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.

Small Ankryin 1 (sAnk1) is a 17kD transmembrane protein that plays a role in stabilizing the network sarcoplasmic reticulum in skeletal muscle (Ackermann et al., 2011). Recent studies have shown that sAnk1 can bind to and regulate sarco(endo)plasmic reticulum Ca2+-ATPase1 (SERCA1) activity (Desmond et al., 2015). SERCA1 transports Ca2+ against its gradient into the SR after muscle contraction. SERCA is inhibited by sarcolipin (SLN) in fast twitch skeletal muscle and atrial cardiac muscle and by phospholamban (PLN) in slow twitch muscle and ventricular cardiac muscle. Like SLN and PLN, sAnk1 also interacts with SERCA at least in part through its transmembrane domain (Asahi et al., 2003; Hutter et al., 2002; Desmond et al., 2015). The interaction of SERCA with SLN and PLN has been studied individually and together, but the effects of sAnk1 and its regulatory activity have only recently started to be addressed (Desmond et al., 2015, 2017). Here I show that sAnk1 can interact with PLN or SLN independently of SERCA1. sAnk1 forms a three-way complex with SLN and SERCA1 that ablates SLN inhibition (Desmond et al., 2017). sAnk1 can also form a three-way complex with PLN and SERCA1 that abolishes all inhibition. I show that the complexes that sAnk1 forms with SLN or PLN and SERCA1 are distinct, suggesting unique roles for each protein in SERCA regulation. I also examined sAnk1 and SERCA in several CNS tissues, and found that sAnk1 is not expressed in neurons, but that it is expressed in astrocytes, where it has the potential to bind and regulate SERCA2B. Studying the multi-protein complex of SERCA, sAnk1, SLN, and/or PLN can help us better understand physiological SERCA regulation. This knowledge can lead to better treatment for diseases related to misregulation of calcium, including muscular dystrophies and potentially some neuropathies.

Compartment syndrome is characterized as an excess in swelling leading to an increase in pressure in a limited space and a lower extremity fasciotomy is performed to mitigate the effects. A two-incision fasciotomy is performed on the medial and lateral sides of the leg, accessing the posterior superficial, deep superficial, anterior and lateral compartments respectively. Ongoing studies have shown the lateral compartment is commonly decompressed incorrectly. This error has led to the hypothesis that there is variability in septum position and that using the fibula as a landmark can lead to erroneous incision placement in patients. CTA scans were analyzed to assess septum position. Findings indicate that the septum position shifts anteriorly progressing distally down the leg, indicating variability at different points in the leg. If surgeons do not take septum variability into consideration when decompressing the lateral compartment, this can lead to incorrect decompression of the lateral compartments.

Background: Drug resistance is one of the most prevalent causes of death in advanced prostate cancer patients. Combination therapies that target cancer cells via different mechanisms to overcome resistance have gained increased attention in recent years. However, the optimal drug combinations and the underlying mechanisms are yet to be fully explored. Aim and methods: The aim of this study is to investigate drugs that inhibit the growth of cells that are resistant to standard chemo and androgen deprivation therapy, and determine the underlying mechanisms of their action. To achieve this aim, we established cell lines that are resistant to this standard combination drug treatment and tested new compounds to overcome this “double drug” resistance. Results: Our results show that combination of enzalutamide (ENZ) and docetaxel (DTX) effectively inhibit the growth of prostate cancer cells that are resistant to either DTX or ENZ alone. The downregulation of transcription factor E2F1 plays a crucial role in cellular inhibition in response to the combined therapy. Notably, the androgen receptor (AR) variant AR3 (a.k.a. AR-V7), but not AR full length (AR-FL), positively regulates E2F1 expression in these cells. Specifically, E2F1 regulates AR3 and forms a positive regulatory feed-forward loop. Moreover, this drug combination treatment also results in DNA double strand break via the E2F1-AR3 signaling axis. Importantly, we established new drug-resistant cell lines that are resistant to ENZ+DTX combination therapy and found that the expression of both AR3 and E2F1 was restored in these double drug-resistant cells. Furthermore, we identified that auranofin, an FDA-approved drug for the treatment of rheumatoid arthritis, overcame the drug resistance and inhibited the growth of drug-resistant prostate cancer cells both in vitro and in vivo. Conclusion and significance: This proof-of-principle study demonstrates that targeting the E2F1/AR3 feedforward loop via a combination therapy or a multi-targeting drug could circumvent castration resistance in prostate cancer.

