During development, cardiomyocytes (CMs) undergo a hypertrophic growth phase to generate load for the heart to pump sufficient blood. However, in the early stages of pathological hypertrophy, stress-induced signal transduction promotes the addition of new contractile units through poorly understood mechanisms to maintain tensional homeostasis. Microtubules provide mechanical resistance in CMs. Our previous data shows that inhibition of contraction by expression of D65A cTnC (a point mutation on the calcium binding site of troponin C) results in complete myofibrillar disarray, with muscle stress fibers emerging in the cellular periphery. Surprisingly, when given topological cues, these cells show aligned myofibrils in the absence of contraction. Thus, the mechanism behind the maintenance of myofibril and passive tension in non-contractile CMs is not explained. My goal was to determine the role of microtubules in maintaining tensional homeostasis in response to change in internal tension in CMs. Here, wild-type (WT) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) were transduced to express cardiac troponin C (cTnC) with point mutations L48Q (hyper-contractile), I61Q (hypo-contractile), and D65A (non-contraction) to study the effect of varying levels of contractility or internal load on microtubules. The percent coverage of microtubules (staining within the CMs for cTnC L48Q, WT, I61Q, D65A) was 42.32%, 50.20%, 64.09%, 75.70% respectively (n=300). Complementary proteomics data have indicated that protein levels related to microtubule proliferation (MAP4) and assembly (Tubb3, Tubb4b, Tuba1a) were elevated in CMs expressing cTnC D65A. This new data gives us insight into how the microtubule remodeling in non-contractile and dysfunctional cardiomyocytes maintains tension in early stages and its possible role in myofibril formation.

With the impending implications of climate change and global warming, space colonization in the near future will be necessary for the long-term survival of humans. We are examining how exposure to space affects biological aspects of humans by studying a model organism using a CubeSat. A CubeSat is a miniaturized satellite used in space research, built to a set of standardized measurements (10 cm per side), allowing small research projects to be launched simultaneously. We asked the questions: what would the experimental design look like investigating microgravity and UV radiation’s impact on the model organism C. elegans and what is a feasible experiment to design on a CubeSat? In this project we developed this research question to design a molecular biosciences project on a CubeSat. As part of this project, we conducted a literature review to determine 1) how small-scale satellites can be used for this kind of research, 2) what model organism is best suited for our project, and 3) how we can build upon the existing body of knowledge. The literature review, done in consultation with experts in the field, focused on the effects of microgravity and UV radiation on living organisms in the space environment. We didn’t limit our model organism research to C. elegans in the literature review. We used the constraints of the CubeSat to determine our data sampling methods and developed a research question. We anticipate research from our literature review will help us determine the next steps to take in designing our project. We hope to continue this research in preparation for implementing our work in collaboration with UW CubeSat. Research projects and experiments done in CubeSats like this one can advance the research and help address challenges our Earth faces with a rapidly expanding human population and ongoing climate change.

To expand my experience with the human brain beyond dissections, I worked in the Intelligent Networks Laboratory (INL) in UW Bothell’s Computing and Software System (CSS) Division to simulate activity in a biological neural network. INL has a computer software system called “Graphitti” that allows users to run simulations of large biological neural networks. The specific simulations I analyzed were of growth and development in cultures of initially dissociated cortical neurons, which produce bursts of activity that propagate as waves across the network. Networks were grown over 28 simulated days on a 100 by 100 neuron grid, producing 500,000 synaptic connections. Two previous student papers suggest that this bursting activity is stable with 10% inhibitory neurons distributed across the simulated culture. With the original raw data stored in Matlab software (www.mathworks.com), I ran other code to pull burst location, speed, and distance from four simulations with different neuron firing rates and percentage of inhibitory neurons. In one simulation, burst origin locations were scattered across the network, indicating bursting behavior throughout the network. Two other simulations had a few, stable origin locations, suggesting a discernible pattern in bursting behavior. The burst propagation speed graphs for each network demonstrated growth and stabilization of activity by the end of the simulations. Working with this neural network has provided me with a fresh and "live" perspective on the connectivity of neurons. By understanding the dynamics of the network under these conditions, the data inspires further research investigating the connectivity effects of neurodegenerative diseases such as Parkinson's Disease.

