Bacteria can inherit genes through two modes of transmission: vertical inheritance, in which a cell receives genes from its parent cell during division, or horizontal gene transfer, in which genes are passed laterally between two unrelated cells. Extrachromosomal DNA, called plasmids, can be transmitted from a bacterial cell to a neighboring cell of the same or different species through a form of horizontal gene transfer known as conjugation. These conjugative plasmids can encode for antibiotic resistance genes which allows a host cell to produce proteins that degrade antibiotics. In an environment with these drugs, a host cell with this plasmid would be more likely to survive, compared to a plasmid-free cell, due to the benefits of the antibiotic resistance gene. Additionally, these antibiotic resistance genes can often acquire mutations which increases the level of antibiotic resistance for the host cell. However, these acquired mutations may be more beneficial (i.e., higher resistance for the host cell) in one bacterial species than the same mutation in another species. In other words, the effect of mutation on the plasmid-encoded antibiotic resistance gene may be contingent upon the host species. Given that antibiotic resistance genes are often encoded on conjugative plasmids that are shared among species, my project is investigating how protein evolution may be affected due to plasmid genes existing in multiple bacterial hosts. I performed a series of competitions between three bacterial species containing plasmids with versions of an antibiotic resistance gene to determine which versions of the gene outcompete others in an environment containing antibiotics. In doing so, I can determine how this particular gene may evolve when various bacterial hosts exist in a microbial community. The results of this experiment will improve our understanding of plasmid biology and the evolution of antibiotic resistance genes in diverse communities of bacteria.

Many human diseases, including cancer metastasis, and developmental disorders, involve sheets of cells moving together. This concerted process, called collective cell migration (CCM), is critical to maintaining the integrity of the sheet of cells during movement. Maintaining cell sheet integrity is dependent on individual cells retaining their shape while still connected to neighboring cells. To study collective cell migration, we use Drosophila melanogaster (fruit fly) embryogenesis. Dorsal closure (DC), an event in Drosophila embryogenesis, is an example of CCM. During DC, two epithelial layers wrap around the embryo. Because CCM’s core mechanics are conserved, studying DC provides an opportunity to indirectly study human diseases/disorders function in a model that has fewer genetic redundancies and is easy to manipulate. Adherens junctions (AJs), are the site of adhesion between individual cells, and what allows for dynamic rearrangements like CCM by strategically increasing or decreasing adhesion. Despite the importance of this process and its relevance to many diseases, CCM is highly complex and how the scaffolding protein Canoe stabilizes adherens junctions under high stress is not entirely understood. We are interested in how junctions between cells are stabilized during a dynamic movement like in CCM. Using a maternally driven Gal4/UAS system, we created moderate canoe knockdowns. We then performed targeted, immunofluorescence antibody staining of embryos during DC. We expect that the loss of Canoe will lead to increases in cell-sheet tension as well as the compensation by other stabilizing proteins like Polychaetoid. Our research improves our understanding of stabilizing proteins at AJs and CCM and may lead to the more effective treatment of disrupted CCM human diseases/disorders like wound healing, palate formation, and neural tube closure.

  My research focuses on ‘tauopathy’—the pathological effects of misfolding of the tau protein—using the fruit fly, Drosophila melanogaster, as a model system. In particular, I am interested in how the expression of tau affects locomotive function. Tau is a protein found in both humans and flies that is associated with stabilizing neuronal microtubules. However, under certain physiological conditions, human tau proteins form neurotoxic aggregates in the brain. This neuronal damage leads to dementia, a hallmark of Alzheimer’s Disease (AD). Aging comes with a multitude of age-related functional deficits, including decline in locomotor function. Some individuals with AD pathology are never diagnosed, however, because the cognitive impairment they experience is not sufficient for a clinical diagnosis of dementia, the most common symptom of AD. Consequently, it is imperative we understand AD as more than dementia. I study the climbing abilities of transgenic tau and control flies through negative geotaxis assays. Negative geotaxis refers to the tendency of flies to move vertically upward when startled. For my project, I am measuring the climbing performance of the flies to see the effects of tau on locomotor function as flies age. I hypothesize that the neurotoxic tau aggregates that form will interfere with neural activity involved in the flies’ motor function, resulting in decreased climbing performance. This research holds an abundance of biomedical implications and a capacity to better the lives of many. By using motor function to flag at-risk individuals early in their lives, they have the opportunity to participate in preclinical AD clinical trials that may prevent them from developing AD pathology later on. It is important that we, as a scientific community, strive to better understand the effects of aging and tauopathies, as it is through this understanding we can provide elderly individuals with care that transforms their quality of life.

