Using simple behavioral analysis (SimBA) and Deep Lab Cut (DLC), we can create predictive behavior classifiers using pose estimation (PE) data obtained through DLC. PE is a computerized technique to track and predict the location of mice by training the video dataset with labeled frames using specific regions of interest (ROIs). With this, we can create machine-learning (ML) predictive classifiers of complex social behavior in SimBA. Social behaviors and interactions are difficult to manually track due to their rapid successions. To overcome this, I plan to use ML classification using our SimBA pipeline for behavioral classification allowing us to exceed human performance and increase throughput and consistency. I plan to create accurate classifiers for social behaviors that I will use to analyze the behavioral motifs of mice undergoing an operant social stress procedure. First, we train male and female C57BL/6J mice to self-administer (SA) their same sex cage mate. Experimental mice are then subjected to either physical stress for males or witness stress for females. Following social stress, non-reinforced SA is used to assess social reward seeking. Next, social interaction (SI) tests are performed to document time spent approaching the familiar same-sex conspecific cage mates and the aggressive CD-1 mice. All behavior was recorded, and transferred to DLC, followed by frame extraction. Using these frames, we trained the operant behavioral dataset to track the orientation of the mice. Next, we evaluate the dataset for a low error margin as observed by a continuous plateau of iteration loss. Although not complete, I expect to create behavioral classifiers for mice during social decision making in a social reward context following social stress inclusive of sex differences. Providing descriptive statistics of both movement and probability of successive behaviors as they occur in real-time.

Neuropsychiatric disorders pose a difficult challenge for healthcare providers. Treatments for such disorders vary in efficacy and come with detrimental costs for patients and their communities. Historically, preclinical animal models have failed to incorporate the nuances of volitional human social behavior. This project used chronic social defeat stress to induce depression-like behaviors in male and female mice, this was followed by self-administered social interactions within an operant chamber in which lever presses were reinforced by social contact. The goal is to develop preclinical animal models that can be assessed to identify mechanisms responsible for stress-induced social motivation. The mice will be injected with a nuclear localized tag (oNLS) and viral retrograde tracer rAAV2-retro-GFP. Male and female mice will train to self-administer social interaction with a sex and age-matched housing partner over the course of ten 12-trial sessions. Next, experimental male and female mice will be subjected to physical and witness defeats followed by operant social self-administration. Before and after the 10-day operant social stress sessions, we will test social reward seeking via non-reinforced self-administration of social reward followed by a progressive ratio test. Brain tissue will be collected and prepared for immunohistochemistry and iDISCO+ whole-brain clearing for cfos labelling. We predict results will show differential cfos activity in sexually dimorphic brain regions such as the hippocampus, prefrontal cortex, amygdala and the bed nucleus of stria terminalis. We determine that operant social stress can be used to discern differences in social motivation in male and female mice as a result of stress-induced factors. There is great potential in using whole-brain activity mapping to identify brain structures activated during social reward following social stress, as this can also serve as a technical resource for the field by identifying relevant non-canonical brain regions and circuits that govern such behaviors.

The visual system is a critical portion of the sensory arsenal in many animals, since light is a fundamentally important factor that allows animals to assess and navigate their surroundings. Differences in latitude have measured effects on the quality, quantity, and consistency of daylight, and animals have adapted to these varying light levels in different ways. Research has shown that animals with larger eyes, reflected in their bony orbit dimensions, are able to perceive their environments better in more dimly lit settings. Studies have also suggested that animals at higher latitudes exhibit larger eyes for better visual performance. Our group seeks to understand if there is any significant relationship between latitude and orbital size in Canidae (wolves, foxes, and relatives) due to the difference in light levels at different latitudes. I collected several linear measurements using ImageJ and 3D Slicer from skull 3D scans of 29 species of the Canidae family over a latitude range from -104.07° - 99.02°. Additionally, our team collected data on the average tree coverage, diet, and hunting style of species, to test for additional variables that may affect orbital size. We conducted regressions and ANOVAs among the variables collected. The preliminary results show there is no significant relationship between latitude and orbital size. However, we found that both hunting style and skull length have a significant relationship with orbital volume in Canidae. These results potentially impact the field by allowing us to infer the possible evolutionary trajectories of bony orbital dimensions, and how eye size could allow for more complex hunting styles in Canidae. Future research may seek to validate these findings in other carnivore families.

