The technique of Indirect Calorimetry (IC) allows for the non-invasive and continuous measurement of metabolically relevant functions of an animal. The Michael Schwartz lab uses IC techniques in the investigation of how brain regions, neuronal populations, and the larger neuronal circuitry that they connect with, defend either blood glucose or body weight. Taking IC measurements of treatment mice allows for quantification of glucose consumption and energy homeostasis. My project was to develop a programming pipeline for the analysis of circadian metrics from rodent populations placed on a 14-hour light and 10-hour dark cycle and placed on control or high-fat high-sucrose diets. The circadian cycling of oxygen consumption and respiratory energy ratio was quantified, daily maxima and minima were measured, and latency from dark cycle onset to peak was measured. Using C#, I generated a parsing software that could combine data from experiments, recalibrate time to the day-night cycle used, and pull out these parameters for future research.

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted pathogen that is estimated to infect around 23 million people per year and there are not any approved vaccines that are therapeutic (that act in infected subjects) or preventative (that would prevent an infection). Most vaccines that we use today rely on injecting antigens subcutaneously or intramuscularly in order to elicit an adaptive immune response. However, with mucosal pathogens, mucosal immunity might be preferential because having local immunity in the site of the first pathogen exposure has the best chance at preventing the spread of infection beyond the pathogen portal of entry. My hypothesis is that a mucosal immunization would prime memory cells to reside in vaginal tissues and provide better protection for HSV-2 than other routes of immunization. From preliminary data, intranasal immunization was found to be effective in protecting mice from an HSV-2 infection. I have also characterized the role of CD8+ T cells in preventing infection in order to provide insight into the role of mucosal immunization for HSV-2. This work contributes to the efforts to make an effective vaccine to prevent HSV-2 mediated disease. 

Venezuelan Equine Encephalitis Virus (VEEV) is an arthropod-borne virus spread by mosquitos. It causes a range of acute disease from mild flu-like symptoms to more severe illnesses such as encephalitis. Certain species of rodents serve as a reservoir host for VEEV and display no symptoms, whereas horses, as amplification hosts, often exhibit lethal symptoms, suggesting that differential immune responses in these hosts contribute to differences in pathogenesis. Because reservoir and amplification hosts exhibit distinct pathogenesis profiles, this suggests VEEV replicates differently in these hosts and may be able to better evade the equine innate immune system. We have shown that changes in 3’ untranslated region (UTR) sequence and structure affect the ability of VEEV to evade host innate immune sensors such as IFIT2 and RIG-I. We generated mutant viruses which encode 3’-UTR sequences from either pathogenic or attenuated strains of VEEV and are using these to investigate the role of RNA structures in the 3’ UTR in limiting VEEV replication in equine cells. By stimulating the interferon response in equine fibroblast cells (NBL-6 cells) using a synthetic double-stranded RNA analog, Poly I:C, we predict equine antiviral responses will inhibit VEEV replication. Furthermore, we hypothesize pathogenic chimeras of VEEV will replicate more efficiently in Poly I:C-treated cells than attenuated chimeras. These studies will expand our fundamental understanding of the molecular mechanism of how RNA structure in the VEEV genome contributes to host innate immune evasion and has implications in future antiviral therapeutics and vaccination strategies.

Plant hormones are necessary for their growth, development, and overall function. Gibberellins (GAs) are a class of plant hormones that are used for cell elongation and growth. The GA pathway has been genetically manipulated in many crops to enhance agricultural yields. Research has also found that under elevated CO2 concentration conditions, GA dwarf plants revert to their less productive wild-type forms. This is very problematic for the agriculture industry because this reversion can potentially reduce their yields by half. Through my research, I hope to test whether modulation of GA signaling pathways can create crops that are more productive and better adapted to climate change. Interventions like this are needed because climate change is occurring more rapidly than plants are able to adapt to. Rewiring of the GA pathway is a potentially significant solution to this problem because we can modify plants in a manner that is likely transferable across many species. By using a novel genetic tool called a GA-sensitive Hormone Activated Cas9-based Repressor (GA HACR), we can modulate targeted genes in the GA hormone response pathway to turn down their transcriptional activity. The GA HACR targets specific genes through guide RNAs to repress the gene with complementary DNA sequence. The GA HACR is degraded in the presence of GA, which allows them to have a natural response to the activating hormone signal. We hypothesize that by targeting the HACRs to genes involved in GA biosynthesis (GA20 oxidase) and GA response (GID1 genes), we can modulate root growth of these plants. We are currently growing plants with ideal dwarf GA phenotypes in a CO2-enriched growth chamber to simulate future climate conditions and test how their growth responds. In this way, I will determine whether genetic intervention targeting the GA is a feasible strategy.