In 2020, the COVID-19 pandemic caused students living in rural areas to experience exacerbated educational disparities. This included familial financial stresses, which also pushed many migrant students living in rural communities to prioritize work over school. The pandemic shed light on educational disparities featured in rural public-school education systems. The purpose of the study was to examine how the education trajectory of students in rural communities had been affected by the social and economic impacts of COVID-19. To accomplish this purpose, we examined the extent to which familial needs impacted students’ post-high school educational plans, how financial strain influenced their post-graduation choices, and how students practiced resourcefulness and resilience despite experiences of economic hardship. In this community-based qualitative research project, we conducted semi-structured interviews with Eastern Washington high school seniors who are 18 years of age or older and used a phenomenological thematic analysis to gather themes related to our research questions. As part of the research, we collaborated with a community advisory committee composed of teachers and recent high school graduates from Eastern Washington communities to develop the project’s research methods and to ensure the analyses and interpretation of interviews are reflective of the students’ experiences. We predicted that students will plan to alter their post-high school paths to accommodate their families’ needs. Anti-racist, strength-based frameworks were used to make academic support recommendations for students in rural communities. Ultimately, our study can help inform collaboration with community members to find solutions so we can best support students and encourage them as they navigate pathways after high school graduations.

Substance use disorders are shockingly prevalent in the United States, with the Centers for Disease Control and Prevention estimating that in 2019 alone, nearly 50,000 people died from opioid-involved overdoses. Neuropeptide S and its receptor have been previously implicated in drug-seeking behavior, making it an important component in understanding the biological functions underlying addiction. However, such findings have not been localized to any specific region. We set out to investigate the connection between the locus coeruleus, a region with a known population of neuropeptide S producing cells, and the orbitofrontal cortex, a region known to express neuropeptide S receptors. By utilizing an NPSR1-cre mouse line and cre-dependent viral expression, we introduced GCaMP, a fluorescent calcium sensor, into NPSR1 expressing neurons in the orbitofrontal cortex. This enabled us to record calcium fluorescence in vivo as a proxy for neuronal activity. This technique was paired with various behavioral paradigms to explore the endogenous activity of these neurons in natural reward-seeking, social interaction, and fear conditioning. Our results demonstrate that these neurons are activated during cue and food reward-delivery, various social rewards, and foot shock. Aligning these findings with previous research that has demonstrated neuropeptide S’s involvement in drug reward-seeking behavior, we believe these neuropeptide S receptor-expressing neurons in the orbitofrontal cortex could be implicated in drug-seeking behaviors. These findings contribute to the understanding of the neural circuitry involved in substance use disorders, which is integral in continuing the development of treatment options for patients.

Understanding the neural circuit mechanism underlying aversive memory formation is important for developing new treatments for patients with post-traumatic stress disorder (PTSD). The nociceptive circuit involved in such aversive memory formation remains incompletely characterized. Nociceptin opioid peptide receptors (NOPRs) in this circuit are expressed throughout the brain and have been implicated in nociceptive responses, anxiety, and aversive fear memory. To model aversive memory formation, we used a cued fear conditioning paradigm in mice. A mouse first learns to associate a tone and foot shock on day 1 and is assessed by its behavioral freezing response to tone only on day 2. We observed that using a high dose of a NOPR agonist on day 1 of the cued fear conditioning was associated with decreased arousal and prevented associative learning on day 2. Then, using thermal pain assays, we demonstrated a weakly anti-nociceptive effect after administration of the NOPR agonist, suggesting the disrupted fear learning is likely not due to loss of sensory transmission. Because disruption of fear learning was associated with decreased arousal, we screened for brain regions involved in the sedation from NOPR activation by selectively expressing NOPRs using a viral vector in different brain areas in a mouse lacking NOPRs. Interestingly, we found activating NOPRs in the parabrachial nucleus (PBN) in the brainstem was sufficient to produce sedation. Additionally, in situ RNA hybridization showed strong co-localization of oprl1 (NOPR gene) expressing cells and calca, a genetic marker encoding CGRP of a small group of cells in the ventrolateral PBN. Given previous literature has shown disrupted fear learning by inhibiting neural activity of those CGRP neurons, we hypothesize that NOPR-expressing PBN neurons may play an important role in aversive memory formation as well. Future investigations will explore the necessity and sufficiency of NOPR-expressing PBN neurons in aversive memory formation.

