Fluorescent biosensors are a vital tool in the goal to decipher the complexity of neural networks. Genetically encoded fluorescent indicators (GEFIs) are protein-based sensors 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. Our project aims to tackle this problem by developing a high-throughput sensor engineering platform that can effectively generate and screen unbiased genetic libraries of GEFIs in mammalian cells. Our platform can identify high performing sensor variants on a custom microarray and effectively isolate and recover their genetic material. Our new platform will be used to develop a sensor for the μ-Opioid receptor (MOR), which is a G-protein coupled receptor that is involved in opioid addiction. Our experiments have already developed a MOR sensor that surpasses the standard in the literature and we will continue to optimize it 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.

As dam removals become more frequent across the United States and globally, understanding kelp forest response to coastal dam removals, in particular, is critical for managing nearshore habitat. Dam removals impact flow and sediment regimes of rivers, both of which can influence coastal kelp forests. The removal of the Elwha River, WA dams released ~19 Mt of sediment into marine ecosystems. This sediment flux dramatically influenced the turbidity of the nearshore water column and permanently increased the amount of sediment released from the river. The objectives of this study were two-fold. First, I developed methods to remotely sense and quantify the abundance of canopy forming kelps from aerial images. Next, I determined how changes in suspended sediment concentration and light attenuation during and following a major dam removal event in the Elwha River impacted the growth of nearshore bull kelp, Nereocystis luetkeana. By incorporating remote sensing to quantify the relative abundance of bull kelp in the area, my study observed that following dam removal, bull kelp abundance declined in the study area. This was an unexpected finding because I calculated that suspended sediment concentration decreased following dam removal, thus improving conditions for kelp growth. Although suspended sediment concentration decreased after the dam removals, other factors, such as an increase in herbivorous predation, could have played a large role in suppressing bull kelp abundance. The supervised classification scheme that I developed for remotely monitoring kelp abundance will make analyzing larger areas feasible in future studies, which may help to better identify regional trends in kelp abundance. Marine aerial monitoring remains an important avenue to better predict and manage future kelp forest response to dramatic changes in the ecosystem.

Humans hear through the conversion of pressure waves vibrating in the inner ear into chemical signals released by activated hair cells. Loss of function of hair cells due to damage can result in permanent hearing loss. Zebrafish are one of the model organisms used to study hair cells, since zebrafish have hair cells similar to humans. Unlike humans, zebrafish can regenerate their hair cells after damage. Regeneration occurs when support cells differentiate into hair cells. Determining how zebrafish support cells differentiate into hair cells is important to understand if human support cells can be induced to differentiate in a similar way. This requires comparing the structural differences between support cells and hair cells. One important aspect of neuronal signaling is the release of calcium ions from the cell’s endoplasmic reticulum (ER). This experiment looked to answer whether the ER in zebrafish hair and support cells was quantifiably different. Since structure mediates function and support cells do not signal to neurons, their ER structure should not be equivalent to the hair cells’. To test this, I manually segmented and reconstructed serial block-face scanning electron microscope (SBF-SEM) images of both cell types’ ER into 3D. Using SBF-SEM as a reconstruction method allowed for more detailed visualization of both the cell volume and the ER, in contrast to other methods such as confocal microscopy. Support cells had an ER volume of 50.51 µm3, while hair cells had an ER volume of 29.09 µm3. Support cells had a higher ER to cell volume and ER surface area to volume ratio than hair cells. It can be concluded that ER structures of support and hair cells are quantifiably different. Quantifying ER differences between support and hair cells is an important step toward discovering solutions to deafness caused by damage to human hair cells.

Humans are incapable of regenerating a majority of their major tissues following traumatic injury. Xenopus tadpoles have the ability to regenerate a variety of complex tissues quickly following tail amputation, but lose this regenerative competency during metamorphosis. Though tadpoles have been extensively used to study regeneration, we do not yet understand the roles that stress signals from injury play in directing regenerative gene expression. We have found that inhibition of the stress responsive transcription factor Hypoxia Inducible Factor 1α (Hif1α) with the Hif1α inhibitors 2-methoxyestradiol (2ME) and Echinomycin (Ech) prevents regeneration. In particular, inhibition of Hif1α decreases Wnt mediated gene expression. Wnt is known to be one of the primary signaling processes necessary for proper Xenopus regeneration, specifically tail regeneration. While we have shown that Hif1α and Wnt regulate expression of similar gene programs in regeneration, we predicted that there are unique processes that Hif1α regulates to facilitate growth. To investigate how Hif1α and Wnt regulate regeneration, we utilized the two Hif1α antagonists, as well as the Wnt antagonist, IWR-1 (IWR) which I have previously shown inhibits regeneration. To determine Hif1α and Wnt regulated genes, we performed RNA-sequencing 24 hours post amputation. I then identified genes downregulated in Hif1α inhibited tadpoles, which are likely Hif1α dependent. I then removed genes downregulated by Wnt in order to isolate genes uniquely regulated by Hif1α. With the 250 genes uniquely regulated by Hif1α, I used PANTHER to perform gene set enrichment based on Gene Ontology terms. The main biological process of interest found to be regulated by Hif1α during regeneration was DNA replication. By determining how Hif1α uniquely regulates DNA replication during regeneration, we will continue to enhance our understanding of the roles that stress signals play in directing how regenerating cells meet increased proliferative demands post traumatic injury.

