To navigate their surroundings, migratory cells respond constantly to many signals in their environment, including both chemical and electric cues. These signals are produced locally by other cells, pathogens, and in the context of electrical signals, by disruption to the normal ionic balance across cell boundaries. Disruption to this ionic balance will create a local electric field to which immune cells will respond to guide their movement and prevent infection. How cells sense or respond to this electrical cue is not known. To better understand this phenomenon, we are using HL-60 cells that are a migratory neutrophil-like human leukemia cell line, which we have found migrates to the cathodal pole of an applied DC electric field. We have identified a number of gene candidates related to glycosylation, the modification of proteins with the addition of sugar molecules, that reduce the directionality of HL-60 cells in an electric field. Using CRISPR interference to create cell lines with reduced expression (knockdown) for eleven of the gene candidates, we are studying how the loss of these genes alter migration. We used video microscopy to track their migration in 3D at different intensity levels of current to see how the loss of these genes affected cell movement when cells are exposed to an electric field. All of these knockdown lines showed marked change in the cell's response, with less persistence towards the cathode at higher currents than control HL-60 cells. Of these eleven, knockdown of UXS1, a gene that encodes for UDP-xylose that is used in the attachment of long sugar chains (glycosaminoglycans) to certain proteins on the cell surface, showed the greatest effect. Our results suggest that UXS1 is critical for neutrophils' ability to sense or respond to DC electric fields. 

Melusin, a chaperone protein expressed in cardiac tissue, induces a protective hypertrophic response in response to chronic mechanical stress. This protective hypertrophic response prevents the progression of cardiomyopathy into heart failure. In previous work done in wild-type (WT) and melusin knockout (melKO) mice, the absence of melusin was correlated with a hypertrophic response indicative of heart failure. I plan to investigate the biomechanical role of melusin in humans using human-engineered heart tissues (EHTs) created from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that lack melusin and their isogenic controls. EHTs are more representative of the human heart, making them an ideal model for studying the role of melusin in humans. I hypothesize that WT EHTs subjected to mechanical stress, i.e., high afterload, will outperform the melKO EHTs. To measure this, I will increase the stiffness of the EHT posts and measure contractile force. I have successfully differentiated high-purity WT and melKO cardiomyocytes from iPSCs, essential for creating healthy EHTs. I will cast both WT and melKO tissues on a bed of silicone posts that can be stiffened to varying extents to induce different amounts of mechanical stress on the cells. I will conduct a western blot on EHTs from all treatment groups to determine the level of melusin expression and examine the expression of Heat Shock Proteins (Hsp) 70 and 90, due to their coregulation with melusin. The EHTs that undergo mechanical stress are expected to express melusin and these results will work to establish whether melusin expression in humans is activated by mechanical stress. I will measure and compare the contractile force between the WT and melKO tissues. Improving our understanding of the role of melusin in humans can lead to further research into therapies and treatments for heart failure.

With cardiovascular disease being the leading cause of death worldwide, new and improved disease models are required to facilitate the research and production of new treatments. In this research, we are developing improved methods for modeling hypertension in engineered heart tissues (EHTs) to investigate resulting tissue remodeling at the tissue and cellular levels. We developed a model of hypertension using 3D-printed polylactic acid braces that enable stiffness adjustment of the flexible polydimethylsiloxane (PDMS) EHT platform. The braces were validated by mechanical testing to quantify their stiffening effect. Braces were shown to increase stiffness according to beam bending theory, with bracing half of the post’s length resulting in a 7-fold increase in stiffness. Next, we applied braces to tissues that only contain stromal cells, which are responsible for remodeling the extracellular matrix (ECM). The next steps are to quantify how stiffness affects ECM remodeling, including tissue-wide effects such as changes in tissue length and width, and micro-scale effects such as changes in cell migration, apoptosis, and cytoskeletal structure, which are quantified in 3D using IHC and confocal microscopy. We hypothesize that hypertension results in tissue thinning and lengthening, as well as decreased cell density, increased apoptosis, and increased expression of cytoskeletal markers. Next, the braces are applied to EHTs containing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The effect of hypertension on EHT remodeling and hiPSC-CM maturation is then quantified using a custom MATLAB image processing suite. Finally, increasing systolic resistance of novel varieties of EHTs that incorporate hiPSC-derived cardiac fibroblasts (hiPSC-CFs) alongside cardiomyocytes enables evaluation of fibrotic remodeling. We hypothesize that hypertension promotes a hypertrophic response in both hiPSC-CMs and hiPSC-CFs in EHTs, displaying increased sarcomerization and fibrosis respectively. Developing this improved hypertension model will accelerate cardiac regenerative medicine research, and provide new approaches for drug discovery.