 Δ⁹-tetrahydrocannabinol (THC) is the primary psychoactive compound found in Cannabis sativa. In mice, intraperitoneal (i.p.) injections of THC, produce a characteristic triad of behavioral responses: hypolocomotion, hypothermia, and analgesia. However, injections of THC do not accurately represent how humans typically administer THC, which primarily consists of inhalation and oral consumption. Thus, we have developed and optimized a paradigm of oral THC consumption in mice to better model a typical route of administration used by humans. Our model balances an acute consummatory window with a highly palatable, chocolate-flavored gelatin. This incentivizes mice to voluntarily consume enough THC to produce measurable cannabimimetic behaviors. Over a 3-day exposure paradigm we habituated mice to the gelatin where they had ad libitum access for 2 hours each day. We introduced THC into the gelatin and measured the triad of behaviors immediately following consumption to determine whether voluntary oral consumption induces the acute cannabimimetic behaviors. We found significant hypolocomotion, hypothermia, and analgesia at our highest concentration. Next, to determine whether these behaviors are caused by THC’s action at the primary endocannabinoid receptor, CB₁R, we treated mice with the inverse agonist SR1 prior to the behavioral tests. SR1 blocked the cannabimimetic behaviors induced by the consumption of THC-gelatin, suggesting the effects are CB₁R-dependent. To finalize this model, we have adapted our oral consumption paradigm to an acoustic startle behavioral model. Following our consumption paradigm, mice are subjected to tones of varying decibels and their startle response is measured. Moving forward we will continue acoustic startle testing to confirm preliminary data and expand the doses tested. Overall, these data verify that our model effectively induces cannabimimetic behaviors and can be used for future behavioral studies investigating a more translational route of administration compared to i.p.

Binge eating disorder is a debilitating disease which can arise from many kinds of traumas, pains, and stresses of life. Previous characterization of a binge-eating model developed by our lab shows that mice will consume greater quantities of high palatable diet (HPD) following exposure to specific types of psychological stressors including forced swim and foot shock compared to mice exposed to such psychological stressors. It was found that the claustrum of the brain had increased neural activity following bouts of binge-eating. One aspect of my research required me to quantify the density of neural activation in the claustrum from its most rostral to caudal area. We found that stressed mice displayed significantly higher levels of claustrum neural activation compared to controls. For the behavioral pattern we wanted to rule out influence of energy expenditure in the stress paradigm. Mice were given access to running wheels for an hour and then received access to HPD. Mice who displayed high levels of running had similar food intake to that of mice who did not display running activity. This suggests that psychological stress is an underlying component in this model for stress eating. As an ongoing project we are utilizing 1-photon imaging in the claustrum to monitor single cell activity across no stress vs. stress sessions and subsequent feeding behavior. We have thus far found increased neural activity in response to onset of a feeding bout in no stress conditions and we are investigating how stress modulates the effect of neurons tracked across time. This research potentially has great impact on the scientific community’s knowledge behind why psychological stressors contribute to binge-eating behaviors and could one day have astounding translational benefits for treating humans with binge-eating disorder.

