Plasmodium falciparum is a eukaryotic parasite that causes severe malaria and contributed to 405,000 deaths worldwide in 2018. Victims of severe malaria are predominantly sub-Saharan African children, who typically present with symptoms of severe anemia or unarousable coma. The pathogenesis of severe malaria is poorly understood but mediated by the expression of adhesive variant surface antigens (VSAs) on infected red blood cells. VSAs are involved in sequestration and rosetting, unique virulence processes that allow the parasite to evade host immune responses and prevent clearance in the spleen. A relatively unstudied family of VSAs, the repetitive interspersed family (RIFIN) proteins, have recently been found to be important in rosetting and host immune suppression. RIFINs also appear to be targets for protective immunity; humoral immune responses against RIFINs have been correlated with asymptomatic infections. In this dissertation, I applied a multi-faceted approach using protein and peptide microarrays, transcriptomics, and reverse vaccinology to identify appealing RIFIN candidates for inclusion in a future severe malaria vaccine. I show that serological responses against epitopes within the semi-conserved domain of RIFINs associated with severe malaria reflected age-related malaria exposure. Sequencing and identifying specific rif genes expressed in clinical infections have not been feasible. I have addressed these challenges by adapting a novel bioinformatic pipeline and developing an HMM-based tool to process, assemble, classify, and subtype RIFIN sequences from peripheral blood samples. This takes advantage of a targeted probe capture method that I determined yields more abundant, full-length RIFIN sequences than other library enrichment approaches. Finally, I performed a comprehensive genomic survey of RIFIN gene repertoires using publicly available whole genome data of sixteen P. falciparum isolates to identify highly conserved, strain-transcendent sequences. Together, these results provide insights and powerful tools that can advance our understanding of the role RIFINs play in severe malaria pathogenesis and the development of naturally-acquired immunity to severe malaria. This work will aid efforts to determine targets for vaccines to protect children from the deadliest consequences of malaria.

Changes in are observed during breast tumor formation and progression, which promote malignant phenotypes in both normal mammary epithelial cells and breast cancer cells. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. This work applied time-lapse confocal microscopy and quantitative methods to define the rapid mechanically-stimulated calcium signaling mechanisms that occur in breast epithelial cells. While most tumor cell studies focus on long-term effects of mechanical stimulation (>24h), the current approach detected an immediate initiation of cytosolic calcium signals within 2 seconds after transient mechanical stimulation. Two novel methods were developed to describe this response and underlying mechanisms: a) the real-time scratch assay and b) scratch on low elastic dishes (SLED). The real-time scratch assay revealed an ATP/P2Y2/Ca2+ signaling axis in response to scratch with implications as a path toward EMT. The second method developed was SLED, a non-damaging application of mechanical stress to breast epithelial cells with physiologic implications as the elasticity of the cell substrate better matched that of the mammary gland in vivo than the typical glass or plastic experimental dishes. New data obtained using this novel approach revealed that normal breast epithelial cells are mechanically sensitive, responding to mechanical stimuli through a two-part calcium signaling mechanism. An immediate, robust rise in intracellular calcium (within seconds) was observed followed by a persistent extracellular calcium influx (up to 30 minutes). This persistent calcium was sustained via microtubule-dependent mechano-activation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on TRPM8 channels to prolong calcium signaling. Disruption of this conserved mechanobiology mechanism was possible through oncogenic activation such that, among common oncogenic mutations, constitutively-active KRas suppressed this signaling pathway. Therefore, certain oncogenes render cells mechanically unresponsive which could have interesting implications for the evolution of cancer cell mechanosensing in the tumor microenvironment as cells acquire new mutations. In addition, altered expression of the ROS generator (NOX2) and the ROS-responsive channel (TRPM8) are indicators of reduced overall survival in ER-negative breast cancer, suggesting that this mechano-pathway identified in breast epithelial cells may also be modified in patients in vivo.

Natural Killer T (NKT) cells play an important role in cancer surveillance and can reduce lymphoma burden in vivo; however, a hallmark of cancer is its ability to evade immune surveillance. Our goals were to elucidate novel mechanisms utilized by B cell lymphoma to evade NKT cell-mediated immune surveillance and determine the prognostic potential of assessing NKT cell function in lymphoma patients. We found that knockdown of sphingosine kinase 1 (SK1) in human lymphoma cells results in a significant increase in CD1d-mediated NKT cell activation. Lipidomic and co-culture studies identified cardiolipin as being upregulated in SK1 knockdown cells and implicated cardiolipin as an NKT cell-specific cancer neoantigen. We also sought to determine the efficacy of NKT cell-based therapy on survival and the induction of anti-tumor immune responses in a mouse model of B cell lymphoma. We found that activation of NKT cells via early administration of α-galactosylceramide (α-GalCer) only provided modest protection. Our data suggest that the lack of protection is due, at least in part, to the expansion of myeloid-derived suppressor cells in α-GalCer-treated tumor bearing mice. Lastly, we sought to identify novel immunological biomarkers in lymphoma patients. It was found that lymphoma patients have a reduction in NKT cell function compared to healthy donors. Furthermore, lymphoma patients have significantly higher levels of both pro- and anti-inflammatory cytokines in their sera compared to healthy donors. In addition, lymphoma patients who experience relapse have significantly reduced NKT cell function in the blood, compared to lymphoma patients who did not relapse. Collectively, our studies demonstrate the multifaceted role NKT cells play in immune responses to B cell lymphoma and will help inform the next generation of cancer immunotherapy.