The alpine zone has been underrepresented in herbarium collections due to its difficulty in access and short growing season. Despite its underrepresentation, the alpine zone presents a unique opportunity to study climate change impacts due to species; limited ability to migrate to more suitable habitats. For this study, we are examining the Cascades Range from the Canadian border to Mount Adams. Our primary objective is to understand the distribution patterns of alpine species richness and how it is influenced by latitude and elevation. Our secondary objective is to see if these patterns in phytogeography correlate to species; life history characteristics of dispersal, pollination mode, and flower color. To research these questions, we created a species list of Washington's alpine plants using 50 Peaks Project data, historical herbarium records, and literature references. To assess latitudinal course patterns of species richness along the Cascades range, we created three relatively equal zones and scored the presence of each species in it. For statistical analysis, the total number of species per zone will be tallied and Chi-square analysis will be performed to test for significant differences in species richness. We will use regression analysis to quantify the relationships between latitude and the number of peaks, and latitude and average elevation. To compare life history traits across the three zones, we will analyze frequency distribution of those traits. Our preliminary results for latitudinal patterns indicate that the North Cascades have the most species while the Southern and Central Cascades are nearly tied. The final results from this study will inform the selection of future collecting locations and future analysis for species richness among peaks for the 50 Peaks Project. Preliminary Run through the Burke Herbarium, the 50 Peaks Project collects plant specimens to document diversity and distribution in Washington's Cascades Range alpine zone.

 As climate change worsens, flooding from extreme weather and sea-level rise continues to threaten coastal populations and energy infrastructure. Small, isolated island states like Puerto Rico have weaker electrical grids and are especially vulnerable. Coastal habitats such as mangroves and coral reefs buffer shorelines and offer natural protection against these storms. To identify where habitats reduce the risk of flooding and erosion, we used a spatial model that takes in biophysical data and estimates an exposure variable for every 250m of coastline. The model shows that habitats safeguard 250,000 people living on vulnerable coastlines. Most of these people live in major port cities with substations and fuel terminals that deliver power to the entire island. As urbanization and global warming further degrade coastal habitats, Puerto Rico loses its best defense against tropical storms. Our results highlight the importance of sustainable development planning, especially as the island invests in its renewable energy transition. The spatial model can help prioritize which vulnerable communities receive resilience funding and where to avoid siting tourism to preserve ecosystems. Our model also reveals degraded habitats that could be targeted for ecological restoration. In future projects, we will apply the model to other states to explore relationships between communities, energy, and climate across multiple land and seascapes.

It is well-established that mutations have impacts on an organism’s fitness; however, the fitness effects of mutations are not static, and can vary depending on environmental contexts, such as the species in which a mutation is found. Evolution of the same gene in different species could thus lead to the evolution of different phenotypes, as different species would favour different sets of mutations. If that gene could be exchanged between species, it could lead to increased evolutionary possibilities, as high-fitness genotypes that require a prerequisite deleterious mutation in one species could become accessible if the mutation is not deleterious in another. Our research examines how the presence of two bacterial hosts, Escherichia coli and Klebsiella pneumoniae, could affect the evolution of an antibiotic resistance-conferring TEM-1 β-lactamase gene located on a conjugative plasmid. If different hosts confer different mutational effects to TEM-1, the process of horizontal gene transfer (HGT) that allows mutations to be shared between species could open up more mutational possibilities than those accessible in either single-species population alone. We tested this hypothesis through three rounds of experimental evolution in the presence of the antibiotic cefotaxime, where we evolved two single-species E. coli and K. pneumoniae populations and one multi-species population where HGT was simulated with a shared plasmid pool. We are now reconstructing the genotypes found in all three populations after each round to assess how much antibiotic resistance they confer in both species, and hope to see if the genotypes acquired under HGT treatment provide higher resistance compared to the single-species populations. Our results have practical implications for the predictability and nature of antibiotic resistance development in the real world, a current global health crisis, and potentially motivate further study in predicting resistance emergence in clinically encountered multi-species populations.