The locus coeruleus (LC) is a small nucleus of noradrenergic neurons in the pons, which, despite its size, has broad projections throughout the central nervous system (CNS). Functionally, the LC is believed to be involved in various critical functions, including in the physiological responses to stress and mediating arousal. Previous investigations have demonstrated that optogenetic activation of the LC using channelrhodopsin at a tonic frequency promotes wakefulness in rodents. While this observation causally implicates LC function in wakefulness, it is still not known how the LC is endogenously controlled to mediate arousal. One potential candidate in this control involves the peptide nociceptin and its cognate receptor, the nociceptin opioid peptide receptor (NOPR), both highly expressed around the LC. To investigate, we conducted two pharmacological experiments using the NOPR agonist Ro64-6198 to investigate its effects on locomotion and on the activity of LC noradrenergic neurons. We found that Ro64-6198 (10 mg/kg) strongly reduced open-field locomotor activity compared to vehicle treatment. Using in vivo 2-photon calcium imaging (GCaMP6s), we found that Ro64-6198 (5 mg/kg) profoundly reduced LC noradrenergic neuron activity. Wakefulness appeared reduced in both in vivo experiments. To determine where the endogenous nociceptin signal to the LC originates, we performed an intracranial injection of a Cre-dependent retrograde virus (AAV2-DIO-eYFP) into the LC of a mouse expressing Cre recombinase in nociceptin-expressing neurons. We identified a long-range nociceptinergic projection from the bed nucleus of the stria terminalis (BNST). In order to evaluate how the activity of these BNST neurons are affected by wakefulness, we conducted home-cage fiber photometry recordings. Together, these studies suggest that nociceptin acting on LC noradrenergic neurons reduces arousal, and that the endogenous sources of nociceptin come from the BNST. These experiments shed new light on an understudied endogenous opioid system that may be a druggable target for sleep disorders.

Substance use disorders contribute to mortality nationwide and are associated with aversive and appetitive behaviors the ventral hippocampus (vCA1) is known to process. The neuromodulators dopamine (DA) and norepinephrine (NE) are also involved, but their release dynamics have not been thoroughly studied in this region. The dorsal hippocampus (dCA1) has similarly weak innervation from the ventral tegmental area (VTA), the major DA source, and the locus coeruleus (LC), the major NE source, and preliminary data in this region suggest an inhibitory effect on DA during salient aversive stimuli and minimal DA release during anxiogenic behaviors. To quantify monoamine release in the vCA1 and determine whether LC-sourced DA can be released there, we intracranially injected Dbh-cre positive mice with one of two biosensors, dlight for DA or GRABNE for NE, in the vCA1, and chrimson, a cre-dependent red-shifted channelrhodopsin, in the LC. A fiber optic lens for optogenetic stimulation and recording was implanted in the vCA1. We recorded dynamics during aversive and appetitive behaviors and found that DA and NE are released in the vCA1 during salient aversive stimuli, and that DA is released and NE release is inhibited during appetitive behaviors. We stimulated chrimson-transfected LC noradrenergic axon terminals while recording neuromodulator dynamics in the vCA1, and found that when stimulated for three seconds at 20 Hz, GRABNE fluorescence increases before slowly returning to baseline, while dlight fluorescence spikes quickly over 10 seconds, then exhibits a secondary, less steep, wave over the next 20 seconds. This secondary wave of DA is not observed in preliminary dCA1 data, indicating that it is unique to this region. In future studies we will investigate the role of the VTA in this secondary release with pharmacological inhibition experiments. These findings reveal distinct monoamine release in the vCA1 during aversive and appetitive behaviors. (Funded by NIMH-R01MH112355)

