RAB-2 is a small GTPase protein in the Rab family, which are known to function in dense-core vesicle (DCV) trafficking. RAB-2 functions like a molecular switch; its active form is bound to GTP, and its inactive form is bound to GDP. Activation of RAB-2 is promoted by guanine nucleotide exchange factor (GEF) proteins and intrinsic GTPase activity is dramatically expedited by GTPase-activating proteins (GAPs). The ability to switch between both forms is important to DCV maturation. RUND-1 is a known specific effector of RAB-2 that promotes the GTPase activity RAB-2, but less so than a GAP. In Caenorhabditis elegans, animals expressing RUND-1 (T237P), present a more severe locomotion defect than the null mutation. Given that RUND-1 has an effect RAB-2 GTPase activity and that the rund-1 (T237P) mutation presents a more severe defect in C. elegans, I hypothesize that the T237P mutation will either greatly promote or obstruct RAB-2 GTPase activity compared to wild-type RUND-1. To do this, I have purified RAB-2 via an intein tag and will purify the mutant protein similarly. I will use malachite green assay to measure inorganic phosphate release from GTP hydrolysis. These experiments will help help us understand the effects of the rund-1 (T237P) mutation on RAB-2 GTPase activity.

We use genes to survive and reproduce, but this means that genes can hold our survival hostage to ensure their own propagation, without providing any benefit to us. Selfish genes are a brutal, and poorly-characterized, demonstration of this concept. Instead of producing beneficial proteins, they produce nonessential proteins that prevent individuals who carry the selfish gene from successfully reproducing with non-carriers. One such example, the PEEL-1/ZEEL-1 system, is natively found in C. elegans. In this system, PEEL-1 is a toxin protein that kills cells when it is expressed without the antitoxin protein ZEEL-1. My aim is to determine the mechanism of toxicity employed by PEEL-1. AlphaFold predictions suggest that PEEL-1 contains an amphipathic helix. The amphipathic property of this region is hypothesized to play a critical role in PEEL-1 toxicity. To test this hypothesis, I am conducting a Deep Mutational Scanning (DMS) on the amphipathic helix of PEEL-1, with the goal of identifying key polar or nonpolar residues in this region that are essential to PEEL-1 toxicity. First, I generate a library of single-residue PEEL-1 mutants. Second, I transfect these constructs into HEK293T cells. I sample this initial pool of cells for sequencing, to identify which PEEL-1 mutants the pool carries, and in what proportions. Third, I induce expression, exposing each cell to the effects of the PEEL-1 mutant they carry. Only the cells expressing loss-of-function mutants survive. Now, I sequence this final surviving pool of cells, similarly to the initial pool. Mutations that drastically alter the polarity of the residue and thus break the overall amphipathic structure of the region are expected to be overrepresented in the surviving pool. This result would provide a broader understanding of the various methods of cell death in nature and provide novel insight into how animal-derived selfish genetic systems function.

Over the past several years, the city council of Oxford, England has developed an environmental policy project named the Zero Emissions Zone (ZEZ) which seeks to establish an area in the city center where vehicles will be either prohibited or fined for through travel. The council conducted many studies on where the policy would most effectively reduce carbon emissions from private vehicle use and considered existing pedestrian-only areas for potential use. The policy has proven controversial in the community. There has not been significant research on the popularity of the policy and how environmental policies like the ZEZ affect the political beliefs of Oxford community members. I hypothesized that the ZEZ would be a popular policy on the whole with the primary opposition coming from those who utilize private vehicles as their primary mode of transportation in Oxford and that approval or disapproval of this policy will significantly affect one’s perceptions of the city council and political parties. Through an anonymous online survey, I collected data from respondents over the age of 18 who either work, live, or study in Oxford or the surrounding suburbs. Participants answered questions regarding their political opinions, views on the ZEZ policy, and how the policy has affected their politics. These questions sought to illuminate a connection between views on environmental planning policies and political perceptions in general. I expect the results to show that younger respondents, particularly those whose political beliefs are to the left of center, will approve of this policy, and by extension the city council, more so than older respondents. This research has implications for Oxford City Council and any others interested or intending to install or expand similar zones so that they may understand what impact it may have on local politics. 

