains in the field to further our understanding of parasite resistance mechanisms and combat the spread of drug-resistant malaria. In Chapter 3, we show that earlier reports of PPQ resistance in Yunnan Province, China could be explained by the unique China C PfCRT variant. Using gene editing, we reveal that this variant confers a loss of fitness and parasite re-sensitization to the chemically related former first-line antimalarial CQ, while acquiring PPQ resistance via drug efflux. We employ biochemical assays to measure mutant PfCRT-mediated drug transport and molecular dynamics simulations with the recently solved PfCRT structure to assess changes in the central drug-binding cavity. This study provides impetus for adding CQ into an antimalarial treatment regimen where PPQ has lost efficacy. In response to widespread treatment failures, PPQ was removed as a first-line partner drug. Recently, additional mutations have been observed on the highly-resistant Dd2+F145I PfCRT isoform. These mutations developed in parasites in long-term in vitro culture or in Southeast Asian field isolates. In Chapter 4, I characterized the impact of these mutations on parasite fitness and antimalarial susceptibility by editing asexual blood stage parasites to express these mutant PfCRT haplotypes. Competitive growth assays with a GFP-expressing reporter line revealed that these additional mutations reduce the fitness defect imposed by F145I, likely the primary driver of their emergence. I found that these mutations differentially impact parasite susceptibility to PPQ and CQ in in vitro dose-response assays. I used proteoliposome-based drug uptake studies, molecular dynamic simulations, and peptidomics to detail the molecular features of drug resistance and parasite physiology of these lines. These experiments provide insight into parasite responses to the changing drug selective pressures in SE Asia to inform treatment strategies in this region moving forward. In Chapter 5, I sought to determine whether Asian PPQ-resistant PfCRT mutations could also mediate PPQ resistance on African PfCRT haplotypes. Using zinc-finger nuclease-based gene editing, I introduce the most common African mutant pfcrt alleles with a SE Asian PfCRT mutation into Dd2 parasites. In PPQ survival assays, these mutations only confer high-grade PPQ resistance (defined as ≥10% survival at 200 nM) on the FCB PfCRT background. I assessed the susceptibility of these gene-edited isogenic lines to other clinical antimalarials and the relative fitness of these engineered lines with in vitro assays. These experiments clearly show that there is a genetic path to PPQ resistance in African parasites; however, they also suggest that fitness costs associated with these mutations may hinder the spread of resistance. Our data provide important insights into PPQ resistance. In chapter 6, these findings are summarized along with future studies to strengthen and expand on the findings presented herein.

Plasmodium falciparum, the primary causative agent of malaria in humans, is responsible for life-threatening infections and disease in many tropical regions of the world. In 2018, there were over 228 million cases and 405,000 deaths due to malaria infection, according to World Health Organization estimates. While there has been recent progress in decreasing mortality rates attributed to malaria, the emergence of widespread antimalarial drug resistance in recent decades has endangered such progress. Therefore, there is an urgent need for new antimalarial drugs with a novel mechanism of action. Plasmodium kinases could serve as attractive drug targets due to their essential functions in all stages of the parasite’s life cycle. Plasmodium falciparum Aurora-related kinases (PfArks) have essential regulatory roles in all stages of the parasite’s asexual intraerythrocytic life cycle. As a result, it is hypothesized that PfArks are excellent potential molecular targets for novel antimalarial development. The intent of this study was to identify potent and selective inhibitors of Plasmodium from an Aurora kinase-focused commercial inhibitor library of 3,000 compounds. An initial phenotypic screen was performed at a fixed inhibitor concentration of one micromolar to identify novel compounds with potency against the P. falciparum chloroquine-resistant Dd2 strain. From this library, we have identified multiple compounds with submicromolar antiplasmodial activity in asexual intraerythrocytic life cycle stages and adequate selectivity. Additionally, this project aimed further to characterize the cellular mechanism of action of hit compounds. Multiple compounds were found to exhibit inhibitory effects against early intraerythrocytic asexual life cycle stages as well as liver stages. At this time, one hit compound (DC-6275) was found to inhibit asexual intraerythrocytic as well as liver stage parasites in addition to PfArk1 amongst other Plasmodium protein kinases tested. Overall, we believe that these identified compounds have great potential to serve as scaffolds for future antimalarial drug development.