Aging is the most prevalent risk factor behind many common diseases such as cancer, cardiovascular disease, and various neurodegenerative diseases. The changes that take place with age are complex and affect most of the human body; age-related changes in gait, in particular, have been found to be associated with the onset of disease. While it is generally understood that there is an effect of aging on walking speed in humans, the mechanisms underlying age-related locomotor impairment have not yet been fully characterized. The focus of my research is using Drosophila melanogaster as a model to investigate such mechanisms. My hypothesis is that D. melanogaster exhibit similar age-related changes in gait as those seen in humans, which includes a decrease in walking velocity and duration, and a significant change in limb coordination over their lifespan. I followed cohorts of D. melanogaster over their lifespans and recorded videos of their walking in an arena. I then used these videos to study factors such as walking velocity and duration by analyzing the trajectories of each fly. In addition, I investigated more in-depth limb coordination of individual flies by analyzing the movement of each individual leg. Previous studies using D. melanogaster have found a significant decrease in climbing behavior with age, but have not looked at gait on a finer scale. Therefore, upon completion of this study, I expect to see a decrease in limb coordination and population walking velocity as the flies age. With the findings from this study, I hope to establish a foundation for how gait changes with age in D. melanogaster and to further use gait analysis to help predict the onset of disease and to distinguish between diseased and healthy flies.

Alzheimer’s Disease (AD) is a neurological disease that causes memory loss and neurodegeneration, and is one of the leading causes of death within the United States. Alzheimer’s is linked to the presence of neurofibrillary tangles within the brain, generated by hyperphosphorylation and aggregation of a protein called tau. However, the specific impact that tau has on biochemical pathways in neurons is largely unknown. My project attempts to fill this gap. I compared samples of wild type fruit flies, Drosophila melanogaster, with a strain which expresses the human tau gene in neurons. Despite the difference in human and fly brains, determining the affected biochemical pathway in flies could suggest similar effects in humans. I am using metabolomics, which quantifies the levels of approximately one hundred metabolites, to try to identify the biochemical pathways that are affected by expression of tau in the neurons. Head and body samples of these two fly strains were frozen at 12 days of age and assayed for a targeted set of metabolites. I analyzed these metabolome data using statistical methods in Python to search for potential differences between wild type and tau flies. Any metabolites found to be different will then be analyzed to see if they tend to represent similar biochemical pathways, allowing us to speculate about the effect of tau on those pathways, both in flies and humans. Future work aims to analyze fly brains and individual neurons, thus mapping the affected metabolic pathways more precisely. Importantly, metabolic pathways are often identical between organisms, allowing us to speculate on the effect tau has on similar pathways within humans, thus this work will inform our understanding of the mechanism of AD in humans.

Seed dispersal is a crucial phase of plant lifecycles. Effective dispersal is important to the ecosystem as a whole because it affects composition of the community, ecological succession, and response to climate change. Given the importance of seed dispersal, understanding the factors that contribute to the evolution of varied dispersal modes and promote convergence on specific dispersal strategies is particularly important to understanding plant ecology. We think habitat might be one of these important factors. Here, to explore the relationship between habitat and dispersal mode, we are studying the onion grasses (Melica), a small genus of perennial grasses primarily distributed in temperate regions, and their relatives. These grasses are found in a wide variety of habitats and possess a remarkable diversity of seed dispersal strategies. These traits make them a useful case study for better understanding the factors that influence the evolution of dispersal strategy. We are testing the hypothesis that evolution in traits associated with seed dispersal is correlated with changes in habitat. In particular, we hypothesize that the evolution of wind dispersed seeds follows transitions into open habitats. Seed dispersal structures (diaspores) were collected from 28 grass species (14 Melica and 14 outgroup). To assess wind dispersal potential, we quantify falling velocity by filming seed descent at 1000 fps. Lower falling velocities are associated with higher wind dispersal potential. Diaspores were photographed and the images were used to measure surface roughness, which is associated with adhesive dispersal potential. Habitat data were obtained for each species via a literature survey. These data, along with several other traits associated with seed dispersal processes, were mapped onto the evolutionary tree of the onion grasses. Initial results indicate that convergence upon wind dispersal may be in part driven by convergence upon disturbed habitat types.