The composition of metropolitan governance has many effects on land use decisions, budget allocations, housing development, transportation planning, and racial, economic, and social equity in urban areas. However, there has been little academic inquiry into the effect of regional governance structure on transportation accessibility. This paper seeks to examine statistical linkages between regional governance fragmentation and trends toward and away from greater transportation accessibility in metropolitan areas. I perform a comparative statistical analysis of 47 of the 50 largest Metropolitan Statistical Areas, examining census data from 2002 to 2022 and transit accessibility data from the University of Minnesota Accessibility Observatory from 2014 to 2021 to examine this relationship. The causal factor I investigate is metropolitan governance fragmentation, which I capture through a Governance Fragmentation Index (GFI). The dependent variable, transportation accessibility, is captured through an Accessibility Gap Index, which categorizes transportation access through accessibility levels throughout each Metropolitan Statistical Area, utilizing data from the Accessibility Observatory from 2014 to 2021. My analysis controls for potential confounding variables, such as geographic area, population size, poverty levels, and region. I expect to find that lower levels of governance fragmentation in a Metropolitan Statistical Area will be associated with greater gains in transportation accessibility. Whether or not a significant relationship is identified, the research conducted will contribute to literature and ongoing research surrounding metropolitan governance and transportation accessibility.

Déjà Vu is an emotional experience that occurs less as we age and is most frequent when traveling, having vivid dreams, or brain injury. Research of déjà vu may prove vital for advancing interdisciplinary research, our interpreting of the world, and answering, arguably, many of life's greatest mysteries: human consciousness and even comprehension of life & death. Societal views of déjà vu have often labeled it abnormal, mysterious, and magical. As déjà vu transitioned from the realms of literature to scientific inquiry in the late 1800s, it captured the interest of the rapidly developing psychological and neurological disciplines. This project seeks to help clarify this issue by exploring how the explanations of déjà vu shifted from literary and artistic interpretations to an empirical research question. In this project, a literature review of historical and contemporary studies of déjà vu will be conducted to help map the conceptual overlaps, theoretical shifts, and methodological progression of research on déjà vu. Understanding how déjà vu's reconfiguration is an empirical question will help us better understand the entangled relationships between the body and experience (i.e., embodied experiences). Additionally, advancing interdisciplinary research about the experience of déjà vu may be vital to advancing phenomenological and scientific (e.g., neuroscience) understanding of many of life's greatest mysteries, from differences in human perception to diverse experiences of lived reality.

Reward-seeking behavior, crucial for survival, is hijacked by various neuropsychiatric disorders, notably substance use disorder. Prior work implicated reward-seeking with the dorsomedial striatum (DMS), a brain structure expressing the endogenous opioid dynorphin (dyn) in 50% of its neurons. Previous studies also linked dyn, signaling through the kappa opioid receptor (KOR), with escalation and reinstatement in drug-seeking, however, the exact mechanisms of dyn-KOR signaling remain unknown. Recent experiments from the Bruchas lab show that supraphysiological release of dyn in the DMS with excitatory optogenetics elicited reward-seeking behavior, demonstrating that dyn-KOR is sufficient for reward-seeking behavior, but not that it is necessary. To provide evidence of necessity, I plan to inhibit dyn-KOR signaling in the DMS by expressing the novel inhibitory optogenetic tool Platynereis dumerilii (PdCO) that we recently showed to be useful for such studies (Wietek et al., Nat Methods, 2024). PdCO is expressed in a target neuronal population and begins inhibiting under 473 nm light. Hence, in this study, PdCO will be injected into the DMS and expressed selectively in dyn neurons. Following recovery, mice will learn a self-administration procedure to study reward-seeking behavior, where sucrose is delivered when mice poke their nose in the correct 1 of 2 holes, with a 5s light cue before reward delivery. Following learning, I will inhibit dyn release in a counterbalanced fashion by activating PdCO using 20 Hz pulsed 473 nm light. I anticipate that inhibiting dyn release would result in reduced sucrose consumption. Finally, I plan to use an extinction learning procedure, where dyn release is inhibited during perceived reward delivery. Here, I hypothesize that inhibition will accelerate extinction learning. By leveraging the specificity of optogenetics on various procedures, this study begins to isolate the exact mechanisms by which dyn-KOR signaling impacts reward-seeking behavior with potential insights for substance use disorder interventions.