Autosomal dominant polycystic kidney disease is caused by the loss of function of either two transmembrane proteins, polycystin-1 or polycystin-2. In renal epithelia, the consequence of polycystin loss is the formation of progressive, focal, fluid-filled cysts. However, the function and associated downstream signaling pathways specific to the polycystins have not been defined. Therefore, a new in vitro tubuloid model was designed to investigate the proximate cellular changes in renal epithelial cells following inactivation of Pkd2, the gene that encodes for polycystin-2. This model system reinforced the relevance of proteins associated with cell junctions, adhesions, and matrix in the cyst mechanism. The impact of this model was further supported through morphometrical analysis of epithelial compartmentalization in human ADPKD tissue, demonstrating an altered apical compartment in emerging cysts compared to noncystic tubules. Seeking connection between the junctions and disrupted apical compartment led to investigation of ezrin, a master scaffold in the apical compartment in renal epithelial cells. Ezrin plays a critical role in regulation of polarity, cytoskeleton organization, and protein trafficking, and the downstream consequences of its disruption have not been elucidated. Investigation into the initiating events of cystogenesis in ADPKD revealed a dramatic change in ezrin, following loss of PC2 in our tubuloid model, cystic mouse model, and pathological human ADPKD tissue. Based on this novel regulatory relationship between PC2 and ezrin, as well as the antecedent loss of ezrin to cyst formation in mice, ezrin was overexpressed in the pkd2 morpholino zebrafish model. Increased expression of ezrin diminished the formation of pronephric cysts. This lead to the design of a cyst rescue mouse model, which has exhibited promising preliminary data for cyst area reduction with additional ezrin. The disruption of ezrin in Pkd2 inducible in vitro and in vivo model systems, changes in ADPKD patient tissue, and rescue of pronephric cysts in the pkd2 MO suggest there is a role of ezrin in renal cystogenesis. Understanding the relationship of ezrin, with PC2 in renal epithelial cells will help elucidate the mechanism of ADPKD cystogenesis and define important downstream pathways necessary for epithelial functions.

After repeated P. falciparum infections, individuals in high-transmission areas acquire clinical immunity to malaria. However, the genes important in determining allele-specific immunity are not entirely known. Previous genome-wide approaches explored signatures of selection in the parasite genome to identify targets of clinical immunity; however, these approaches did not account for individual level allele-specific immunity. Here we take a whole-genome approach to identify genes that may be involved in acquisition of allele-specific immunity to malaria by analyzing parasite genomes collected from infected individuals in Malawi. However, obtaining whole genome sequence data from clinical samples is one of the major hurdles in the field of malaria genomics. In order to obtain whole genome sequence data from non-leukocyte depleted, low parasitemia samples, we optimized a selective-whole genome amplification (sWGA) by filtering the DNA prior to sWGA, to generate high coverage, whole genome sequence data from P. falciparum clinical samples with low amounts of parasite DNA. Using this optimized approach, we successfully performed whole-genome sequencing on 202 parasite isolates. We compared parasite genomes from individuals with varying levels of clinical immunity, defined using an individual’s proportion of symptomatic infections during the course of the study, hypothesizing that individuals with higher immunity become symptomatically ill due to infection with parasites with less common alleles. Using FST, we identified 161 SNPs to be genetically differentiated between the two groups and the median allele frequency was significantly lower at these sites in individuals in higher immunity group compared to the lower immunity group. We also examined pairs of parasites collected at different time points from the same individuals and identified 225 loci in 174 genes that vary within same individuals more often than expected by chance. Using both of these approaches, we identified 25 genes that encode likely targets of immunity, including a known antigen, CLAG8. Further analysis of clag8 global diversity showed evidence of immune selection in the C-terminal region, supporting the use of this approach in identification of new vaccine targets. Identifying and further analyzing these genomic regions will provide insights into mechanisms involved in allele-specific acquired immunity.