Our project is mainly focusing on hemichordates, they are marine invertebrates, related to echinoderms. While no chordate is able to regenerate all regions of its central nervous system after deathly injury. Ptychodera flava, a hemichordate, exhibits an exceptional ability to regenerate its entire central nervous system and anterior structures in as little as two weeks. We are studying this process to understand the underlying gene networks involved in neural regeneration. I am currently investigating the expression of transcription factors involved in neural development with homeodomain-containing genes Pax6, Six3, DLX, Frizzled, Chordin, Pou, and Msx-2, which have been shown to be upregulated during P. flava regeneration through transcriptome analyses. I have previously cloned the isolated PCR products into plasmids to make RNA probes for in situ hybridization. I am generating RNA probes, performing in situ hybridization. I will use RT-PCR to examine when there is a high RNA expression at various stages of regeneration, then I will perform in situ hybridization on P. flava tissue samples at these different stages. I will also be sectioning these samples after in situ hybridization and staining to image the cellular and tissue structures to aid in seeing where these genes are expressed in specific tissues during regeneration. My research goal is to ascertain the gene networks underlying regeneration and then to compare these to the expression of the same genes during embryonic development in P. flava. Finally, we can ask whether these genes exhibit similar gene networks that have been reported during development in direct-developing hemichordates. Further experiments will be examining if these gene networks are necessary and sufficient in regeneration by knocking-out or overexpressing these specific genes at different stages of regeneration. By using P. flava as a model to study transcriptional regulation during regeneration, we are aiming to identify the genetic and morphological mechanisms to achieve sufficient central nervous system and whole-body regeneration in a stem deuterostome, which might also give us the insight into potential key regulator factors during human regeneration.

The skin is a highly innervated sensory organ, providing our bodies with the vital ability to understand and respond to external stimuli, such as pain, temperature and touch. However, many injuries to the skin result in the severance of somatosensory axons, causing temporary or permanent loss of feeling. To reestablish innervation, skin and neuronal cells launch wound healing responses. Although these responses are known to occur after an injury, the exact biological pathways and cellular components involved remain poorly defined. This project aims to characterize the transcriptional responses to skin injury using zebrafish (Danio rerio) as a model organism. Unlike human skin where complete regeneration is not observed, zebrafish have almost perfect regenerative abilities. To address our question, we plucked fish scales to induce a rapid regenerative response in somatosensory neurons and skin. I previously conducted RNA-seq analysis on populations of neurons and skin cells over the course of an induced injury response and curated a list of differentially expressed genes. Using gene ontology annotations to inform on these transcriptional changes, I have begun to identify the enrichment of well defined pathways, processes, and cellular components that are up or down regulated during the injury response. Thus far, I have identified a number of upregulated biological processes associated with changes in the extracellular matrix of skin-resident cells. The identification of enriched biological processes will help guide future experimental designs that study the effects of manipulations in these processes. With a deeper understanding of genes and mechanisms involved in zebrafish skin repair, I hope to unlock regenerative secrets that could apply to the treatment of human tactile maladies. 

DNA glycation is the DNA damage induced by reactive carbonyls (such as methylglyoxal and glyoxal) in plants and mammals. Glycation damage is quantitatively as important as oxidative damage. It is one of the major in vivo DNA damage sources associated with increased mutation frequency, DNA strand breaks, and cytotoxicity. In humans, glycation damage may contribute to Parkinson’s disease, cancer, and oxidative stress-induced diseases. Glycation damage in plant organelle (Chloroplasts and Mitochondria) DNA remains poorly understood. We recently showed that the demise of plastid DNA (ptDNA) and mitochondrial DNA (mtDNA) during maize seedling development is associated with an increase in DNA damage resulting from oxidative stress caused by reactive oxygen species. As oxidative and glycation stress are closely linked in plants, we hypothesize that glycation might be one of the causes of ptDNA and mtDNA damage during development. Glycation damage can be prevented by the activity of the protein deglycase DJ-1, also known as Parkinson's Disease Protein 7 (PARK7). Our objective is to quantify glycation lesions in the organelles of maize plant tissues in the presence and absence of DJ-1. Our approach involves the quantification of glycation and deglycation in ptDNA and mtDNA using PCR analysis and the enzyme-linked immunosorbent assay (ELISA). This research should better understand glycation damage in plant organelles and might lead to insights concerning human pathologies and neurodegenerative disease caused by glycation.