HSV-2 is a sexually transmitted disease of global importance, with an estimated infected global population of over 500 million people, as estimated by the WHO. HSV-2 is a lifelong infection which results in painful, recurrent lesions in the genital area. Previous research has shown that high numbers of regulatory T cells (suppressive immune cells important for maintaining tolerance) are present in the mucosal tissue alongside traditional CD4+ and CD8+ T cells (T cells important for an adaptive immune response), and their role is not fully understood. The reporter strain “Nedel” is a strain of HSV-2 with an inserted mNeonGreen fluorescent marker, allowing us to visualize HSV-2 infection. Our current project is to characterize the viral kinetics of the reporter strain of HSV-2 and compare it with WT HSV-2, as well as visualize the virus in infected cells via immunofluorescence and flow cytometry. We have infected mice intravaginally with both strains of virus, collected vaginal washes for plaque assays (to determine viral load) and harvested their vaginal tracts for flow cytometry. Our first round of experiments have shown that while the general frequency of immune cells were comparable, there was significantly lower activation of these cells in the Nedel-infected mice as compared with the WT-infected mice. In ongoing experiments, we have infected the mice with a higher dose of Nedel virus in an effort to make infection more comparable to WT, and we will be harvesting vaginal tracts for flow cytometry analysis to see whether we can visualize the mNeonGreen. Our overall goal is to utilize the Nedel HSV-2 strain to investigate the role of regulatory T cells in modulating anti-viral immune response, with a broader goal of finding treatments and an effective vaccine for HSV-2.

Antiseizure medications (ASM) may contribute to adverse fetal outcomes in pregnant women with epilepsy (WWE). Folate processing (methylenetetrahydrofolate reductase, MTHFR) gene abnormalities are common in women with epilepsy and depression. The enzyme product of this gene is a component of the metabolic pathway that makes folate bioavailable through methylation, critical for maintaining adequate serum folate levels. Folate is known to be crucial for fetal development, namely in the prevention of spina bifida and other neural tube defects (NTDs). The teratogenesis of some ASM in combination with genetic polymorphisms put WWE at higher risk for infertility, miscarriage, and/or major fetal malformations. L-methylfolate supplements may bypass deficiencies in the MTHFR-mediated folate metabolism pathway, yet their use in WWE during gestation or on fetal development is not well studied. We hypothesized that supplementation with L-methylfolate and methylcobalamin (methylated B12) may support better perinatal and fetal outcomes in pregnant WWE. We examined pregnancy histories of three WWE who supplemented with either folate or L-methylfolate and methylcobalamin (methylated B12) during pregnancies. Their pregnancy outcomes (both in conception and gestation) as well as mood stability improved with supplementation. L-methylfolate and methylcobalamin supplementation merits further study in WWE who have MTHFR mutations, fertility, recurrent miscarriage and/or depression histories.

  Constructed response short answer assessments provide greater insight into student understanding than multiple choice evaluation, but involve time-intensive grading. To increase scoring efficiency, we worked with the Automated Analysis of Constructive Responses (AACR) Research Group to use supervised machine learning to generate a computer scoring program for a biology constructed response formative assessment question. However, ensuring accurate and unbiased scoring is necessary before this technology enters classrooms. Due to underrepresentation of first-generation and minority students in STEM classrooms, and the assessment rubric’s potential specificity to University of Washington curriculum, I hypothesized a decreased scoring accuracy for first-generation and minority student responses and for students not attending the University of Washington. Responses for the constructed response formative assessment question were collected from five institutions, including public universities and community colleges, and were scored by myself, another trained human scorer, and the scoring program. Previous research found that this question, human-scored, shows no bias by student demographic (i.e., there is no differential item functioning). Responses were de-identified prior to human scoring, and human scores were reviewed by the supervising researcher before analysis. I analyzed the scoring program’s accuracy for dependence on students’ reasoning level, GPA, university, timing of assessment, first generation status, race or ethnicity, and gender, using logistic regression and model selection. My analysis found no significant demographic or institutional bias in the scoring program. However, results did indicate a decreased computer scoring accuracy for higher-level reasoning scores (when students had more accurate responses to the question). For this assessment question and scoring program, my results indicate that further training of the program on higher-level responses is needed before scoring bias is eliminated. This bias-analysis research ensures the increased scoring efficiency offered by computer scoring programs does not come with an increase in assessment bias.

