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Epidemiological observations have linked increased host iron with malaria susceptibility. At the same time, blood-stage malaria infection is associated with potentially life-threatening anemia. To improve our understanding of these relationships, this work presents an examination of the mechanisms controlling the upregulation of the hormone hepcidin, the master regulator of iron metabolism, in malaria infection. Chapter 2 presents data from a mouse model of malaria infection which indicate that hepcidin upregulation in malaria infection is associated with increased activity of the sons of mothers against decapentaplegic (Smad) signaling pathway. Although the canonical Smad pathway activators, bone morphogenetic proteins (Bmp) are not increased at the message level following infection, activin B, which has been recently shown to increase hepcidin through the Smad signaling pathway in conditions of inflammation and infection, is upregulated in the livers of malaria-infected mice. Chapter 3 shows that both activin B and the closely related protein activin A upregulate hepcidin in vitro and in vivo. Chapter 3 also explores the effects of the activin-binding protein follistatin in both systems and in the same malaria-infected mouse model as presented in Chapter 2. The work presented in Chapter 4 extends these studies to human infections by demonstrating that activin A protein co-increases with hepcidin in human serum during malaria infection. Taken together, these findings are consistent with a novel role for activin proteins in controlling hepcidin upregulation in the context of malaria infection. This work may form a basis for the development of novel therapeutics that speed recovery from malarial anemia by inhibiting activins’ actions. Chapter 5 examines the role of infected red blood cell-derived microparticles in the initial recognition of a P. falciparum malaria infection, and subsequent hepcidin upregulation. Microparticles stimulate production of cytokines from peripheral blood mononuclear cells (PBMC), which also upregulate activin A message in response to both microparticles and whole infected red blood cells. These data are consistent with a model in which malaria-derived stimuli such as microparticles trigger the systemic release of activin proteins, which then act on the liver to upregulate hepcidin. Evidence has shown that cytokine levels at birth are related to malaria risk. In Chapter 6, hepcidin is measured in cord blood samples from participants in a large-scale clinical study in a malaria-endemic area, and shown to be elevated in cord blood from neonates with a clinical history of placental malaria. Cord blood hepcidin is also compared to birth levels of iron markers and other cytokines, and future clinical outcomes. Finally, the contributions of DNA methylation levels to cord hepcidin and cytokine levels are assessed by comparison of CpG methylation, at sites in genes encoding hepcidin and cytokines, to the serum concentrations of the genes’ protein products. Several intriguing associations are noted which indicate a possible novel role for DNA methylation in the determination of birth cytokine and hepcidin levels. Chapter 7 synthesizes the data presented in this thesis, interprets the possible significance of the major findings, and offers suggestions for future work.

The virulence of Plasmodium falciparum is largely attributed to the ability of asexual blood-stage parasites to cytoadhere to the microvascular endothelium of the human host. This pathogenic behavior is mediated by the primary parasite virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), an understanding of which is crucial to develop interventions to ameliorate the morbidity and mortality of P. falciparum malaria. The work presented in this thesis describes the application of a phenotype-to-genotype experimental approach to identify novel parasite proteins involved in the trafficking and display of PfEMP1. Guided by the overall hypothesis that the in vitro culture-adapted parasite line 3D7 harbors 1 or more genetic determinants of impaired PfEMP1 trafficking, surface PfEMP1 levels were first measured in 3D7, the presumably trafficking-competent parasite line HB3, and 16 unique progeny from an HB3 x 3D7 genetic cross (chapter 2). These phenotypes were then combined with genome-wide SNP data in QTL analysis to identify genetic polymorphisms potentially responsible for the impaired trafficking in 3D7 (chapter 3). A near-significant QTL containing a single protein-coding gene, the putative kinesin Pf3D7_1245600, was identified, characterized, and investigated in CRISPR-Cas9-driven allele-exchange parasite transfection experiments to establish a causal link between the gene and PfEMP1 trafficking (chapter 4). The parasite transfections were unsuccessful, but the potential role of Pf3D7_1245600 in PfEMP1 trafficking was indirectly assessed through the disruption of microtubules with colchicine (chapter 4), which significantly impacted the surface PfEMP1 levels of HB3 but not 3D7. The findings of this thesis suggest that kinesins and microtubules may play previously unconsidered roles in the regulation, production, or trafficking of PfEMP1.