Mycoremediation is a widely researched method using mycelia to clean bacterially contaminated water bodies, such as the Bothell Wetlands, which has been contaminated by crow roosts. This research aims to analyze and quantify the remediative properties of the fungus Stropharia rugosoannulata on the bacteria Escherichia coli, Salmonella enteritidis, Campylobacter jejuni and Klebsiella pneumoniae. Additionally, the remediation of the antibiotic resistance genes (ARG), Tet[A], Tet[B], Tet[M], StrA, StrB, bla_CMY and bla_CTX genes, was examined. One pound rye-seed bags were inoculated with liquid Stropharia. After three weeks, 35g of seeds were used to inoculate 60g of sterile wood chips in four 250mL bottles, for an incubation period of 3 weeks. Including a control bottle containing 60g of wood chips, we added 150mL of wetland water, spiked with 3500 CFU/100mL of the aforementioned bacteria, to each of the 5 bottles that were shaken via rocker. The following water retention times were chosen based on past experiments: 5min, 1hr, 5hrs, and 24hrs. When performing membrane filtration we used duplicate volume of 50ml for both genotypic analysis (qPCR), and for colony counting on selective plates. We analyzed the remediation using colony forming units and quantified using gene copy numbers (qPCR). The remediation of the following ARG was the highest after 1 hour of retention time: StrB (88.6%), Tet[M] (83.5%), and Sul-1 (23.2%), while bla_CMY (100 %) and StrA (73.8%) had the greatest remediation after 24 hours. Results showed no remediation for Tet[A] and Tet[B] for any retention time. The remediation of Campylobacter (83.6%), Salmonella (97.6%) and Klebsiella (93.23%) was the highest after 1hr of retention time, while E.coli (27.61%) showed a greater remediation after 24hrs. The exponential increase in anthropogenic activities promoting bacterial contamination of the ecosystem and the spread of antibiotic resistant bacteria, highlights the urgency to find ways to mitigate them.

It is known that plant-pollinator relationships are central to the proper functioning of agricultural and ecological systems. Of the many navigation pathways pollinators use, floral scent signaling for insects is the most complex yet also the most at-risk from atmospheric human activity. Oenothera pallida, a primrose, interacts with the hawkmoth pollinators Hyles lineata and Manduca sexta, via this scent pathway. Because of their reactivity with floral scent, human-released ozone and NO2 (NOx) are the main perpetrators of scent degradation. To understand the impact of scent degradation on moth responses, I recorded changes of antennal and behavioral responses of these moths to unaltered versus degraded scent, expecting a poorer response to the degraded scent. Moth antennae act as the site of odor reception, bearing sensory hairs that detect odors, allowing the moths to navigate to scent sources. I conducted electroantennographic experiments (EAG) to record the electric signal from the insect antennae in response to each scent blend, with the degraded scent representing the impact of NOx interactions. Following the EAG, I then conducted wind tunnel behavioral experiments to investigate the impact of odor degradation on behavior, and to understand the relationship between antennal and behavioral responses in these moths. I expect that the EAG experiments will have a lower antennal response to NOx degraded scents in comparison to the normal, unaltered scent blend. Likewise, the moths might have reduced behavioral responsiveness to the degraded scent, linking the chemical biology of the scent interaction to the feeding and pollination behavior. This work has broader implications regarding the importance of plant-pollinator relationships, especially when considering environmental and agricultural health as well as the issue of food security in our changing climate.

Molgulidae ascidians are marine invertebrate tunicates, that are the sister group to vertebrates by phylogenomic analyses. We are interested in molgulid ascidians because they have evolved larvae that have lost all of the chordate features. Two main types of molgulids comprise tailed (urodele) larvae and tailless (anural) larvae, and we are using them to understand the evolution of the loss of the tail. Molgulid species are found in at least four distinct clades, three of which have multiple species with tailless larvae. Our current project in the Swalla lab focuses on how three species M. ficus, M. verrucifera, M. complanata are phylogenetically related to the other Molgulid Ascidians. Polymerase Chain Reaction (PCR) was used to isolate the 18S ribosomal RNA genes and a variable D2 loop region of 28S ribosomal RNA genes in these three species. Further sequencing, analysis, and alignment of three species’ genomic DNAs are used to construct molecular phylogenies. Moreover, the observation of larval development and adult morphology are used to characterize each species. For instance, if two species both have tailless larval development and same branchial basket morphology, additional to the identical 18S and 28S rRNA sequences, that suggests these two species are actually one species. If there are more similarities in their development and their genome sequences, they are more likely related to each other, and vice versa. In summary, the phylogenetical relationship of three species M. ficus, M. verrucifera, M. complanata compared to the other molgulid ascidians were determined based on the data of 18S and 28S ribosomal RNA sequences. This enhanced phylogeny allows us to understand the evolution and development of molgulid ascidians.