The mechanistic target of rapamycin, mTOR, functions in the mTORC1 complex with another protein called raptor as a master regulator of eukaryotic cellular metabolism thereby regulating cell growth including from cancer, cell death including after stroke, inflammation, and aging. In a forward genetic screen for hypoxia resistant mutants, the Crowder lab recently identified a missense mutation in the daf-15 gene, which encodes C. elegans raptor. The mutation produces a heat-sensitive reduction of raptor function, hereafter referred to as daf-15(rf). At 20°C, daf-15(rf) is normally hypoxic sensitive, at 22°C very hypoxia resistant, and at 25°C incapable of normal development. Raptor negatively regulates autophagy, a mechanism for breakdown and recycling of proteins and organelles. Activation of autophagy has been found to promote hypoxic survival in C. elegans and higher organisms. Thus, we hypothesized that activation of autophagy was responsible for the hypoxia resistance of our daf-15(rf) mutant. To test this hypothesis, we first asked whether we could detect increased autophagy using fluorescently-tagged autophagy proteins at 22°C in daf-15(rf) but saw no effect compared to wild type. Next, we asked whether a C. elegans transcription factor, HLH-30, that promotes expression of autophagy proteins was activated by daf-15(rf) and found activation at 25°C but not at 22°C. Finally, we tested whether proteins essential for autophagy were also necessary for the hypoxia resistance of daf-15(rf). By generating double mutant strains, we showed that animals with daf-15(rf) but without essential autophagy proteins were still hypoxia resistant. Thus, we conclude that C. elegans raptor regulates hypoxic sensitivity by an autophagy-independent mechanism. These findings demonstrate that raptor can control hypoxic cellular injury by mechanisms distinct from autophagy. Such mechanisms, if identified, could be targeted for treatment of cancer, stroke, and other diseases where hypoxia plays a role.

Mitochondria are the main oxygen consumers in eukaryotic cells and as such are the primary organelles affected by oxygen deprivation, hypoxia. Hypoxia alters the size and shape of mitochondria, called the mitochondrial dynamics, but their role in hypoxic cell death is unknown. The Crowder lab has recently discovered that a mutation in the Mechanistic Target of Rapamycin Complex One (mTORC1) protein Raptor confers hypoxia resistance in the nematode C. elegans. mTORC1 is a master regulator of metabolism and is known to affect certain aspects of mitochondrial biology. Given these two facts, we tested the hypothesis that the hypoxia resistance of the C. elegans Raptor mutant is from alterations of mitochondrial dynamics. First, I showed that hypoxia induces small, rounded mitochondria in C. elegans caused from mitochondrial fission. Second consistent with the hypothesis, I showed that the mitochondria appear to have more normal morphology before and after hypoxia in the Raptor mutant. However, not consistent with the hypothesis, a C. elegans mutant with excess mitochondrial fission was not hypersensitive to hypoxia. Then combining the hyper fission mutant with the Raptor mutant did not diminish the hypoxia resistance produced by reduced Raptor function. Thus, our data demonstrates abrogating mitochondrial fission is not necessary for the hypoxia resistance produced by the Raptor mutant and leads us to reject our hypothesis. By exploring the interaction of mitochondrial fusion and fission with Raptor, we are beginning to understand how these important organelle and metabolic regulators combine to control hypoxic cell death.