Environmental stress and threat influence feeding behavior in animals, but how that interaction occurs is still largely unclear. Neurons in the bed nucleus of the stria terminalis (BNST), part of the extended amygdala, have dense projections to the parabrachial nucleus (PBN) in the brainstem. We have uncovered projections in these neural circuits that link the modulation of feeding and threat assessment in mice. This project aims to investigate and characterize these previously unrecognized neural circuits with the incorporation of a variety of optogenetic, surgical, and histological techniques. We used Cre-dependent anterograde and retrograde viral tracers in order to trace the anatomy of these neural circuits, and found functional projections from inhibitory (GABA) and excitatory (glutamate) populations in the BNST to neurons in the PBN. We also used translating ribosome affinity purification (TRAP) to isolate the mRNA of these projections. This proved useful in separating and identifying the molecular expression profile of different GABAergic and glutamatergic subpopulations. Furthermore, we used a variety of behavioral assays to determine the BNST-PBN circuits’ role in feeding and threat-response behavior. We used fiber photometry to track the activity of GABAergic (vGAT) and glutamatergic (vGLUT2) populations during these behaviors, and found that vGAT and vGLUT2 populations have differing roles in threat and feeding behaviors. vGAT neurons increase their activity during feeding and decrease in response to threat, while vGLUT2 neurons decrease their activity during feeding and increase in response to threat. We also used optogenetic activation of these neurons to determine their causal role in behavior. With activation, vGAT populations drive place preference, operant positive reinforcement, and increased feeding. Conversely, vGLUT2 populations drive place aversion, operant negative reinforcement, and reduced feeding. These findings characterize the distinct nature of BNST-PBN neural circuits and the mechanism behind the evaluation of threatful stimuli and the integration of feeding.

The female reproductive tract must maintain normal reproductive functions while also being able to elicit immune responses to sexually transmitted microbes and viral pathogens. In response to virus exposure, the balance of these two functions can determine the outcome of infection and disease through alteration of vaginal mucosa integrity. Recent in vivo research in our laboratory revealed that vaginal infection by Zika virus (ZIKV), an emerging mosquito-transmitted flavivirus that is also sexually transmitted among humans, induces epithelial cell-specific innate immune response that accelerates a homeostatic form of cell death known as cornification. We hypothesize that specific processes in vaginal epithelial cells mediate cornification in response to ZIKV infection, and that this cornification may alter barrier properties, innate-adaptive immune crosstalk, and ultimately, the degree to which ZIKV disseminates from the female reproductive tract. We therefore analyzed and compared vaginal epithelial cell infection by both African and Asian lineage ZIKV strains using traditional 2-dimensional cell culture and organotypic 3-dimensional culture infection models. Differences in viral infection and replication kinetics, and innate immune response were characterized through RT-qPCR, immunoblot analysis, cell imaging, and viral plaque assay. Ongoing analyses are expected to reveal the application of organotypic 3-dimensional cultures in capturing in vivo qualities of vaginal epithelial cell/ZIKV infection and response compared to 2-dimension cultures. These studies will provide insights for application of vaginal epithelial cell culture models of ZIKV infection that encapsulates the complex functional and structural aspects present in vivo.

 The herpesvirus Epstein-Barr Virus (EBV) targets B-cells and epithelial cells. The virus is transmitted by saliva and commonly associated with infectious mononucleosis. After initial infection, the virus enters a latent stage. The first oncogenic virus identified in humans, it contributes to 1.5% of all cases of human cancers worldwide, specifically cancers of B-cells and epithelial cells, and roughly 140,000 deaths/year. Currently, no treatment is available for EBV-related cancers. Due to the widespread impact of EBV on populations, including a drainage of resources in parts of the world where EBV-associated cancer rates are disproportionately high, an effective treatment will be greatly beneficial. The gH/gL complex and gB, glycoproteins necessary for virus fusion to the host cell, are conserved among herpesviruses, including EBV. Recently isolated, the monoclonal antibodies AMMO1 and AMMO5 inhibit EBV infection by preventing fusion to the host cell. AMMO1 binds to the gH/gL complex and interferes with both epithelial and B-cell infection, while AMMO5 binds to gB and can prevent epithelial cell infection. Emerging evidence suggests certain tumors express these glycoproteins, thus they may be readily targeted for immunotherapy. In this project I created Jurkat leukemic cell line T-cells which express chimeric express chimeric antigen receptors (CARs) using AMMO1 and AMMO5. This CAR should target EBV viral antigens on tumors. First, I used mutagenesis to insert DNA encoding for the AMMO1 or AMMO5 scFV, into a CAR expression plasmid and verify integration by Sanger sequencing. I then used lentiviral delivery to transduce CAR constructs into Jurkat T-cell genomic DNA. Flow cytometry was used to confirm transduction of the T-cells. Future directions with this project include increasing transduction efficiency and transducing the constructs into cytotoxic T-cell so T-cell killing assays can be used to determine the efficacy of the AMMO1 and AMMO5 CAR-T cells.