Plants have evolved to represent a diversity of species, characterized by functional traits that dictate their performance in response to changing environments. One such leaf functional trait that relates to plant ecological strategy and is strongly correlated with photosynthetic rates, is minor leaf vein density (LVD). Our study will assess how ecological strategies within plant communities shifted in response to Earth’s most recent major global warming event, the Middle Miocene Climatic Optimum (MMCO) from 17-14 Ma, where rises in both global temperature and atmospheric CO₂ levels occurred. We hypothesize that global warming led to longer growing seasons and ecological strategies that prioritized persistence over productivity became dominant, and more favorable climates increased the diversity of ecological strategies present within the community. We will be traveling to various museums to photograph fossils that have all their minor veins preserved, collected from sites representing before, during, and after the MMCO in the Pacific Northwest region. Our goal will be to include several species per site, and photograph their fossil leaves under a stereo microscope. From there we will measure LVD using the program ImageJ using standard protocols. Once we have that data, we will calculate measures of the community-level distribution of this trait (mean, variance, kurtosis), and then compare those values between sites, and thus across the MMCO. In support of our hypothesis, we predict to see a lower mean and kurtosis, and higher variance of LVD values in MMCO plant communities, relative to those existing before or after the warming event. Overall this research is important to not only understanding how plant communities responded in ancient times to rising temperature but how plant communities could potentially respond to the rising temperatures in the future.

Plants face an enormous number of environmental stressors, including agriculturally important pests and pathogens. To defend against these biotic stressors, plants rely on pattern recognition receptors (PRRs), which are cell surface proteins that recognize conserved, non-self molecules indicative of attack or infection and initiate pattern triggered immunity (PTI) signaling pathways to mount defense responses. Recently, the first PRR involved in immunity against herbivorous pests was discovered in legumes. INR, a leucine-rich repeat receptor-like protein, mediates defense responses to inceptin, a peptide found in the oral secretions of caterpillars. To better understand the structural aspects of INR that are necessary for its ability to bind to inceptin and associate with downstream signaling components, I am developing a reporter system to screen INR variants to identify mutations that affect its function as a PRR. I have generated several different reporter constructs driving luciferase expression with promoter regions of genes found to be upregulated by inceptin when INR is transgenically expressed in the model organism Nicotiana benthamiana. These constructs vary in strength of expression but only one promoter region shows inducibility by inceptin. However, several constructs also show responses to a different immune elicitor, a peptide fragment of bacterial flagellin, which suggests that these constructs could be used as markers of PTI in plants more broadly. Robust reporters of PTI would not only be useful in understanding the structure and function of INR but may also enable further studies that will inform engineering practices to improve crop resistance to pests and pathogens.

As scientists collect ever larger volumes of data, methods to deal with these data have evolved as well. One of the fields that has thus emerged is the field of network science. Network science has many applications in biological fields as it allows scientists to connect variables of any type in a quantitative way. This versatility makes network science ideal for studies on comorbidity, or the consistent cooccurrence of various diseases in individuals. By analyzing comorbidities, we gain greater insight into interactions between diseases and systems of the body. This helps us understand how potential risk factors such as age, sex, and genotype affect various disease risks as well as the risk of comorbidity. We applied these methods to data on age of diagnosis for over 300 diseases collected from more than 28,000 owner reported surveys through the Dog Aging Project. We constructed comorbidity networks from these data and analyzed these networks using quantitative network statistics which allowed us to compare nodes both in and between networks. We first constructed undirected networks with the nodes representing various diseases to establish and identify pairs of diseases with significantly higher rates of cooccurrence than expected by chance. Using this network, we assigned directions to edges based on temporal data on the relative ages of diagnoses, which allowed us to identify which diseases are precursors to others. We observed how these networks changed through stratifications based on age, sex, and size to identify disease progression through age. In doing so, we hope that we can identify how age affects comorbidity in dogs, which can help researchers identify and develop therapies for lengthening dog lifespans. This knowledge will also provide insight into the mechanisms behind certain disease connections that were previously unknown.