In recent years, the United States has seen an increasing number of opioid overdoses, causing many research studies to focus on opioid drugs of abuse. However, we still lack a fundamental understanding of the opioid receptor that these drugs bind to. The mu-opioid receptor (MOR) is involved in both the pain-relieving and euphoric effects of opioid drugs. The Ventral Tegmental Area (VTA), known to be a major source of dopamine, contains a significant amount of inhibitory gamma-aminobutyric acid (GABA) neurons that express MORs. Previous research has shown that activity at the MOR in the VTA is rewarding and that dopamine projections from the VTA to the Nucleus Accumbens (NAc) are activated to reinforce behavior. However, whether the expression of MORs on GABA neurons in the VTA is important for influencing VTA to NAc dopamine activity during reward seeking behavior is unclear. To investigate the role of the MORs in reward seeking behaviors, I knocked out MORs in the VTA and used dLight, a fluorescent dopamine sensor, to measure dopaminergic release from synapses in the NAc during Operant and Pavlovian conditioning tasks in the absence of these receptors. Pavlovian conditioning utilizes chambers in which a house light turns on and a sucrose pellet is delivered via food hopper to food-restricted mice. In the operant conditioning task, mice must learn to complete a nose poke in order to receive the sucrose pellet reward. These tasks allow me to gain a more complete understanding of how the absence of MORs in the VTA changes behavior and dopamine activity during reward-seeking. This research furthers our understanding of how the MOR affects natural reward and motivated behaviors and is crucial in helping us understand how opioids of abuse alter existing brain circuitry to cause opioid use disorder.

The use of opioid drugs for pain management in postoperative settings has been widespread since the 1860s. However, the risk factor for developing an opioid use disorder (OUD) has increased substantially with its continued use, with addiction rates of more than 10% for those taking opioids. An obstacle to abstinence in opioid addiction are the adverse side effects that occur during cessation of drug use once dependence has formed, including nausea, anxiety, vomiting, and depression. The Nucleus Accumbens (NAc) is part of the mesocorticolimbic reward pathway. Decades of pharmacological studies demonstrate that nearly all abused drugs evoke dopamine release within the NAc, thus altering innate systems for how reward is processed. The activity of NAc neurons is strongly regulated by efferent excitatory input from numerous brain regions. The paraventricular thalamus (PVT) a relatively understudied brain region, regulates behavioral responses to reward and aversive stimuli as well as to drugs of abuse such as morphine. Our preliminary data demonstrate that the activity of these projections is highly regulated by the Cannabinoid 1 Receptor (CB1), which mediates the primary psychoactive effect of cannabis. This is particularly relevant on account of recent clinical findings demonstrating that activation of CB1 can ameliorate the aversive effects of opiate withdrawal. Using fiber photometry (which uses fluorescence emission of the calcium sensitive fluorophore, GCaMP, as a proxy measurement for neural activity), I have shown that this circuit is activated by aversive stimuli and inhibited by rewarding stimuli. Furthermore, treatment with morphine can attenuate the pain-induced activation of this circuit. However, whether cannabinoids can influence this circuit's activity to reduce withdrawal symptoms remains untested. Our research will contribute to our understanding of the neurophysiological basis for opiate withdrawal and how cannabinoids could represent a novel class of therapeutics for the treatment of opiate use disorder.

Addiction is characterized by the compulsive use of substances despite adverse consequences, a process closely linked to dopamine-induced changes in the Nucleus Accumbens (NAc) and its role as the brain's "reward center." The NAc integrates information from various brain regions, including the Paraventricular Thalamus (PVT), to produce motivated behaviors. Recent studies have identified the PVT, especially its anterior segment (aPVT), as a critical hub in addiction neurocircuitry, but findings have been inconsistent, likely due to the PVT's heterogeneity and the specific neurochemical and anatomical properties of its connections to the NAc. Prior research has shown that aPVT neurons, identifiable by neurotensin expression, send excitatory projections to the NAc, which are modulated by endogenous cannabinoids (eCBs). These interactions suggest a complex regulatory mechanism. Preliminary experiments used techniques including transsynaptic viral tracing and in vivo calcium imaging, to study the activity dynamics of NAc neurons, particularly those expressing Proenkephalin (PENK) and receiving aPVT inputs, during reward-seeking tasks. I propose to extend these findings by employing a multidisciplinary approach that combines experimental neuroscience with sophisticated computational analysis. By applying dimensionality reduction techniques, clustering algorithms, and machine learning models to neural and behavioral data, I aim to map the functional connectivity within the NAc and elucidate the roles of specific neuronal ensembles in reward-seeking behavior. This comprehensive analysis will not only clarify the neurobiological underpinnings of addiction but also contribute to the development of targeted therapies for addiction and related disorders, leveraging the unique intersection of computational neuroscience and behavioral analysis.