Phytophthora is a genus of water mold that causes plant disease and spreads quickly. Certain Phytophthora species can wreak havoc among crops and ecosystems. Early detection of the plant pathogen is key to preventing its spread. Based on observed blight on oak trees, we set out to detect Phytophthora in our local watershed, at Clark's Creek Park in Puyallup, Washington. We baited pathogens in the water by placing rhododendron leaves in netted bags, and submerging the bags in one of the park streams for 14 days. We then cultured the infected leaves and extracted deoxyribonucleic acid (DNA) from the cultures. We performed polymerase chain reaction (PCR) to amplify the Phytophthora internal transcribed spacer (ITS) gene, and sent our PCR product out for DNA sequencing. Once we received the results, we used the Basic Local Alignment Search Tool (BLAST), an online tool that matches DNA sequences with available DNA databases. Using BLAST, we identified which species were present in our cultures from Clark’s Creek. We confirmed the presence of two Phytophthora species: P. gonapodyides, P. chlamydospora. These species preferentially infect forest and fruit trees. Scientists and community leaders can use our research to track Phytophthora and focus containment efforts in our local ecosystem.

Documenting plant-animal interactions allows for a better understanding of biodiversity, the behavioral ecology of frugivores, and the coevolution between plants and animals. Bats are important seed dispersers on hundreds of plant species in the tropics, but their patterns of plant use across habitats is still relatively unknown. The overarching goal of my research project is to characterize the interactions between mutualistic fruit bats and plants across different habitats at a site in Costa Rica (La Selva). My hypothesis is that bats will exhibit frequent interactions with Piper plants overall, with more variable interactions across forest Piper species over time because these species have intermittent fruiting peaks. Existing field data consist of 24h camera trap videos and nightly acoustic ultrasonic recordings taken at dozens of forest and gap Piper plants at La Selva during both rainy and dry seasons between 2019-2021. The acoustic data collected is heavy in background noise from organisms and weather conditions; therefore I developed a program in MATLAB to streamline this analysis. I am using RavenPro to identify relevant parameters of bat calls within the filtered files using focal calls as a guide to later identify the species participating in these visitations. I am comparing the number of calls across 10 P. sanctifelicis plants (open habitat) to the number of calls found across 8 P. generalense plants (closed habitat) to observe the frequency of possible fruit-bat interactions across habitats. This project is significant in the field of ecology because of its focus on plant-animal mutualistic interactions: by understanding the frequency of bat visitations to plants across habitats, we can gain a better understanding of the importance of these frugivores for seed dispersal and survival of plants.

Mosasaurs are extinct, enormous lizards that dominated the oceans in the Late Cretaceous, from 90 to 66 million years ago. They were important members of Late Cretaceous marine ecosystems, with some species being the top predators. However, there remain many uncertainties about mosasaur diets, which likely varied considerably among species. Therefore, I aim to investigate mosasaur diets to provide more information on lizard evolution and Cretaceous marine ecosystems. To infer the diets of mosasaurs, I examine the lower jaws and teeth of their closest living relatives, modern lizards, and test for correlations between craniodental morphology and diet. For example, a lizard that eats primarily hard-shelled foods will likely have more robust jaws and teeth than a lizard that primarily consumes insects. To quantify the morphologies of the lower jaw and teeth, I measured the width and height of the jaws at three points, and the height, width, length, curvatures, root length, and cusp numbers of the teeth at the same three points (n = 43 species). I then used the jaw measurements to calculate cross sectional shape values that represent the amount of stress the jaws experience during feeding. Finally, I used phylogenetic regressions and multivariate analyses to test the relationship between jaw/tooth shapes and diets. I find evidence that jawbone heights increase among diets in the following order: insectivores, carnivores, herbivores, and durophagous taxa. Further, bone width is greater in herbivores than in non-herbivorous taxa, and durophagous lizards have the most diverse tooth morphologies. These results provide a foundation for future studies to examine the relationship of jaw/tooth shapes and diet more robustly, with the goal of using modern lizards as analogs for inferring diets of mosasaurs.