Teaching techniques that involve high structure active learning have grown in popularity due to their usefulness in establishing an efficient and equitable learning environment. During this transition, a lot of work has been done to understand the equity impacts of high-stakes testing, particularly moderated through test anxiety and similar forces. Less work has focused on low-stakes points -- course points offered for good faith effort, completion, or participation. Are students as concerned with earning low-stakes points as they are with high-stakes points? The COVID-19 pandemic and the rapid shift to digital learning have provided a natural experiment with regard to course point allocation. Many professors have been forced to make unexpected changes to course organization, including the decision about what assignments to grade for thoughtfulness (low-stakes) and which assignments to grade for accuracy (high-stakes). This project attempts to address demographic and general trends in low-stake performance in a large introductory STEM course before and after the transition to online learning. Through qualitative and quantitative data analysis, we identified characteristics of students who are likely to forgo low-stakes course points in a large, high structure, introductory STEM course. Ultimately, the goal of the project is to recenter the discussion in education research regarding highly structured courses and serve as a reminder that critical low-stakes course points are as critical for student success as are high-stakes course points.

Bergmann’s rule describes the correlation between increased body size and living at higher latitudes, seen in many endothermic animals. However, there is debate over if Bergmann’s rule is applicable to ectotherms. Using an exhaustive review of the literature, we investigated whether the relationship between body size and latitude is consistent with Bergmann’s rule in >100 species of Alpheus snapping shrimps. We found a significant correlation between body size and latitude (i.e., latitudinal midpoint of the range), using both raw species correlations and correlations corrected for phylogeny (phylogenetic independent contrasts). We discuss multiple hypotheses that may explain adherence to Bergmann’s rule in this group of shrimp, and the broader ecological implications of variation in body size during a time of global change.

Skin can detect a wide range of stimuli through the touch system, which is mediated by specific cells and structures. Merkel cells, one of these specialized types of skin cells, sense gentle touch and texture. In mammals, Merkel cells are identified by the expression of the transcription factors Sox2 and Atoh1. Recently, the Rasmussen lab identified Merkel cells in the zebrafish skin that share many characteristics of mammalian Merkel cells, including expression of Sox2 and Atoh1a. Interestingly, a paper by Konig in 2018 also described a novel cell type that expresses serotonin (5-HT), calretinin, and synaptic vesicle glycoprotein 2 (SV2), termed “HCS” cells, within the zebrafish skin. These researchers proposed that HCS cells are a different sensory population than Merkel cells, because of differing visual characteristics between these cells. However, the paper did not present any conclusive evidence, making the relationship between HCS cells and Merkel cells still uncertain. I hypothesized that Merkel cells and HCS cells are actually the same population of cells. I tested my hypothesis using antibody staining and confocal imaging of zebrafish skin. I found that Atoh1a-positive Merkel cells express serotonin and SV2, demonstrating that Merkel cells in zebrafish are the same cells as HCS cells. Overall, my results resolve the identity of recently described sensory cell types in the zebrafish skin. In future research, I hope to use zebrafish Merkel cells as a promising model to better understand touch system development and regeneration.

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.

 Phytoliths are microscopic silica structures produced in plant tissue, and palms (family Arecaceae) are prolific producers of them. The stability of these structures contributes to the abundance of palms within the fossil record, as these structures can fossilize in sediments where leaves cannot, providing a wider range of evidence of the past palm geographic distribution. Palms generally grow in warmer and wetter environments, but certain species can live in more extreme climates (i.e., cold, dry). However, because 90% of palms are currently distributed around the equator and tropical rainforests, they are often used as an indicator of warmer environments in the fossil record. The current understanding of the taxonomic groups below the family level is limited, and it is unclear whether palm phytolith shape can be used to identify different palm groups in the fossil record. The Palm Project hopes to address this gap by testing phytolith classification using a morphometric approach. Over 100 species of known modern Arecaceae (~30 images/species) phytoliths have been imaged with a confocal microscope, and a semi-automated script in ImageJ quantifies the overall shape (globular or hat), density, and size of the phytolith ornamentations. Next, multivariate analysis on the morphometric data attempts to distinguish the phytoliths of various modern subfamilies based on distinct morphological traits, forming a quantitative baseline to describe and classify phytoliths. When combined with the collected ecological data, this could potentially identify signals between the environment of modern palm species and the phytoliths they form. Once the morphological differences between groups of modern palms are quantified, the same analysis can be applied to fossil phytoliths of unknown origins. This could reveal which taxonomic and ecological groups they belong to based on the most similar modern phytolith, giving insight into the phylogeny of Arecaceae and the paleoenvironment the fossils came from.