The consumption of Cannabis has increased with legalization, rising 46% from 2019 to 2020 in the US. The primary psychoactive compound in Cannabis, ᐃ9-tetrahydrocannabinol (THC), modifies motivation and induces hypolocomotive effects that cause patients to stop using medical marijuana. Given the increasing frequency of Cannabis use and the unwanted side effects of THC, I sought to decipher the motivational and locomotive effects of THC on prefrontal cortex (PFC) activity during appetitive Pavlovian conditioning. I utilized biological sensors to measure neural activity (CamKIIa-GCaMP6f for calcium in projection neurons (N=3) and eCB2.0 for total endocannabinoid activity (N=6)) in WT mice aged 8-12 weeks. Neural activity (utilizing fiber photometry) and general behavior was recorded during appetitive Pavlovian conditioning. Here, a house light in the behavioral chamber (conditioned stimulus (CS)), initiated 6s before a sipper (sucrose) extended for 20 seconds (unconditioned stimulus (US)). After a random inter-trial interval of 60, 90, 120, or 150s, another event triggered a reward to consolidate an association between the house light (CS) and the reward (US). Mice experienced this conditioning for 25 minutes every day for 5 days. On day 6, I treated mice with either a moderate dose of THC (5 mg/kg) or vehicle to measure changes in neural and endocannabinoid activity during conditioning. I found that both endocannabinoid and calcium signaling were tightly locked to the CS and the US. Interestingly, trials where THC-treated mice did not interact with the sipper (THC-dependent demotivation), neural activity matched the pattern during training days. These data suggest time-locked neural activity linked to stimuli, separate from the locomotor output, to receive the reward. This study contributes to the understanding of THC’s effects on signaling during motivated versus locomotive behaviors to inform future THC-derived treatment paradigms.

The discovery of fluorescent proteins led to the development of various protein-based biosensors that are vital in the goal to decipher the complexity of neural networks. Genetically encoded fluorescent indicators (GEFIs) are protein-based sensors with cell type specificity that increase in fluorescence upon ligand binding and allow for passive monitoring of neuronal signals. However, the development of such sensors is limited by the slow throughput of traditional protein engineering which has long engineering cycles of new plasmid variants. My project tackles this problem through the development of an optogenetic microwell array screening system (Opto-MASS) that effectively generates and screens unbiased genetic libraries of GEFIs in mammalian cells. The platform identifies high performing sensor variants on a custom microarray and effectively isolates and recovers their genetic material. This new platform was used to develop a sensor for the μ-opioid receptor (MOR), which is a G-protein coupled receptor and is responsible for the pain relieving effects of opioids and addiction. The platform has developed a MOR sensor that surpasses the standard in the literature in response to the synthetic opioid peptide agonist [d-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). I used this platform to engineer a new class of MOR sensors that are ligand-specific to endogenous opioids versus exogenous opioids and optimized the sensors for maximum spatial and temporal precision. The development of a MOR sensor through this iterative process allows researchers to further investigate the molecular mechanisms underlying the pathology of addiction and provides a novel platform for protein engineers to more efficiently develop a wide variety of biosensors.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus that is the etiological agent of Kaposi’s sarcoma (KS), a cancer of endothelial cell origin. Like other herpesviruses, KSHV has distinct latent and lytic replication cycles – during latency there is limited viral gene expression and no KSHV virions are produced, while in the lytic life cycle all viral genes are expressed and new virions are assembled. Both lytic and latent genes are implicated in KSHV’s oncogenic properties. During infection, endothelial cells from both blood and lymphatic vessels undergo changes in signaling pathways and morphology. However, differences in the expression of lytic and latent genes have been described for the different sources of endothelium. We have previously observed that blood endothelial cells (BECs) grown in culture are less susceptible to infection as compared to lymphatic endothelial cells (LECs). Other labs have reported higher levels of lytic gene expression in LECs. I aim to determine if there are differential levels of KSHV lytic gene expression in the BEC and LEC lines in our lab. To determine the levels of lytic replication in BECs and LECs, I isolated RNA at different time points post infection and use RT-qPCR to determine the relative levels of viral lytic genes. I tested different infection rates to determine the role of infection rates on levels of lytic replication. I am also testing different cellular growth conditions, including cell medias to determine if the levels of lytic replication depend on cell proliferation levels. The goal is to determine differences seen in the level of lytic replication in different endothelial cell types in different labs. Understanding the conditions for higher lytic replication of KSHV in endothelial cells could help understand KSHV tumorigenesis as lytic replication is a key factor in the way KSHV causes cancer.