The flexibility of general models, such as flux, provides physiology instructors with a great tool to enhance students’ ability to apply physiology concepts to many different scenarios. The general model of flux is that the magnitude of the gradient divided by the magnitude of the resistance is proportional to the rate of flow. Flux can be used to understand the bulk flow of a wide variety of fluids (e.g., air, blood, sap) as well as diffusion and osmosis across physiological systems. In order to design effective curricula to support students’ mastery of flux, it is necessary to understand the types of reasoning about flux that students bring to class. Our study focuses on students’ use of flux in the specific case of air movement from the atmosphere to lungs while taking a breath. We collected more than 1000 student responses from five institution to a question about how people take a bigger breath. We used a constant comparative analytical approach to uncover recurring patterns in the way students approached this problem and to what extent they utilized flux in their reasoning. We summarized these patterns into a framework describing four levels of student reasoning: Level 4, highest level, explains how air pressure gradient is manipulated to create air flow into lungs by increasing volume and therefore decreasing pressure; Level 3, explains the inverse relationship between volume and pressure OR the concept of pressure gradient between lungs and atmosphere; Level 2, explains that lung volume increases to cause more air flow; Level 1, describes air moving into lungs causes a big breath or describes the anatomical steps in a big breath. Our framework can be used by physiology instructors to improve their teaching. Moving forward we will validate our levels of reasoning through oral interviews conducted for the same question.

 Multiple-choice assessments do not adequately gauge students’ understanding of principle-based reasoning in physiology. Open-ended formative assessments provide a more accurate method to assess students’ reasoning, however, large classrooms and short-staffing can limit their implementation. Utilizing machine learning to develop computer scoring models trained to score according to principle-based reasoning rubrics can make categorization of student responses efficient and feasible. However, potential biases in computer scoring models is yet uninvestigated. The aim of this study is to investigate biases, specifically towards age and accessibility to English in high school, by comparing demographic data with trends in computer and human code differences. We collected demographic data and responses to two physiology questions which were then coded by experts and the computer model. Utilizing generalized linear models including fixed effects to control for potential discrepancies, we investigated relationships between demographic data and computer scoring accuracy. There were no significant differences in computer model accuracy for our demographics of interest. However, the probability of the computer being accurate depended on the reasoning level of the response as categorized by human coders. The computer had a higher probability of being accurate for lower-level responses. When examining how the computer scored if it was incorrect, we found the computer was more likely to score higher levels of reasoning lower than the human categorization and vice versa for lower reasoning levels. While we did not identify any biases due to our demographics of interest, we observed a pattern in the accuracy of the computer depending on the reasoning level of the response. Our investigation provides essential feedback for computer model developers. Improving the computer model will more accurately measure students’ reasoning level and guide physiology education. Instructors from other disciplines will benefit from this research as it proposes a framework for better assessing student understanding.

The COVID-19 pandemic has caused many college courses to shift to an online format. Many instructors have altered their teaching methods to maximize student learning. For example, some active-learning instructors have embedded autopause questions, which appear when the video stops at certain points, in recorded lectures. We wanted to understand how autopause questions could change student behavior in approaching class questions. We hypothesize that if a professor has the video “autopause” before the video provides the answers to these questions, then students will be more likely to generate their own answer prior to hearing the correct answer. We investigated this hypothesis over two quarters of introductory biology III. Students complete “lecture follow alongs (LFAs)” assignments as they watch the video. In one quarter, students were asked to pause the video and answer the questions. In the second quarter, the video autopaused, and the students had to positively affirm they answered the question before the video continued. Only the LFA responses, not the autopause questions, are graded. We are investigating differences in student responses to the LFAs between quarters. In our initial scoring of two LFA sets in summer and autumn quarters, about 50 percent of students copied the instructor’s own answers for about a third of the LFAs, while less than 30 percent copied in an earlier set. Looking at such responses will inform us about how students are approaching the questions and whether they are initially thinking about and forming their own answers. Our results will give insight into the effectiveness of autopause questions in changing student behavior, so that students approach the questions themselves before hearing the correct answer. If autopause questions are able to significantly change student behavior in this manner, then this method can be successfully implemented in other online courses to maximize student learning.