Reactive oxygen species (ROS) such as superoxide (O2–) and hydrogen peroxide (H2O2) are partially reduced oxygen molecules produced during cellular metabolism in all organisms. Plant chloroplasts and mitochondria are major ROS sources because of photosynthesis and aerobic respiration in these organelles. We previously found that the levels of oxidants were higher and antioxidants lower in maize (Zea mays) seedlings grown in the light than in the dark; these changes were accompanied by greater oxidative damage to the DNA in both chloroplasts and mitochondria in light-grown than dark-grown plants. In my research project, we will measure both H2O2 and antioxidants (catalase and peroxidase) after exposing dark-grown plants to light for increasing periods of time. Maize seedlings were grown in continuous darkness followed by 2, 4, 6, 12, and 24 hours of light. We then measured the levels of H2O2 and antioxidants in the chloroplasts and mitochondria isolated from leaf and stalk tissues during seedling development. Our data show the time course of ROS accumulation after the transfer from dark to light growth conditions and should provide a better understanding of the role of light in plant oxidative stress. Specifically, oxidative stress by ROS can be linked to DNA (mitochondria, plastid and organismal genomes) damage and by looking at the gradient of light exposure, it can open doors to discover how plants physiologically limit genetic impairment. This research can be used to assess more cancer-related topics in regards to ROS levels and how scientists can possibly find a solution in plant evolutionary-engineered genetics.

 Cancer recurrence and migration remains a persisting challenge in oncology. As such, accurate models of tumor growth in the laboratory (in vitro) are critical to our continued understanding and treatment of the disease. This project aims to use one such model—the 3-dimensional spheroid—to measure the effects of novel targeted therapy combinations on different cell lines. Two cell lines were explored in this experiment: UM-SCC74A (a tongue squamous cell carcinoma) and HCT116 (a colorectal carcinoma). Spheroids were formed by culturing cells in ultra-low attachment well plates for 24 hours, allowing the cells to coalesce and form a 3-dimensional sphere. An extracellular matrix was added in some trials to further improve the accuracy of the model. Spheroids were cultured in the presence of drug for 48 hours, then allowed to grow in drug-free media following washout. Spheroid size and viability were assessed via imaging and Cell-Titer Glo assays, respectively. The results from both cell lines echoed those obtained in 2-dimensional assays and validated the efficacy of our drug combinations in a 3-dimensional setting. These models could provide an intermediate step between 2-dimensional in vitro experiments and those conducted in live organisms (in vivo). The success of this model in supporting different cell lines affirms the robustness of this approach and its potential for use with a wide variety of cancers.

Microbiomes are now known to provide numerous benefits to hosts, from immunity to reproductive success. Vertical transmission of microbes has been linked to improved offspring success, for example by protecting eggs from pathogens post-oviposition. Infection with pathogenic soil fungi is associated with mortality in soil-incubated eggs, but may be ameliorated by the transmission of beneficial microbes.To determine whether 1) maternal microbes are deposited on eggs during oviposition, and 2) whether they have an antifungal effect that increases hatching success, we conducted challenges between dissected or oviposited eggs from the striped plateau lizard (Sceloporus virgatus) and fungi from failed S. virgatus eggs. Unique fungal and bacterial morphs were isolated from eggs to identify potential pathogenic fungi and antifungal bacteria. Direct challenges were carried out between individual bacteria and fungi to assess antifungal capabilities of bacteria isolated from the S. virgatus eggshell microbiome. Dissected eggs (no contact with the cloaca) had significantly lower hatch success, and hatchlings from dissected eggs were significantly smaller and lighter than those from oviposited eggs, indicating that some element of oviposition is key for hatchling success. One bacterial genus (Enterobacter) was found in the cloacae of females and on eggshells, suggesting that vertical transmission may be occurring. Three fungi were found to dominate the eggshell community of both dissected and oviposited eggs exposed to multiple fungi, suggesting that they may be responsible for egg mortality. At least two of these species of fungi have known pathogenic effects in other reptiles, and may produce antibacterial and antifungal metabolites.These results suggest an antagonistic relationship between fungi and bacteria on S. virgatus eggshells, indicating that bacteria transmitted during oviposition may improve hatch success in this species. Additionally, vertical transmission of beneficial microbes may serve as a form of parental care and occur via a novel pathway, the cloaca.

