Mosquitoes harbor and transmit a variety of pathogens which are dangerous to humans and incur a constant and significant public health cost. Gaining more detailed knowledge about these pathogens, their interactions with the mosquito, and their molecular biology and genetics will allow for new techniques to be developed to prevent harmful effects on humans. These pathogens vary in complexity from viruses like West Nile virus to eukaryotic apicomplexan parasites like Plasmodium falciparum, the human malaria parasite.Culex mosquitoes routinely transfer viruses like West Nile virus in the United States representing a predominant health threat there. These mosquitoes are also routinely infected by a wide variety of other viruses, which have received very little research attention, yet could affect the transmission of pathogens like West Nile virus and St. Louis encephalitis virus. Therefore, in the first chapter of this dissertation work, we performed small RNA sequencing to examine the full virome of field-caught Culex mosquitoes in multiple geographical regions of southern California. These data also allowed us to analyze the interactions between viruses and identify potential pairs of co-infecting or mutually excluding viruses, as well as closely look at the small RNA immune response of Culex mosquitoes against these viruses, expanding the known role of the antiviral piRNA response in Culex. We also identified mosquito miRNAs that may be involved in antiviral immunity or virus infection by comparing highly infected mosquito pools against lowly infected ones. Apart from viruses, single-celled eukaryotic parasites are also transmitted from mosquitoes to humans. One of the deadliest of these pathogens is the malaria parasite, Plasmodium falciparum. Important regulators that propel the life cycle of this parasite also represent potential drug targets, that when identified could lead to new treatment options to lessen the impact of malaria in Africa and across the globe. We performed a wide variety of experiments and studies in this vein. In the second chapter, we examine the role of long non-coding RNAs (lncRNAs) on parasite gene regulation and predict lncRNAs across the genome, categorizing their properties and their essentiality for parasite survival. We also use experimental techniques to determine the binding sites of several of these predicted lncRNAs, and look at the effect of one in particular, lncRNA-14, by knocking it out and using transcriptomic and phenotypic analysis. The third chapter, on the other hand, uses Plasmodium berghei, a mouse malaria parasite, as a model for human malaria and examines parasites proteins SMC2 and SMC4, the homologs of proteins that make up the condensin complex in model eukaryotes. We determine that, as in other eukaryotes, Plasmodium SMC2 and SMC4 form a condensin complex and are key in cell division, particularly in the mosquito stages of the parasite life cycle. ChIP-seq determined that these proteins bind at the centromeres, and transcriptomic and phenotypic analysis revealed the exact roles of these proteins and their importance. Finally, the remaining two chapters examine RNA-dependent and RNA-binding proteins in Plasmodium falciparum, key elements in post-transcriptional regulation, another important aspect of gene regulation in the life cycle. In the fourth chapter, we performed a screen for RNA-dependent proteins using the R-DeeP protocol, obtaining a list of likely proteins and examining RNA-dependent complexes. We also characterized one of the proteins found as an RNA-binding protein using various techniques including enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP-seq). In the fifth chapter, this technique and others were also used to determine the binding sites and functions of two other predicted RNA-binding proteins with RAP (RNA-binding domain abundant in apicomplexans) domains. These RAP proteins were determined to regulate the parasite mitochondrial rRNAs (mitoribosome). All in all, this dissertation work reveals new insights into mosquito-associated pathogens, their interactions with mosquitoes and mosquito immunity, and important molecular components of their life cycles which could be targeted to reduce their impact on humans.

Toxoplasma gondii is a prolific parasite known to infect a wide variety of warm-blooded animals, including humans. The parasite can interconvert between a dormant form of the parasite (bradyzoites), and an acute form of the parasite (tachyzoites), which manifests as Toxoplasmosis, in a process called recrudescence. This process is tightly controlled through a series of changes in gene expression in response to environmental pressure. Histone modifications are effective post-translational modifications, influencing the parasite’s gene expression through different stages of its life cycle. Despite knowing that recrudescence is, in part, epigenetically regulated, little is known about how environmental factors, such as the type of host cell, affect this process. This study uses histone modifications to observe changes in the epigenomic profile of the parasite as they develop within two different host cells, Astrocytes (ASTs) and Human Foreskin Fibroblasts (HFFs). Using Chromatin Immunoprecipitation followed by deep Sequencing (ChIP–seq) against three different histone marks, we revealed a higher abundance of histone H3K4me3 activating marks within ASTs. This observation was supported by an increased binding affinity of H3K4me3 in ASTs and a decline in the number of affinity binding sites in HFF as recrudescence progressed. Of the annotated genes that were identified to have a high affinity with H3K4me3, several had potential roles in invasion, chromatin remodeling, and cell cycle progression.