Postsurgical acute pain hinders children’s recovery from major surgery. Due to enhanced recovery after surgery (ERAS) approaches, patients are now discharged from the hospital sooner, and are managing more of their postsurgical recovery and pain at home. It is estimated that 80% of children undergoing major surgery experience moderate to severe pain two weeks post-surgery. Around 20% go on to develop chronic pain, which affects their long-term health related quality of life. There is currently no method for identifying patients prior to discharge who are at risk for poor acute recovery. This study aimed to determine if a patient’s functional ability as measured by the validated Youth Acute Pain Functional Ability Questionnaire (YAPFAQ) during hospitalization can predict pain and recovery at two-weeks post-surgery. We also aimed to compare the predictive ability of YAPFAQ to hospital pain intensity numerical rating score (PI-NRS) self-report. Adolescents undergoing major musculoskeletal surgery (N=119) completed YAPFAQ daily for up to three days following surgery, and questionnaires assessing health-related quality of life and pain intensity at two-week follow-up. Regression analyses showed that mean YAPFAQ score were significantly associated with both pain intensity and health-related quality of life at two-weeks post-surgery. In comparison, PI-NRS was significantly associated with pain intensity at two weeks, but not with health-related quality of life. The rate of improvement of both YAPFAQ and pain intensity score were not associated with two-week outcomes. YAPFAQ should be considered as a possible tool to alert providers of pediatric patients at risk for poor recovery at home following musculoskeletal surgery.

Heat stress is a preventable exposure that can lead to heat-related illness and death. Agricultural workers are disproportionately exposed to heat from heavy work in hot environments. Though there have been efforts to prevent heat-related illness, few studies have rigorously evaluated multi-level prevention approaches. This study investigates the effects of a Heat Education and Awareness Tools (HEAT) educational intervention on outdoor agricultural workers’ knowledge using a parallel, comparison, group intervention design. We used convenience sampling to recruit 83 workers from six work crews from four different companies in Eastern Washington State. Crews of participating workers were randomized to receive HEAT training versus not receive HEAT training. All workers completed a baseline survey and knowledge assessment. Directly after the HEAT training, workers in the intervention group completed a post-training knowledge assessment. At the end of the season, which was approximately three months for most participants, workers in both groups completed a post-season knowledge assessment. We evaluated the change over time (pre, post-training, post-season) in heat knowledge in workers who were in the intervention group, and we evaluated the change in pre and post-season knowledge scores in the intervention versus comparison group. There were statistically significant differences between pre and post-training and pre and post-season assessments in the intervention group, with higher mean scores at the post-training and post-season time points (p<0.001). This improvement appeared to be driven specifically by improvement in knowledge about the treatment and risk factors for heat-related illness. The difference between pre and post-season scores was greater in the intervention group than in the control group, with greater improvement in the intervention group (p=0.03). This study suggests that HEAT training was effective in improving Washington farmworker heat-related illness knowledge over a summer season. Results of this study will be used to guide heat illness prevention efforts for farmworkers.

Every Spring, a small group of gray whales, nicknamed the “Sounders,” come to Possession Sound and the surrounding waters to feed. Many of these individuals have been documented in this region consistently since the 1990s. Although this group of less than twenty individuals has been the subject of several research studies, little is known about their movement patterns in Possession Sound. Over the past few years, gray whales along the West Coast have been experiencing a mass mortality event. Gray whale research is essential in identifying potential contributing factors. The Sounders are benthic feeders and primarily consume ghost shrimp that live in the sediment throughout the intertidal zone. These gray whales access a handful of shallow areas for feeding which are only accessible during high tides, such as the Snohomish River delta. This research evaluates and combines fifteen years of sighting data from Ocean Research College Academy (ORCA) and thirty years of sighting data from Cascadia Research Collective (CRC) with the intent of identifying spatial patterns and possible correlations with the tides. I am creating ORCA’s sighting catalog with photographs taken during ORCA’s sightings and identifying the individuals present using reference photographs from CRC and compiling any additional available data. In this catalog, sighting data is evaluated on individual whale identification, date, time, location, and additional observational notes such as whale interactions. I am plotting this data and additional data from CRC’s sighting catalog in ArcGIS to create a map of spatial distributions. Preliminary results from ORCA’s sightings suggest that gray whales are most often sighted in Possession Sound between Hat Island and the south end of Jetty Island. Any spatial trends that I identify may lead to a better understanding of gray whale behavior, possibly including gray whale intercommunication, social structure, individual behavior, and local feeding practices.