Gaucher Disease (GD), the most common lysosomal storage disorder, is caused by mutations in the GBA1 gene, which codes for the lysosomal enzyme β-glucocerebrosidase (GCase). GCase breaks down sphingolipids but when it is mutated, it causes the accumulation of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). The common manifestations of GD include hepatosplenomegaly, anemia, thrombocytopenia, skeletal disease, and in case of severe mutations, there are also fatal neurological manifestations. The conventional treatment is not effective in managing the skeletal or neurological manifestations. Hence, a better understanding of the underlying mechanisms that cause GD pathology is required for development of effective therapeutic strategies. The goal of this thesis was to identify the molecular mechanisms responsible for phenotypic alterations in osteoblasts and neuronal cells from GD patients, thereby pinpoint molecular targets for therapeutic intervention. Our laboratory utilizes patient-specific induced pluripotent stem cells (iPSCs) harboring GBA1 mutations to model GD. We have previously differentiated these iPSCs to various cell types and have shown that we can recapitulate the pathologic hallmarks of GD. Thus, in this study, we generated GD-iPSC derived osteoblasts and neuronal cells and found that mutations in GBA1 disrupt the canonical Wnt signaling and lysosomal compartment in these cell types. The phenotypic consequence of this was observed in the form of defective osteoblast differentiation and maturation as well as loss of midbrain/hindbrain neuronal progenitors in the respective cell types. Due to the known lysosomal dysregulation in GD, we then explored the mTOR pathway which is upstream of lysosomal biogenesis. We found hyperactivation of mTOR in GD neuronal cells was mediated by the significant accumulation of GlcSph, a lysolipid of GlcCer. In addition, when we blocked the conversion of GlcCer to GlcSph using acid ceramidase inhibitors, we were able to reverse mTOR hyperactivation and restore lysosomal expression, suggesting that GlcSph is partly, if not fully, responsible for the lysosomal abnormalities observed in GD. In conclusion, our study reveals that activation of canonical Wnt pathway or suppression of mTOR pathway ameliorates the phenotypic abnormalities in GD and identifies b-catenin, mTOR and acid ceramidase as potential therapeutic targets for GD.

Adverse effects from unintended exposure to chemicals have been widely described and have provided the basis for many chemical and environmental management regulations and policies that are intended to protect humans, animals, and/or the environment. The present work focuses on two classes of chemicals that are tested and/or used in training activities at military sites: insensitive munitions (IMs) and per- and polyfluoroalkyl substances (PFAS). As groundwater is a common source of drinking water, accurate risk assessments are critically needed to ensure protection of the environment and public health. IMs are more resistant to accidental, unintentional or incidental detonation than traditional explosives. Incomplete detonation may deposit munition components and by-products on impacted installations and, importantly, mobilize those constituents into groundwater. One component of several IM mixtures is 3-nitro-1,2,4-triazol-5-one (NTO). NTO is especially worrisome because it is highly water soluble and, thus, mobile in the environment. Additionally, laboratory tests with rodents have identified the testes and epididymides as targets of NTO. PFAS are also prevalent and problematic, nearly universal contaminants. PFAS are manufactured for use in paints, cleaning agents, non-stick cookware and food containers, water-impermeable products, and Aqueous Film Forming Foams (AFFFs). PFAS have attracted increased regulatory scrutiny because of their resistance to degradation, ability to bioaccumulate, and growing evidence of toxicity in animals. A considerable body of work has examined the effects of NTO in laboratory rodents. However, there is still uncertainty in the derivation of a safe workplace environmental exposure level (WEEL) for NTO. The present studies evaluate the effects of NTO in the Japanese quail (Coturnix japonica) and in Sprague Dawley rats. In concert, these toxicity data on NTO will improve risk estimation efforts, provide data to explore taxa read-across, and fill a regulatory needed data-gap. The toxicity of PFAS will be evaluated in the native mammalian species, Peromyscus leucopus. Given that the serum elimination half-lives within this class of chemicals can vary greatly from hours to years based on species, sex, and dose, extrapolating between species is difficult and inaccurate. As such, wild mammalian-specific data is novel and valuable for risk estimation in taxa that are directly site-specific.

Hearing loss is the most common sensory impairment, affecting hundreds of millions of people worldwide. Although both men and women are impacted by hearing loss, the incidence rate differs between the two sexes, with the prevalence of bilateral high-frequency hearing loss reported to be 2.7 times higher in males. Recent studies have implicated estrogen as having a protective effect against hearing loss in females. While the localization of the estrogen receptors within the cochlea is known, the roles of estrogen-related receptors in auditory function require further investigation. The aim of this study is to elucidate the localization of estrogen receptors 1 and 2 and estrogen-related receptors α, β, and γ within the inner ear of adult mice using RNAscope. Our results give insight into the molecular mechanism through which estrogen receptors and estrogen-related receptors may grant protection against hearing loss and contribute to the functionality of the inner ear.