Bias in healthcare is often hidden and expressed through unintentionally prejudiced communication between providers and patients. These “implicit biases'' often relate to a patient’s race, gender, or sexual orientation. Implicit biases are automatic attitudes and stereotypes that can operate outside personal awareness and lead to unequal treatment, health disparities, and a lack of patient support. Although much research focuses on implicit bias, the perspectives of those who experience it and the impact it has on these individuals is less explored. Of particular importance are voices of Black, Indigenous, and People of color (BIPOC) and those with marginalized gender identities or sexual orientations (LGBTQ+). These groups have historically suffered from health inequities. For example, research indicates that BIPOC people may be undertreated for pain and LGBTQ+ people may be refused care. The UnBIASED project at the University of Washington and the University of California, San Diego addresses implicit bias in patient-provider communication through computational sensing tools to provide communication feedback. Through 25 interviews with people who identified as BIPOC, LGBTQ+, or both, we explored patients' perspectives on experiencing implicit bias when communicating with healthcare providers. We analyzed interviews through an inductive qualitative approach to understand negative and positive experiences, and identify participants' ideal solutions for improving patient-provider communication. For example, participants suggested having a patient advocate, providing feedback to the provider, and improving providers’ cultural competence. We report on these findings with the goal of describing common pain points and specific sources of dissatisfaction among patients who experience implicit bias. These findings help raise awareness of clinical implicit bias from the perspectives of patients, encourage further research, and suggest patient-driven, patient-centered solutions for how implicit bias can be overcome at personal and institutional levels.

Reactive oxygen species (ROS) are metabolites which play a critical role in biological systems. The deregulation of ROS creation and reduction damages proteins, lipids, and DNAs. Specifically, hydrogen peroxide (H2O2) is a key molecule known for its significant role in the various physiological processes impacted by cellular redox dynamics. However, there is a lack of tools with the sensitivity, dynamic range, and kinetics necessary for researchers to measure intracellular H2O2 levels. Our lab previously developed a genetically encoded fluorescent indicator for the real-time monitoring of H2O2 dynamics to address this need. Genetically encoded fluorescent indicators are protein-based sensors that allow researchers to visualize the activity of important molecules and understand the mechanisms by which critical physiological pathways operate in living systems. These sensors have great advantages as tools for quantifying molecular activity, including high spatiotemporal resolution, subcellular specificity, and minimal invasiveness. Our green-fluorescent H2O2 sensor HRM63 demonstrated 50-times faster onset and 10-times higher sensitivity for H2O2 when compared to the current state-of-art redox sensor HyPerRed – a significant improvement. I strive to expand the fluorescent palette of H2O2 sensors based on the design of HRM63 in order to enable the multiplexed imaging necessary to capture the dynamic pathophysiological processes which involve ROS. I have taken a structure-guided protein design approach and applied molecular cloning techniques to develop and iteratively optimize red-shifted versions of HRM63 with longer excitation/emission wavelengths, which would allow for deeper penetration of light source and fluorescence. I validate the performance of prototype sensors by applying fluorescence microscopy techniques to image their responses to applied concentrations of H2O2 in models of human disease. By optimizing these sensors, we will create invaluable tools for researchers to study the pathophysiology of the wide range of diseases to which ROS are linked, including cardiovascular diseases, neurodegenerative diseases, and cancer.

Dam removals are becoming more common as many dams reach the end of their lifespans. As a consequence, it is important to understand the characteristics of the sediment accumulated behind them for sometimes over 100 years when it is released. These sediments can contain organic matter that is deposited in river deltas and reservoirs and can have an impact on the ecosystem. The Elwha River dams were removed starting in 2011, releasing 19Mt of sediment, and also a fraction of organic material that is unknown, some of which made its way to nearshore subtidal deposits. To characterize the relationship specifically between organic content and sediment size in sub-tidal deposits, box cores were collected and examined using x-radiography, wet sieving, and loss on ignition procedures. In subsamples with a lower percent of mud, the percent of organic matter is less than in subsamples that contained more mud. There is also a relationship between both percent mud and organic matter with the distance that the samples were collected from the river, with more mud and organic matter being found farther from the river mouth. These relationships can be explained by the settling velocity of flocculated sediment because organic matter better combines with mud particles and flocs sink slower than sediment of a larger size. This study is useful for future dam removals to determine what range of impacts it might have downstream.