 Birth control is an important tool to prevent unwanted pregnancies. However, many people who might benefit choose not to use birth control, contributing to a range of negative outcomes, including unwanted pregnancies and sexually transmitted infections. Many factors affect people’s perceptions of birth control options, and ultimately influence their use of contraceptives. Previous research shows racial differences in rates of birth control utilization, but little work has explored why these racial differences exist and if and how culture contributes to birth control attitudes and utilization cross-racially. Furthermore, most previous research in this area focuses exclusively on race, typically reporting only on Black, Hispanic, and White women, with little or no intersectional analysis. The present study investigates how culture influences birth control attitudes, considering both a broader range of racial categories and the impact of multiple intersecting identities on culture. Our goal is to gain insight into the health care decision processes of intersectionally marginalized patients. We accomplish this with a cross-sectional qualitative study. We first pre-screen potential interviewees to recruit participants with diverse cultural backgrounds. Selected participants will participate in structured 1:1 interviews, answering questions about how their cultural background (including race, ethnicity, religion, and family) influences birth control attitudes. We will conduct thematic analysis to determine what aspects of culture impact birth control attitudes. Shedding light on how culture influences the perception and use of birth control provides insight into a broader range of patient populations, allowing for improved contraceptive counseling and education in the medical setting. Recruiting a diverse sample will illuminate the lived experiences of individuals who are typically excluded from research and scholarship, allowing future advancements in birth control to be more representative and sensitive, with the knowledge of all cultural experiences in mind rather than just a select few.

People living with epilepsy (PWE) often have a poorer quality of life (QoL) compared to the general population. Anti-seizure medicines (ASMs) are used to control seizures in PWE but are often associated with side-effects that lead to reduced adherence. Poor adherence is associated with reduced seizure control which can also negatively impact QoL. A rat model of acquired epilepsy was used to evaluate how poor adherence to the ASM, perampanel (PER), impacts an animal’s engagement with environmental enrichment, which is provided to promote species-specific behaviors and general well-being. Cardboard enrichment was provided to single-housed male Sprague Dawley rats with acquired epilepsy and I scored their level of engagement at the start of each day: i.e.,1 being no engagement; 4 being completely engaged. Animals were observed for 8 weeks: 4 weeks without PER and 4 weeks with PER in a fully adherent (100%) or variably nonadherent (50%) dosing paradigm (10 mg/kg/day, p.o.). I recorded data on the number of days till first engagement with the enrichment, days till max, and max enrichment score. After compiling the data, I found no significant differences in the max enrichment score or days till max score amongst the 100% or 50% treatment groups. Interestingly, when compared to their pretreatment baseline, fully adherent rats took less time to initially engage with their enrichment, i.e., 6-9 days, compared to 9-12 days, respectively. In contrast, nonadherent rats did not show a similar improvement in their enrichment behavior when treatment was initiated. These results suggest that fully adherent rats were more willing to interact with their enrichment whereas, poor medication adherence may have a direct negative impact on QoL. Further investigation is necessary to determine if this is due to a difference in seizure control between the groups.