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The human malaria parasite, one of the deadliest infectious agents in the world, still contributes significantly to the global burden of disease. In 2017, an estimated 214 million cases of infection and over 400,000 malaria-related deaths were reported, a majority of which are caused by the most lethal human malaria parasite, Plasmodium falciparum. Given the absence of an FDA-approved vaccine and parasite resistance to all current antimalarial drugs there is a desperate need for new therapeutic approaches.

Plasmodium falciparum has a complex life cycle that requires coordinated gene expression regulation to allow host cell invasion, transmission and immune evasion. However, this cascade of transcripts is unlikely to be regulated by the limited number of identified parasite-specific transcription factors. Increasing evidence now suggests a major role for epigenetic mechanisms in gene expression in the parasite. Therefore, in this dissertation work, we further explore genome architecture, epigenome, proteome and transcriptome including long-non-coding RNAs (lncRNAs) to better understand the relationship between chromatin structure, genome organization and transcriptional regulation in malaria parasites.

In the first chapter, we explore genome organization in human Plasmodium parasite stages including the transmission stages from human to mosquito (gametocytes) and from mosquito to human (sporozoites). Our work demonstrates that genome organization is an important regulator for several parasite-specific gene families involved in pathogenesis and immune evasion, erythrocyte and liver cell invasion, sexual differentiation, and master regulators of gene expression. In the second chapter, we investigated genome organization in five malaria parasites and two related apicomplexan parasites with the goal to identify common features of genome organization and possible connections between genome architecture and pathogenicity. We show that in all malaria parasites, genome organization is dominated by the clustering of Plasmodium-specific gene families in 3D space. Our data highlight the importance of spatial genome organization in gene regulation and control of virulence in malaria parasites.

In the subsequent chapters, we aim to identify molecular components, specifically proteins and lncRNAs, that maintain and regulate chromatin structure in the malaria parasite. To investigate parasite proteins and protein complexes maintaining and regulating nuclear architecture, we undertook comparative genomics analysis using twelve distinct eukaryotic genomes. We identified conserved and apicomplexan parasite-specific chromatin-associated domains (CADs) and proteins (CAPs). We validated two of our candidate proteins including a novel plant-related protein that is functionally analogous to animal nuclear lamina proteins and might have a role in heterochromatin organization. Finally, we also explore the role of lncRNAs in P. falciparum. In eukaryotes, lncRNAs have been shown to be pivotal regulators of genome structure and gene expression. To investigate the regulatory roles of lncRNAs in P. falciparum, we first explored the intergenic distribution of lncRNA using deep sequencing in nuclear and cytoplasmic subcellular locations. We then validate the subcellular localization and stage-specific expression of several putative lncRNAs at single cell resolution using fluorescence in situ hybridization (FISH) technology. Additionally, we explore the genome-wide occupancy of several candidate nuclear lncRNAs using Chromatin Isolation by RNA Purification followed by deep sequencing (ChIRP-seq) technology. Data analysis revealed that lncRNA occupancy sites within the parasite genome are focal and sequence-specific with a particular enrichment for several parasite-specific gene families, including those involved in pathogenesis, erythrocyte remodeling, and regulation of sexual differentiation. Discovery of these proteins and lncRNAs are the starting point for further exploration of mechanisms regulating chromatin structure and genome architecture in these deadly parasites.

Collectively, our data highlight the importance of spatial genome organization as a mechanism of transcriptional regulation in malaria parasites, and our work directly addresses one of the central outstanding questions in Plasmodium biology, namely, how a parasite with approximately 6,000 genes manages to control gene expression in a coordinated fashion using a limited number of transcription factors.

The unicellular protozoan, Plasmodium falciparum, is the human malaria-causing parasite responsible for over half a million deaths each year. P. falciparum has a complex life cycle, alternating between human and invertebrate hosts, and is capable of not only evading the host immune responses but also developing resistance to all clinically administered treatments. Consequently, malaria remains a persistent global health concern. Recent advances in omics-based technologies, improved sequencing platforms, and enhanced CRISPR-based gene editing tools have opened new avenues for understanding the parasite biology, identifying new therapeutic strategies and combating the spread of malaria.This report delves into the systems-wide approaches employed to explore the drug mechanism of action of a new compound, MED6-189, a new analogue of the kalihinol family of isocyanoterpene natural products, effective against drug-sensitive and -resistant P. falciparum strains. We also use advance multi-omics strategies to identify and functionally characterize new potential drug targets involved in gene regulation. Those included targeting proteins coding for chromatin remodeling complexes or better understanding the role of long non-coding RNAs (lncRNA) in the parasite life cycle progression. In essence, the goal of my PhD thesis was to identify promising and alternative targets to curb the spread of malaria, either through the development of new antimalarial agents or the discovery of novel, unexploited potential targets critical for the parasite's survival.

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