mTOR, the mechanistic target of rapamycin, is a serine/threonine kinase that regulates protein synthesis, cell growth, and metabolism in response to nutrients and energy in most eukaryotes. mTOR consists of two distinct complexes, mTORC1 and mTORC2. These complexes can be further divided into three components: mTORC1 consists of mTOR, Raptor, and mLST8, and mTORC2 consists of mTOR, Rictor, and mLST8. mTORC1 is critical in metazoan development and has been implicated in aging, cancer, diabetes, cardiovascular disease, and hypoxia. Previously, the Crowder lab conducted a mutant screen in C. elegans for hypoxia resistant mutations, and identified a missense reduction of function mutation in the daf-15 gene, the C.elegan ortholog of Raptor. A unique feature of this mutation is that the function of Raptor can be turned on and off by varying temperature. It has normal hypoxia resistance at 20 degrees, increased resistance between 21-22, and developmentally arrests at 25 degrees. I and the other authors made use of this conditional developmental arrest phenotype to screen for genetic suppressors. Using genetic mapping, sequencing, and complementation testing, we have identified multiple mutations in three different genes responsible for restoring Raptor function. One of the genes identified in this manner was rnf-126. Results show mutations in rnf-126 suppress the Raptor mutation. A null mutation in rnf-126 similarly suppressed the Raptor mutation. Previous work has implicated mammalian rnf-126 in degradation of the mTORC1 complex in cancer cells, suggesting that reduced levels of daf-15 may produce hypoxia resistance. We tested this hypothesis using auxin-mediated degradation of daf-15, finding that auxin-treated animals are hypoxia resistant. Current work by myself and others will further investigate how rnf-126 controls Raptor function and hypoxia sensitivity. Elaborating the function of this gene will define novel mechanisms whereby Raptor and mTORC1 controls metabolism, hypoxic injury, and development.

 The locus coeruleus (LC), a nucleus in the pons of the brainstem, is the primary source of norepinephrine (NE) in the brain and is responsible for regulating arousal and stress responses. The LC also has roles in pain, avoidance and reinforcement learning, exploration, and feeding. How LC neurons are mediated to generate these distinct responses has yet to be defined. One area of interest is the peri-LC, which sends local GABAergic projections to the LC and is thought to modulate physiological arousal through direct inhibition of the LC. The peri-LC is composed of various populations of GABAergic neurons that may contribute to the inhibition of the LC. We were interested in exploring the role of neurokinin1/substance P, nociception, and enkephalin GABAergic neurons. Using vGAT-cre, Tac1-cre, Noci-cre, and Penk-cre mice strains, we injected an excitatory DREADD into the peri-LC to be able to activate genetically defined neural subpopulations within the peri-LC. I ran the mice through open field and elevated zero maze testing to assess anxiety levels. The activation of all peri-LC^GABA neurons led to a decrease in anxiety-like behavior, which is consistent with an inhibition of the LC and a decrease in LC^NE release. In contrast, the activation of peri-LC^Tac1 neurons increased anxiety-like behavior, while the activation of peri-LC^Noci and peri-LC^Penk neurons had no behavioral effect in these assays. Together, these results suggest that the different peri-LC^GABA subpopulations likely represent distinct nodes that differentially integrate novel stimuli to influence distinct behaviors. Understanding how the peri-LC regulates LC function can give insight into the neurobiological basis of arousal and stress-related disorders, which informs our understanding of neuropsychiatric pathologies.

Gravitational waves are ripples in the fabric of space-time caused by the rotation and merging of black holes deep in our universe. On earth, these waves cause a minute strain that can be measured with the Laser Interferometer Gravitational-wave Observatory (LIGO). We are developing a calibrator that exerts an oscillating gravitational force on the test mass of LIGO to precisely calibrate the strain sensitivity of the interferometer. The calibrator consists of four motor-driven rotors which are placed around the test mass. The four motors have to run at a constant speed and have to maintain an exact phase relationship. In this research, I designed motor-controller software in Python, which rotates the motors with constant speed with a phase uncertainty of less than 2 degrees. The system uses one of the motors as the reference and converts its encoder position changes to frequency. A Proportional-Integral-Derivative (PID) loop locks the encoder’s frequency to a reference frequency. Then, the three witness encoders are locked to the lead encoder. The gravitational force from the calibrator is calculated using a Python program from Prof. Gundlach's team. The code decomposes the calibrator into 3D points and applies multipole expansions to accurately compute the force at the center of LIGO’s test mass. The gravitational calibrator will help to reduce uncertainties in LIGO’s strain readouts.