Treatment of individuals with HIV using antiretroviral therapy (ART) is highly effective, but effective clinical management depends on maintaining therapeutic drug concentrations. Antiretroviral (ARV) drug concentrations in patients with HIV can vary due to differences in drug metabolism, medication adherence, or interactions between multiple drugs. These individuals may have subtherapeutic or supratherapeutic drug concentrations, putting them at risk of treatment failure, acquisition of drug resistance, and risk of hospitalization or death. Current measurement of ARV concentration is done through liquid chromatography tandem mass spectrometry, which requires expensive equipment and requires a labor-intensive protocol. This restricts accessibility to specialized laboratories, making it difficult for persons with HIV to have routine measurements of ARV drug concentrations. The goal of the project is to develop an assay that is simple to perform and uses standard equipment to increase access to routine clinic-based drug level monitoring to improve HIV care. We designed an assay using a 2-step process of DNA strand transfer and quantitative polymerase chain reaction (qPCR) to quantify integrase strand transfer inhibitors (INSTIs). We tested for dolutegravir (DTG) and cabotegravir (CAB) in both buffer and plasma -- the latter to simulate patient blood samples. We were able to demonstrate that the assay could quantify clinically relevant drug concentrations of DTG and CAB. By developing an assay that can be readily integrated into most clinical laboratories, we will contribute to increasing access to routine HIV drug level monitoring to improve clinical HIV care and maintaining viral suppression in persons with HIV.

As of 2021, there were approximately 38.4 million people living with HIV who require routine viral load testing. Viral load testing returns a quantitative measure of viral concentrations and is indicative of antiretroviral therapy efficacy and adherence compliance, with lower viral loads correlated to better health outcomes. Quantitative polymerase chain reaction (qPCR) is the gold standard for measuring viral load, but is not accessible to many clinics and patients around the world due to its long assay times and requirements of specialized equipment and highly trained personnel. As a result, qPCR is limited to centralized testing facilities far from the point-of-care (POC), leading to delayed results or loss of follow-up. Our group has addressed this by developing an HIV viral load test using recombinase polymerase amplification (RPA), which has a 20 minute sample-to-answer time and is more appropriate for POC settings. Our test involves the formation of discrete fluorescent nucleation sites which can be counted to estimate the viral load. However, our test fails to accurately quantify higher HIV viral loads (>3,000cps/rxn). We have difficulties distinguishing between individual sites at these higher copy numbers due to sites merging. In this project, I address the limited dynamic range of this test by performing RPA between two glass slides and investigating the effects of different slide thicknesses and concentrations of polyethylene glycol (PEG), a crowding agent used in RPA reactions. I perform nucleation site analysis using computer vision techniques to measure nucleation site radius and intensity and study how these factors affect site diffusion and amplification. By analyzing nucleation site behavior, we demonstrate potential for an HIV viral load test with a higher dynamic range and gain a better understanding for RPA nucleation site formation, ultimately helping to improve access to testing and treatment for people living with HIV.

The periodontal ligament is a connective tissue that anchors teeth to the bony socket and is crucial in providing nutrition for the survival and functioning of the human body through the mastication of food by teeth. Because the periodontal ligament is anatomically sealed off from the oral cavity, no non-invasive techniques currently exist to investigate it in-vivo, making the development of sound in vitro models critical for periodontal research. With periodontal disease affecting over 743 million people worldwide, in-vitro research to develop regenerative therapies to replace diseased periodontal tissue is urgently needed. To achieve this, we have developed a novel 3D in-vitro model, which closely mimics the in-vivo periodontal ligament. The 3D tissues are fabricated by inverting an array of silicone posts into silicone molds containing a cell-collagen gel mixture in a 24-well plate. Once the tissues have been incubated and the collagen polymerizes, magnetic mechanical force stretches tissues on posts. The post deflection is used to calculate tissue stiffness and contractility. Preliminary data shows a reduction of contractile force in the tissue constructs after 24 hours of mechanical stretching. I expect to find similar outcomes through additional experimentation. Understanding the periodontal ligament’s response to mechanical force is crucial for its effective restoration and ensuring that it is mechanically sound. The novel in-vitro 3D model that we have developed provides a valuable opportunity to better comprehend the ligament's response to these forces. By performing further experiments with this model, we can gain a deeper understanding of the periodontal ligament, allowing for informed decisions when it comes to replacement and repair in patients. This model offers controlled and repeatable experiments, providing more accurate insights into the biology of the periodontal ligament and contributing to the advancement of periodontal disease treatment.