No description available.

No description available.

Malaria parasite (Plasmodium spp.) infection in the mosquito Anopheles stephensi induces significant expression of A. stephensi nitric oxide synthase (AsNOS) in the midgut epithelium as early as 6 h post-infection and intermittently thereafter. This induction results in the synthesis of inflammatory levels of nitric oxide (NO) in the blood-filled midgut that limit parasite development. However, the Plasmodium-derived factors that can induce AsNOS expression and the signaling pathways responsible for transduction in A. stephensi have not been identified until completion of the work described herein. In my studies, I have determined that P. falciparum glycosylphosphatidylinositol (PfGPIs) can induce AsNOS expression in A. stephensi cells in vitro and in the midgut epithelium in vivo. Based on related work in mammals, I hypothesized that parasite-derived AsNOS-inducing factors signal through the insulin signaling pathway and the NF-kappaB-dependent Toll and Immune deficiency (Imd) signaling pathways. In support of this hypothesis, I have determined that signaling by P. falciparum merozoites and PfGPIs is mediated through A. stephensi protein kinase B (Akt/PKB) and DSOR1 (mitogen activated protein kinase kinase, MEK)/Extracellular signal-regulated protein kinase (ERK), kinases which are associated with the insulin signaling pathway. However, signaling by P. falciparum and PfGPIs is distinctively different from signaling by insulin and these parasite signals are not insulin-mimetic to A. stephensi cells. In other studies, treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, reduced AsNOS expression by P. falciparum merozoites in A. stephensi cells. This result suggested the involvement of Toll and Imd pathways in parasite signaling of mosquito cells. Knockout of Pelle, a proximal signaling protein in the Toll pathway, increased AsNOS expression following parasite stimulation, suggesting that the Toll pathway may negatively regulate signaling by Plasmodium-derived AsNOS-inducing factors. In contrast, knockout of TGF-beta-activated kinase 1 (Tak1), a proximal signaling protein in the Imd pathway, reduced AsNOS expression by 20% relative to the control, suggesting that the Imd pathway is required for signaling by Plasmodium-derived AsNOS-inducing factors. Despite the NO-rich environment of the midgut, Plasmodium development is not completely inhibited. This observation suggests that Plasmodium may have efficient detoxification systems during sexual development in A. stephensi. To identify Plasmodium defense genes that may defend parasites against nitrosative stress caused by AsNOS induction, expression of several antioxidant defense genes known to function in nitrosative stress defense in a variety of organisms were examined during sporogonic development. Notably, increased expression levels of P. falciparum peroxiredoxins containing 1 or 2 cysteines (1-cys or 2-cys PfPrx) were associated with periods of parasite development just prior to and during parasite penetration of midgut epithelium, an event associated with significant AsNOS induction in the midgut. The provision of N omega-L-arginine (L-NAME), a known inhibitor of NOS enzyme activity, to A. stephensi with Plasmodium culture by artificial bloodmeal significantly reduced expression of 1-cys and 2-cys PfPrx indicating that these gene products may function to protect parasites against nitrosative stress induced by AsNOS. / Ph. D.