Severe anemia is the most prevalent life-threatening complication of malaria infection. In addition to destruction of red blood cells (RBC), decreased RBC production or erythropoietic suppression has been shown to contribute to malarial anemia. The mechanism of this suppression is unknown, but it is considered to be multifactorial since erythropoietic suppression can be observed in the presence of both inflammatory mediators and parasite-derived factors. Experiments presented in this thesis aimed at determining the role of host cytokines released in response to blood-stage malaria infection and parasite-derived factors in erythropoietic suppression underlying the development of malarial anemia. Pro-inflammatory cytokines released during malaria infection have been proposed to play a central role in erythroid suppression. To dissect the discrete roles of these cytokines in the processes leading to anemia, mice were treated with CpG-oligodeoxynucleotides (CpG-ODN) which, like malaria infection in humans and experimental mouse models, induces an acute type 1 pro-inflammatory response. CpG-ODN treatment induced anemia, which was associated with suppressed erythropoiesis and reduced RBC survival. Importantly, CpG-ODN-induced IFN-gamma was found to be the major factor mediating erythropoietic suppression but not decreased RBC survival. We also studied the roles of Th1, Th2 and anti-inflammatory cytokines produced in response to Plasmodium chabaudi AS infection in the development of erythropoietic suppression during blood-stage malaria. Signal transducer and activator of transcription (STAT)6, required for signaling of the Th2 cytokines IL-4 and IL-13, was shown to play a critical role in malarial anemia by inhibiting the proliferation and differentiation of erythroid cells. We also observed that suppressed erythropoiesis is a general feature in mice infected with various rodent Plasmodium species that differ in their clinical manifestations and immune responses. Since parasite-derived factors have been shown to contribute to malarial pathogenesis including anemia, the contribution of P. falciparum - and P. yoelii-derived products to erythropoietic suppression was investigated. Both Plasmodium-derived and synthetic hemozoin (Hz) suppressed the proliferation but not the maturation of erythroid progenitor cells in vitro. However, P. yoelii-derived Hz but not synthetic Hz induced transient anemia in mice. These findings provide novel insights into the complex interactions between the parasite and host immune system and the regulation of erythropoiesis during severe malarial anemia.

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Drug resistance is a significant challenge in the fight against malaria. Importantly, reduced efficacy has been reported against artemether (ATM)/Lumefantrine (LM) (LM-ATM), amodiaquine (AQ)/artesunate (AS) (AQ-AS), two important combination treatment regimens in Africa, and against piperaquine (PQ), a drug which has been evaluated as a potential alternative in Africa, in combination with dihydroarteminisin (DHA). Chloroquine (CQ) resistance in P.falciparum is associated with two main transporters PfCRT and PfMDR1. I investigated the mechanisms of resistance to PQ, LM and AQ, with the overall goal of identifying molecular markers that can be used to track resistance. I used CQ as a reference. The key antimalarial drugs were highly active against clinical isolates from Kilifi, Kenya with median inhibitory concentrations (IC₅₀s) of <5nM for DHA and <55 nM for CQ, AQ, PQ, LM and DEAQ (desethylamodiaquine, the active metabolite of AQ). pfcrt-76 and pfmdr1-86 mutations were associated with AQ, DEAQ and LM but not DHA or PQ activity. Interestingly, > 20% of analysed isolates had decreased susceptibility to LM (IC₅₀ >100nM); these isolates were the most susceptible to CQ and carried wild type genotypes at pfcrt-76 and pfmdr1-86. I observed that CQ resistance had been declining in Kilifi since 1993 (prior to CQ withdrawal) to 2006 (7 years after its withdrawal), similar to observations in Malawi. My results support the hypothesis that susceptibility to antimalarial drugs returns when drug pressure is removed, and suggest that the use of LM-ATM may hasten the return of CQ susceptibility. Continued monitoring of drug susceptibility is crucial. pfcrt-76 and pfmdr1-86 may be useful molecular markers of LM-ATM efficacy in Kilifi and other African sites. Using a microarray approach, I identified additional genes (including various transporters) that may contribute to LM resistance. I recommend further studies to clarify the exact roles of the identified genes.