Chronic pain is the most common cause of disability. Progress in research to alleviate pain is hampered by the fact that metrics for studying pain in animal models are controversial. Rodents highly value social interactions, preferring them even over drugs of abuse or other hedonic rewards. Here, I tested the hypothesis that pain will reduce preference for social interaction, thereby offering a novel tool to quantify pain behaviors. After training rats to self-administer social interaction, I found that acute pain causes devaluation of social interaction. This devaluation was specific to social interaction, because after training rats to self-administer food, acute pain elicited no change in valuation for food self-administration. My findings display the importance of social interaction in pain behaviors, and suggest a novel metric for pain studies.

Excitation-contraction coupling (ECC) allows muscle to translate an action potential (AP) into muscle contraction. Optical methods for measuring membrane potential changes can expand our understanding of excitable cell function. Applying the potentiometric dye Di-8-ANEPPS and high-speed confocal microscopy to flexor digitorum brevis (FDB) muscle fibers from adult mice we non-invasively measure the electrical properties of these fibers. We determined action potential conduction velocity by comparing the time course of action potentials initiated at either end of muscle fibers by using alternate polarity electric field stimulation. Action potentials propagated longitudinally at a velocity of 0.39 ± 0.02 m/s. Conduction velocity of calcium transients, using mag-fluo-4, a low-affinity calcium indicator, was 0.37 ± 0.03 m/s, similar to Di-8-ANEPPS. We used mag-fluo-4 to examine whether our approach could capture conduction changes due to ionic concentrations, fiber length, and a lack of dystrophin, and found that we could. A lumped component equivalent electrical circuit model of the muscle fiber’s passive properties reproduced the observed passive responses of muscle fibers. This method using dyes allows the study the action potential propagation in a non-invasive manner in FDB fibers under differing physiological conditions and in various disease states. In skeletal muscle, AP sensing is governed by Cav1.1. Cav1.1 has 4 voltage sensor domains (VSDs) located in the membrane that that move in response to changes in membrane potential. During an AP these VSDs shift, initiating calcium release. We have not yet elucidated VSD movement in Cav1.1. Here we transfect mouse FDBs with a version of Cav1.1 that has a cysteine near the VSD. This allows us to attach a thiol-reactive fluorescent probe near the VSD and track its movement. This did not noticeably affect trafficking or function of Cav1.1. In resting conditions, our cysteine of interest should be embedded in the membrane. Upon depolarization, this cysteine shifts out of the membrane, allowing us to detect its movement as a change in fluorescence after changes in fluorophore environment. Using field stimulation fluorometry, we observed the shifts of Cav1.1 VSDs in response to field stimulation and analyzed how they correspond to Ca2+ release in native tissue.

Cigarette smoking is the leading cause of preventable death in the United States. The nicotine withdrawal syndrome (NWS) remains a barrier to successful smoking cessation; however, current pharmacological treatments minimally impact sustained abstinence. An emerging class of non-invasive neuromodulation devices, such as transcranial direct current stimulation (tDCS), have been proposed as novel therapeutics for smoking cessation. tDCS has the potential to modulate brain circuits by application of weak currents through the scalp; its use builds upon recent advances in mapping the large-scale network organization of the brain. Functional magnetic resonance imaging (fMRI) functional connectivity (FC) studies have identified three networks as particularly vulnerable to disruption in psychopathology: the Executive Control Network (ECN), Salience Network (SN), and Default Mode Network (DMN). The NWS has been hypothesized to be mediated by reduced FC within the ECN, and between ECN–SN; and increased FC within the DMN, and between DMN–SN. It is hypothesized that tDCS, applied to cortical nodes of the ECN (e.g. dorsolateral prefrontal cortex) and DMN (e.g. ventromedial prefrontal cortex), may remediate NWS network dysregulation. Network effects of tDCS were assessed by simultaneous task-based fMRI. 15 smokers (in sated and withdrawal states) and 28 matched nonsmokers participated in a double-blind, randomized crossover design of three tDCS conditions: anodal left-dlPFC/cathodal right-vmPFC (“An-dlPFC”), polarity reversed (“An-vmPFC”), and Sham. Although single-session (25min, 2mA) tDCS did not evoke task behavior changes, An-dlPFC tDCS robustly suppressed DMN nodes during a working memory task, and enhanced anterior cingulate activity (SN node) during a conflict monitoring task. DMN suppression within smokers was more pronounced during the sated (vs. withdrawn) state. Given that DMN and SN are hypothesized to be dysregulated in nicotine and other addictions, these data quantitatively support the hypothesis that tDCS may modify large-scale circuits implicated in addictive disease. Additionally, the observation of state-dependent tDCS effects in smokers suggests that tDCS may be most efficacious when combined with standard smoking cessation therapies. This work contributes a translational approach to assessment of tDCS, an emerging intervention at the crossroads of basic neuroscience research and clinical therapeutics in addiction and psychiatric disease.