As computers have progressively gotten smaller and more powerful, we approach a physical barrier to further progress. Due to this, expanding resources have been devoted to developing quantum computing where the obstacles to progression are not physical. This technological advancement would have significant implications for various technological fields. Entities with access to this new technology would not only be able to benefit from it but would be capable of abusing it. These misuses could pose significant ethical risks, such as undermining the security and privacy of data and creating new forms of financial inequality. This literature review reveals the incoming disruption from quantum computing and the dissension between government, the public, and industry to ensure the responsible and beneficial use of quantum computing. Industry wants to develop and commercialize quantum computing as quickly as possible to gain an advantage in applicable industries. Government wants to balance the opportunities and risks that quantum computing poses for national and global security, but at the cost of expedited development. Though regulation and policy usually lag behind technical development, quantum computing's significant force will compel government and industry to adjust policy. The key future implications of this research are to develop and implement cross-disciplinary ethical principles and standards for designing and adopting quantum computing technologies. Achieving this by anticipating the possible impacts of quantum computing on existing systems and infrastructures, and developing strategies and policies to mitigate the risks and maximize the benefits. To ultimately support and promote research and innovation in quantum computing that defines a common good, and seeks to fulfill it.

The relationship between genius and mental illness has long fascinated scholars and captured the public imagination. While early notions of this connection can be traced to ancient Greek philosophers, modern scientific research explores possible psychological and neurological explanations. This literature review provides an overview of past studies, both historical and recent, that offer explanations for this connection. Various causes for the creativity-mental illness connection were investigated, including influence from stereotypical representations of successful artists and genetics that correlate with both artistry and psychosis. Additional citations were gathered through an interdisciplinary lens, including and posthumous assessments of artists who exemplify this connection. Drawing inspiration from researchers such as KR Jamison, this research references iconic figures in the arts and sciences, such as Van Gogh and Isaac Newton. These case studies exemplify various manifestations of the link between creativity and mental illness. While early research anticipated a direct correlation between creativity and mental illness, modern results do not show a definitive connection. Rather, many sources indicate a strong connection with varying explanations. The differences in existing theories have led to gaps in our understanding of the nuances of this topic. By synthesizing many viewpoints and evidence from prior studies, more definite theories regarding associations’ validity and complexities can be created. Additionally, this knowledge could inform progress in domains from cognitive science to therapeutic interventions and art therapy. Future research should aim to bridge the gaps between past studies, potentially by combining past research methods and utilizing more diverse subject populations.The implications of any associations identified could pave the way for future research in this area, as well as revisions to treatments for people struggling with mental illness.
 

Kaposi’s Sarcoma (KS) is among the most common tumors in central Africa and is a prevalent AIDS-associated malignancy. Kaposi’s Sarcoma-associated herpesvirus (KSHV) is the etiologic agent of KS. While all herpesviruses are capable of both lytic and latent replication programs, KSHV is predominantly in the latent state in the main KS tumor cell, the spindle cell - a cell expressing markers of the endothelium. There is limited viral gene expression during latency so it is difficult to target the virus directly. Therefore, our approach is to target host cellular requirements for KSHV latent infection. Previously, the Lagunoff Lab performed a genome wide CRISPR-Cas9 screen targeting over 18,000 human genes to identify cellular genes essential only to cells latently infected with KSHV. ACADS and CHD1 are two genes identified as some of the top hits from the screen. ACADS encodes a tetrameric mitochondrial flavoprotein, which catalyzes the first step of mitochondrial beta-oxidation. CHD1, or chromodomain helicase DNA binding protein 1 alters gene expression by chromatin modification. I hypothesize that ACADS and CHD1 are required for survival of latently infected KSHV cells but not uninfected cells. To test this hypothesis, I created knockout tert-immortalized microvascular endothelial (TIME) cells of each gene with CRISPR-Cas9 and plasmids containing guide RNAs used in the original screen. Then, I infected control and knockout cells with either KSHV or mock, and conducted trypan blue assay at 72 hours post infection to measure percent of live cells. Preliminary data suggests an increase in cell death for KSHV infected ACADS knockout cells compared to the control cells. In future experiments, I expect a significant decrease in the percentage of live cells in the ACADs and CHD1 knockout cells compared to the control and uninfected cells.