Many genes involved in different cell differentiation processes are known, however it is more challenging to know the history of transcription in each individual cell within a new organ. To fill that gap, I am building and testing a genetically-encoded recorder that uses two serine integrases to follow expression in developing roots of the model plant Arabidopsis thaliana. An integrase is a protein that binds to unique DNA regions, called integrase sites. Once bound, the integrase can perform an inversion or excision of the DNA between the integrase sites, depending on how the sites are oriented relative to one another. In a one-integrase design, we put a constitutive promoter between two fluorescent reporter genes, flanked by integrase sites. When the integrase is expressed under the control of a promoter for a developmental gene, there is a switch between the reporters only in the cells that are undergoing that developmental program. I am now using molecular cloning techniques to build a recorder that can track the expression of two different genes, using two different integrases. My integrase-based recorder will provide insights into when and where genes are “switched” on and off to promote cell specification. In the future, reporter genes can be replaced by genes that control development, and, in this way, we can engineer plants with different root structures. Control over root architecture could lead to plants that are more resilient to heat and drought.

Endophytes are microbes that reside within plants, forming a mutualistic relationship where they improve host physiology in exchange for plant carbohydrates. Inoculating plants with a cultivated consortia of endophytes originally isolated from trees in plant family Salicaceae is proposed as a sustainable strategy for increasing crop yield, plant health, and climate change resilience. However, this plant-microbe relationship may turn parasitic if endophytes become over abundant and their carbohydrate demand exceeds the plant’s capacity. Inoculation method is an important yet underexplored factor in predicting the outcome of this relationship and endophyte colonization success. This study investigates how inoculation methods influence the number of endophytes that colonize different plant tissues. A consortium of Salicaceae endophytes was introduced into hybrid poplar plants grown in sand through different inoculation methods. The methods tested are (1) no inoculation, (2) a pre-planting root soak inoculation, (3) a pre-planting unrooted stem soak inoculation, and (4) a post-planting media soak inoculation. After growth, endophyte density and distribution will be estimated by separately culturing endophytes isolated from leaf, stem, and root tissues and counting their colony forming units per gram. The carbon cost of hosting endophytes will be estimated through in vitro and in planta respiration measurements from the sampled tissue. Epidermal wounding from shoot cutting in the stem-soak inoculation method may increase the amount of entry sites for endophyte colonization. Therefore, it is expected that plants receiving inoculum via stem-soak will show increased endophyte distribution and density and higher respiration rates when compared with other inoculation methods. Comparisons of estimated in planta endophyte densities with plant physiological measurements will establish the relationship between endophyte abundance and host health in poplar. Further research will be needed to create a more generalized model of the effect of individual endophyte distribution and density on the benefits and costs to plant health.  

Endophytes are mutualistic microbes that promote plant growth and stress tolerance by improving host nutrient uptake and producing phytohormones. Plants grown under drought or nutrient limitations have benefited from inoculation with endophytes isolated from wild poplar. Previously, tissue culture-grown poplar has received a pre-planting inoculation to successfully promote growth under abiotic stress. However, a lag in plant growth and initial negative physiological and morphological response has also been observed in plants inoculated early in their development. These results suggest that a favorable plant-endophyte interaction may depend on the method of inoculation. To further understand the best method to maximize plant growth promotion with endophyte inoculation, I have explored how the timing of inoculum delivery impacts plant physiological processes. In a greenhouse experiment, hybrid poplar plants were split into three treatment groups: an uninoculated control group, a pre-planting group inoculated with an endophyte consortium via a root soak, and a post-planting group inoculated via a soil soak. Measuring changes in root system architecture, plant height, and biomass will determine how the plants allocate their resources in response to inoculation treatments. Leaf chlorophyll fluorescence will be measured to assess the stress put on plants from the association with endophytes, while measurements of leaf photosynthetic and root respiration rates will be used as a proxy for changes in the host’s carbon budget. I anticipate that allowing plants to acclimate to greenhouse conditions before inoculation will allow them to have greater above- and belowground productivity due to the photosynthetic cost of establishing symbiosis. Host plant physiology will be correlated with additional measurements of endophyte density and distribution throughout the plant to relate inoculation timing to estimated intracellular endophyte concentrations. This information will provide a greater understanding of the initial costs and benefits of endophyte-plant symbiosis and optimal timing of inoculation.