Both vertebrates and anopheline mosquitoes inhibit <i>Plasmodium</i> spp. (malaria parasite) development via induction of nitric oxide (·NO) synthase. Expression of <i>Anopheles stephensi</i> ·NO synthase (<i>AsNOS</i>) is induced in the midgut epithelium beginning at 6 h following a <i>Plasmodium berghei</i>-infected blood meal. ·NO reacts readily with other biocompounds forming a variety of reactive nitrogen intermediates (RNIs) that may impose a nitrosative stress. These RNIs are proposed to be responsible for the AsNOS-dependent inhibition of <i>Plasmodium</i> development. In my studies, I identified several RNIs that are induced in the blood-filled midgut in response to <i>Plasmodium</i> infection. Stable end products of ·NO (NO₃⁻ and NO₂⁻), measured using a modified Griess assay, are elevated in infected midguts at 24 h post-blood meal (pBM). Further studies using chemical reduction-chemiluminescence with Hg displacement showed that infected midguts contained elevated levels of potentially toxic higher oxides of nitrogen (NO<SUB>x</SUB>), but <i>S</i>-nitrosothiol (SNO) and nitrite levels did not differ between infected and uninfected midguts at 12.5 and 24 h pBM. Thus, nitrates contributed to elevated NO<SUB>x</SUB> levels. SNO-biotin switch westerns indicated that <i>S</i>-nitrosated midgut proteins change over the course of blood meal digestion, but not in response to infection. Photolysis-chemiluminescence was used to release and detect bound ·NO from compounds in blood-filled midguts dissected from 0-33 h pBM. Results showed increased ·NO levels in <i>Plasmodium</i>-infected midgut lysates beginning at 8 h, with significant increases at 12.5-13.5 h and 24-25.5 h pBM and peak levels at 20-24 h. Photolyzed ·NO is derived from SNOs and metal nitrosyls. Since SNO concentrations did not change in response to infection, I proposed that metal nitrosyls, specifically Fe nitrosyl hemoglobin (nitrosylHb) based on the concentration of hemoglobin, were elevated in the infected midgut. At 12-24 h pBM, levels of midgut RNIs in infected mosquitoes were typical of levels measured during mammalian septic inflammation. The inverse relationship between AsNOS activity and parasite abundance indicates that nitrosative stress has a detrimental effect on parasite development. However, nitrosative stress may impact mosquito tissues as well in a manner analogous to mammalian tissue damage during inflammation. Elevated levels of nitrotyrosine (NTYR), a marker for nitrosative stress in many mammalian disease states, were detected in tissues of parasite-infected <i>A. stephensi</i> at 24 h pBM. Greater nitration of tyrosine residues was detected in the blood bolus, midgut epithelium, eggs and fat body. In the midgut, Hb remained in an oxygenated state for the duration of blood digestion. The reaction between ·NO and oxyhemoglobin (oxyHb) can result in the formation of nitrate and methemoglobin (metHb). Although nitrate levels were elevated in response to parasite infection, there was little to no metHb present in the mosquito midgut. The simultaneous presence of nitrates, nitrosylHb, oxyHb, and NTYR, together with a lack of elevated nitrites and metHb, suggested that alternative reaction mechanisms involving â ¢NO had occurred in the reducing environment of the midgut. In addition, I proposed that nitroxyl and peroxynitrite participated in reactions that yielded observed midgut RNIs. To cope with the parasite-induced nitrosative stress, cellular defenses in the mosquito may be induced to minimize self damage. I proposed that peroxiredoxins (Prx), enzymes that can detoxify peroxides and peroxynitrite, may protect <i>A. stephensi</i> from nitrosative stress. Six Prx genes were identified in the <i>A. gambiae</i> genome based on homology with known <i>D. melanogaster</i> Prxs. I identified one <i>A. stephensi</i> Prx, AsPrx, that shared 78% amino acid identity with a <i>D. melanogaster</i> 2-Cys Prx known to protect fly cells against various oxidative stresses. <i>AsPrx</i> was expressed in the midgut epithelium and is encoded by a single-copy, intronless gene. Quantitative RT-PCR analyses confirmed that induction of <i>AsPrx</i> expression in the midgut was correlated with malaria parasite infection and nitrosative stress. To determine whether AsPrx could protect against RNI- and ROS-mediated cell death, transient transfection protocols were established for AsPrx overexpression in <i>D. melanogaster</i> (S2) and <i>A. stephensi</i> (MSQ43) cells and for <i>AsPrx</i> gene silencing using RNA interference in MSQ43 cells. Viability assays in MSQ43 cells showed that AsPrx conferred protection against hydrogen peroxide, ·NO, nitroxyl and peroxynitrite. These data suggested that the ·NO-mediated defense response is toxic to both host and parasite. However, AsPrx may shift the balance in favor of the mosquito. / Ph. D.