A key neuromodulatory system involved in anxiety disorders is the locus coeruleus noradrenergic system (LC-NE), which projects broadly throughout the central nervous system. The LC is stress responsive and tonic activation of the LC and its projections to the BLA is anxiogenic. Previously, the Bruchas Lab has used two-photon calcium imaging to show that a powerful stressor (predator odor) increased synchronous activity of LC neurons. They also found that mimicking this predator odor evoked activity with optogenetics altered the activity of individual neurons downstream in the BLA in a β-adrenergic receptor (β-AR) dependent manner. Although these data support the LC's involvement in promoting aversion and increasing anxiety-like behavior, the specific neurotransmitter, neuronal cell types, and receptors responsible for these effects remain unidentified. Therefore in hopes of identifying these specific signaling molecules and neuronal cell types and receptors, I first used fiber photometry and a novel biosensor (GRABNE2m) to detect norepinephrine (NE) release in the BLA while mice were exposed to a predator odor. I found that predator odor produced robust increases in NE release in the BLA compared to control odor (n=5, 3 male, 2 female) Further, we found that optogenetic activation of terminals from the LC to the BLA produced very similar levels of NE release compared to what was evoked by predator odor. To determine the cell type and receptor that is sensing this stress-induced NE release, I used a CRISPR/SaCas9 virus, developed in collaboration with Dr. Larry Zweifel’s lab, to knock-down β2-adrenergic receptors (β2-ARs) in glutamatergic BLA neurons to test their causal role in stress-induced anxiety-like behavior. CRISPR knockdown of β2-ARs in the BLA blocked several stress-induced anxiety-like behaviors (n=4, 4 female). By understanding the circuit-based mechanisms of how stress-induced anxiety is regulated, researchers could identify potential targets for therapeutic treatments of anxiety disorders.

Cannabis use has dramatically increased in response to legalization in the U.S., with U.S. sales jumping 46% from 2019 to 2020. ᐃ9-tetrahydrocannabinol (THC) is the primary psychoactive compound in Cannabis, and it has been shown to modify learning and motivation amongst regular users. Learning and motivation are key central processes primarily organized by the prefrontal cortex (PFC) brain region. I sought to test effects of THC on PFC activity during appetitive Pavlovian conditioning in mice- a behavior in which a subject learns to pair two stimuli together over time. Doing so provided much needed insight into learning and motivation under the effect of THC. THC acts on the endocannabinoid CB1 receptor, a presynaptic signaling protein responsible for modulating neural activity throughout the brain, with robust expression in the PFC. To monitor neural activity during behavioral trials, we implanted optic fibers into the PFC and virally expressed biological sensors. We used VGLUT1-Cre mice with a Cre-dependent GCaMP6f sensor to selectively target pyramidal glutamatergic activity during conditioning. We also utilized machine learning tracking software, SLEAP, to analyze behavior through video recordings. In our conditioning paradigm, animals were presented with a houselight and a sucrose reward, which they consolidated an association between after many trials. The mice experienced 5 days of Pavlovian conditioning, and I injected a moderate i.p. dose of THC (5 mg/kg) to one cohort, while another was given a vehicle before undergoing further trials. Our preliminary results showed that glutamatergic activity correlated with learning and association to the cue over time. We expected and observed that THC decreased the signals across the animals and reduced motivation. We categorized THC-induced behavior using SLEAP, a program tracking the mouse’s body parts to capture real-time movement, and found that locomotion decreased and resting behaviors increased in the THC cohort.