 The diversification of many vertebrate groups was spurred by the use of novel food resources, and jaw functional morphology provides clues about the adaptations associated with dietary diversification. The external dimensions along the mandible reflect strength to resist bite forces, which are in turn associated with physical properties of the diet. Variation in these dimensions along the jaw and between different species therefore may reflect adaptations of the jaw to specific diets. We applied this biomechanical framework to investigate the relationship between jaw robustness and diverse diet types in bats. Using mandibles of more than 60 species, we quantified the external dimensions at interdental gaps to generate mandibular strength profiles. The strength profiles of frugivorous, insectivorous, and omnivorous bats showed similar patterns, with a trend of increasing jaw depth toward posterior teeth. All diet types showed a high level of variation in jaw shape along the toothrow, suggesting differences in the functional roles of different teeth. Insectivores showed the greatest within-guild variation in jaw shape, while nectarivores had noticeably gracile symphyses. Further, insectivorous bats showed relatively deep jaws at the canine and premolars, which may be associated with the use for prey capture, while frugivores have relatively deep jaws at the posterior molars, possibly linked to adaptations for crushing seeds and pulp. These results suggest that mandible strength profiles reflect dietary adaptations in bats.

 Solid tumor cancers are capable of releasing chemicals that are detectable in body fluids. These cancers can cause severe acidosis within the patient, resulting in the change of structure and function of Complement serum proteins, membrane-bound regulators, and receptors that interact with various cells and mediators. A better understanding of the mechanistic interaction between the complement system and tumor-induced acidosis can provide a new direction in cancer immunotherapy research. Here, we investigate the effect of pH on binding between Heparin, Complement Factor H (CFH), and C3b. We found that in controlled in vitro assays conducted through biolayer interferometry, CFH and Heparin bind better to C3b in pH 6.0 but only in lower concentrations of CFH. As for pH 7.4, the binding between CFH, Heparin, and C3b is lower in comparison; however, the binding between only CFH and Heparin is higher at pH 7.4 than at pH 6.0. Our results demonstrate how Heparin, Complement Factor H, and C3b are likely to function in a tumor microenvironment where the pH is generally lower. We anticipate this research to be a starting point for investigating the role of the Complement System in tumor growth and researching the implications of its biological actions with respect to the development of anticancer therapy.

Weight loss (WL) is recommended for people with obesity to mitigate cardiometabolic risk, but its effect becomes limited when reaching a WL-plateau (WL-PL), when the rate of WL becomes minimal despite efforts to continue losing weight. The biological basis for the WL-PL is not fully understood. The goal of this study is 1) to test two diet-regimens in the development of a WL-PL in mice with diet-induced obesity (DIO), and 2) to identify subsequent changes in mitochondrial function. We hypothesized that despite similarities in caloric intake, higher fat content will make high-fat diet (HFD-CR20) mice protect their adiposity and reach a WL-PL sooner than low fat diet (LFD-CR20) mice. We also hypothesized that mitochondrial function will be reduced in mice that have reached a WL-PL. To test hypothesis 1, individually housed DIO mice were divided into two groups, and were provided with 80% of their ad libitum caloric intake with either a high-fat, or low-fat diet daily for ~2.5 weeks. Body weights were recorded daily. A WL-PL was identified by weight stability (<0.5% change BW/day) following weight loss. HFD-CR20 mice reached a WL-PL phase after 10 days of caloric restriction. LFD-CR20 mice did not achieve a plateau within the study time frame. LFD-CR20 mice lost more weight than HFD-CR20 (-10.8% ± 2.2 vs. -5.2% ± 1.8, p<0.05 respectively), which was attributed to a greater loss in adiposity, measured by an EchoMRI, (-23.3 g ± 6.0 vs. -3.3 g ± 3.0, p<0.05). To test hypothesis 2, mitochondrial function was assessed by high resolution respirometry at the study endpoint. We will further analyze this data to identify differences in mitochondrial function attributed to the WL-PL. This work will improve our understanding on the biological mechanisms behind resistance to weight loss to help advance obesity treatments in humans.