Mutations in ß-myosin heavy chain (MHC) are implicated in familial cardiomyopathies. Such mutations can modify myosin motor function or modulate the number of heads participating in contraction; changes at the protein level in turn affect sarcomere performance and ultimately lead to pathologic cardiac remodeling. Our aim is to better understand the pathogenesis of familial dilated cardiomyopathy (DCM) by studying how the R369Q mutation, which resides on loop 4 of the ß-MHC structure and participates in the interaction with actin, modulates the structure and dynamics of pre-powerstroke myosin. Previously, we have employed molecular dynamics to simulate and study the pre-powerstroke state at atomistic scale. Measurements of root mean square deviation (RMSD) and root mean square fluctuation (RMSF) for each of the six simulations demonstrated that R369Q myosin had a higher average RMSD over the course of the simulation and higher RMSF per residue than did wild type; this suggests that the overall structure of the R369Q ß-MHC is less structurally stable than the wild type. Additionally, we observed a significant loss of several contacts between Switch I and a flexible loop in the Upper 50 kDa Domain in the R369Q structure. Thus, we postulate that the R369Q mutation introduces structural instability into the myosin structure and disrupts the inter-protein communication of structural changes that drives the ATPase cycle. Here, we expand on these results with analysis of a stem cell-derived cardiomyocyte model and Brownian dynamics simulations. Analysis of myofibrils, the contractile organelles of cardiomyocytes, from cultured cells will confirm my previous computational work and give further insight into nucleotide-binding pocket coordination while Brownian dynamics will reveal changes in actomyosin interactions. A multifaceted analysis of the R369Q mutation from computational and experimental approaches will consolidate the mechanism of the mutation, providing a foundation for design of a therapeutic to ameliorate contractile dysfunction.

Tumors generally spread and remain in one individual; however, transmissible cancer differs. Detected among Tasmanian devil and dog populations, transmissible cancer is defined as whole cancer cells spreading among individuals in a population. Recently, Bivalve Transmissible Neoplasia, a transmissible cancer found among bivalve species, was discovered among soft-shell clams (Mya arenaria) on the East Coast of the USA and Canada; however, it has not been reported in any populations of soft-shell clams on the West Coast. Preliminary data showed that BTN was present in at least one West Coast soft-shell clam population. We focused on testing for BTN among soft-shell clams sampled from multiple sites in Washington State and determining which sub-lineage of BTN can be found among the West Coast soft-shell clams. I extracted DNA from the hemolymph of 37 soft-shell clams from Similk Bay to test for the presence of BTN using quantitative PCR (qPCR). The results indicated that soft-shell clams from Similk Bay tested negative for BTN despite an exposed population nearby in South Skagit Bay. I also analyzed DNA from soft-shell clam BTN from South Skagit Bay and Triangle Cove, Washington, to identify specific genetic markers to diagnose the specific sub-lineage the Soft-shell clams derived from - East Coast, USA or Prince Edward Island, Canada. An SNV in the COI gene specific to the Canadian sub-lineage of BTN was not found, and insertion sites of the Steamer retrotransposon specific to the USA sub-lineage of BTN were present among the West Coast soft-shell clams BTN samples, showing that the BTN on the West Coast arose from the USA sub-lineage. Given these results, analysis of more sites will provide information on how far the USA BTN sub-lineage has spread among West Coast areas and possibly how neoplastic cells can transmit among softshell clams across a body of water.

VirScan, a revolutionary technology based on Phage Immunoprecipitation Sequencing (PhIP-Seq), allows the interrogation of antibody responses to all known human viruses using a small blood volume, providing information on an individual's previous viral exposures. This study aims to provide a comprehensive data quality assessment system for VirScan, which will improve its reliability and interpretability by routinely assessing VirScan data quality at both the sample, assay (N=96 samples in replicate), and sequencing batch levels (N=192 samples in replicate). The study focuses on creating standards and thresholds for data quality at all three levels, considering aspects such as aligned reads, read depth, percent of epitopes discovered, and correlation of sequence counts between replicates. The assay/batch-level analysis provides metrics like the mean, median, standard deviation, and range of mapped reads and correlations for count and peptide detection, evaluating consistency, accuracy, and comparability across assays and batches. Further, these criteria can effectively categorize sample quality into Good, Questionable, and Failed, identifying samples that may need to be repeated or excluded from analysis. These quality calls were all encoded within an R Shiny App, enabling a user-friendly and flexible interpretation of VirScan data. Implementing this systematic quality control strategy will considerably improve the usability of VirScan in research and clinical contexts, allowing for more trustworthy interpretations of an individual's viral exposure history while also contributing to a better knowledge of immune response dynamics.