Obesity is a condition characterized by excessive fat accumulation, resulting in increased risk for chronic diseases like cardiovascular disease and diabetes. Weight loss can effectively reduce the burden of cardiometabolic risk factors, but weight loss maintenance is difficult to achieve. Mitochondria are key organelles within cells that are responsible for the breakdown of substrates to produce energy. Mitochondrial dysfunction is implicated in obesity, but little is known about the role of mitochondrial dysfunction in weight loss maintenance. Additionally, females are often underrepresented in obesity research, partly attributed to female mice being more resistant to develop obesity compared to males. The aim of this study is to compare mitochondrial function in liver, adipose tissue, and skeletal muscle, following weight loss in female mice with diet-induced obesity (DIO). We hypothesized that obesity would result in a reduction of mitochondrial function across tissues, and weight loss to further reduce it. We provided CB6F1 female mice with a high fat diet, where 87% of them developed DIO. DIO mice were separated into two groups: one underwent 20% caloric restriction for 4 weeks (HFD-CR), and the other group remained on ad libitum high fat diet for the same intervention (HFD-AL). A healthy weight control group was maintained on a regular chow diet. Their weight and food intake were recorded daily. Body composition was assessed twice, before and after the 4-week intervention period. We had an unexpected finding, where mice lost 5-10% of their body weight prior to the intervention period. HFD-AL mice regained lost weight at study endpoint, while the weight of HFD-CR mice remained weight reduced until study endpoint. CR mice had lower adipose tissue mass compared to HFD- AL mice. Future analyses will include comparisons of mitochondrial content and function in different tissues. Findings will provide more insight into the effects of weight maintenance and regain on mitochondrial function.

Kānaka Maoli (Native Hawaiian) health practices have been significantly impacted by colonialism, beginning with the illegalization of practices like hula, lā’au lapa’au, ‘ÅŒlelo Hawai’i (Hawaiian language), and the growing inaccessibility of cultural foods. Increasing attention has been paid to creating culturally-grounded interventions that address these disparities, which have proven to be effective in increasing health outcomes in Indigenous communities. This study aims to understand the potential benefits of Papa and Pohaku, a culturally-grounded family intervention created and led by esteemed elder Uncle Earl Kawa’a through Keiki O Ka ‘Ä€ina (KKA). KKA is a non-profit organization built to perpetuate Kānaka Maoli culture for ‘ohana (families) and keiki (children). Uncle Earl Kawa'a, a respected kupuna (elder), leads courses on creating a papa (pounding board) and pohaku ku’i ‘ai (stone pounder) to promote healing and wellbeing through grounding participants in cultural practices. To understand the impacts of the intervention, two focus groups were conducted with participants and KKA staff. Questions explored the benefits of participating in Papa and Pohaku, specifically its impact on relationships and traditional Hawaiian knowledge. Our team used Collaborative Qualitative Analysis, which is a structured and rigorous method of conducting inductive thematic analysis. All research team members identified as Indigenous, consisting of one faculty advisor, one doctoral student, and two undergraduate researchers. Respondents reported the intervention positively impacted the following: 1) participants’ pilina (relationships) with their ‘ohana, partners, and with other participants; 2) appreciation for the huaka’i (journey), or process, and commitment to future growth; and 3) understanding of and connection to mo’oemeheu (Kānaka culture). The findings of this study indicate the various benefits of culturally-grounded family-based interventions and a greater need for the availability of culturally-grounded interventions for Indigenous communities.

Obesity is associated with mitochondrial dysfunction. A study previously conducted in our laboratory produced preliminary data suggesting a reduction in T-cell mitochondrial function in subjects with obesity relative to healthy weight controls. The purpose of this present study is to determine whether changes in T-cell mitochondrial function (MITO) reflect MITO changes occurring in liver and skeletal muscle, which are known for having greater influence in glucose homeostasis and energy expenditure. We hypothesize that mice with diet-induced obesity (DIO) will exhibit reduced MITO in T-cells that will correlate to a decline in MITO in liver and skeletal muscle. Adult male C57Bl/6J mice were divided into two groups–the control group was fed a standard chow diet whereas the experimental group was fed a high-fat diet for fifteen weeks. Body weight and food intake were measured every week. Body composition was performed at the endpoint. MITO was measured via high-resolution respirometry in permeabilized liver tissue, skeletal muscle fibers, and splenic T-cells. DIO mice had higher body mass than standard CHOW [52.8g±1.8 vs. 35.1g±2.6] that was explained by an increase in fat mass [19.9g±0.9 vs. 6.1g±1.8] and lean mass [31.5g±1.7 vs. 26.1g±1.1]. We found that differences between DIO and CHOW in mitochondrial leak respiration, maximal oxidative capacity, maximal electron transport chain, and ADP sensitivity were not the same across all tissues. We will proceed to determine which aspects of MITO are correlated between different tissues and assess if variations in respiration are associated with differences in mitochondrial content. An improved understanding of how DIO affects different types of cells regarding oxidative capacity might provide key insights into the development of therapeutics and other preventative approaches to improve immunity and cardiovascular fitness in obesity.