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.

  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.

 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.

Seed dispersal is an important component in the lifecycle of plants and aids in establishment of successive generations. Flowering plants have developed multiple ways of dispersing their progeny, including via wind, a strategy known as anemochory. In this presentation, I evaluate and compare wind dispersal potential of ecologically dominant grasses of the tropical savannas of Venezuela, Cerrado region in Brazil, and Serengeti region in Tanzania. Due to increased canopy cover in regions of the Cerrado and Venezuelan savannah relative to the Serengeti, I predict that conditions in the Serengeti would favor wind dispersal. Dispersal structures, known as diaspores, were sampled from specimens obtained from various herbaria. I measured falling velocity as a proxy for wind dispersal ability. I dropped diaspores from a chute and recorded them on high-speed video, which I analyzed to determine the speed of the falling specimen. My data analysis so far has consisted of obtaining and comparing average falling velocity for communities. Contrary to my original prediction, preliminary data suggest that wind dispersal is favored in the Cerrado. This may be due in part to the relative abundance of megafauna in the Serengeti, which would allow for seed dispersal via animal adhesion (epizoochory) or consumption (endozoochory). Diaspores using these dispersal mechanisms may not be as likely to have low falling velocities associated with anemochory. To further evaluate epizoochorous and endozoochorous potential, I am currently analyzing surface roughness using photographs of dispersal units.

It is commonly known that climate has shaped the photosynthesis type, height, and leaves of plants. However, it’s less commonly considered how plants affect climate. Major forest loss events have occurred due to deforestation and tree die off over the past decade. Prior research has shown that which types of plants grow where, and how those plants function, can impact climate both nearby and across larger spatial scales. Because this prior research has focused on theoretical simulations, it remains an open question if the impact of changes in plants can be seen in the real and very noisy climate system. We have compiled maps of actual forest loss as observed by satellites, to create initial conditions for simulation experiments in order to test if the impact of plants can be identified in observations of the atmosphere during the satellite era. We are assessing differences between simulations with and without forest loss to identify how that forest loss impacted the atmosphere. Using these identified impacts we will analyze if these same patterns are found in observed environmental conditions. This project aims to advance our understanding of the effect of forest loss on global climate, atmospheric circulation, and energy balances. Better understanding of this will help us to coordinate efforts to mitigate climate change by planting forests, while minimizing unwanted impacts. Additionally, this allows us to further predict and understand the impact of forest loss.

Many widespread species show morphological variation across the unique habitats and climates they inhabit. For reptiles these morphological adaptations such as limb length or body depth are often correlated with habitat use and substrate types. Additionally, body size trends and the degree of pigmentation of reptiles, driven by thermoregulatory requirements, can correspond with the elevation and climate of populations. The blue ventral coloration of Western Fence Lizards (Sceloporus occidentalis) is a social signaling device and can also be a byproduct of increased melanism in response to thermally challenging environments. I used handheld and photo analysis methods to obtain head, body, and limb measurements in addition to quantifying belly and throat coloration from S. occidentalis in Washington State at a high elevation creek canyon population, a low elevation forest population, and a coastal beach population. I found that high elevation creek canyon lizards used rock perches 69% of the time, low elevation forest lizards used trees 67% of the time, and beach lizards perched on driftwood 97% of the time. Given this distinct substrate use, my morphological data suggests adaptations between populations. Rock lizards had the longest front fore-limbs which could result in increased running speed to optimally navigate their open rocky habitat. Forest lizards had the shortest front fore-limbs possibly due to reduced running reliance, as they specialized in arboreal perches. Driftwood lizards had the skinniest body condition that could optimize them for cracks in driftwood logs they relied on. Beach lizards had bluer and darker bellies than high elevation lizards, and bluer throat patches than the other two sites. My study suggests morphological and signalling adaptations among populations of S. occidentalis at the species’ geographic extreme, and demonstrates how habitat and climate selection pressures create intraspecific variation between populations of widespread species.

Trait-based plant ecology can serve as a means to better understand shifts in ecological strategies within plant communities, and how that affects greater ecosystem processes, such as productivity, before, during, and after a period of major climate change. Apart from modern anthropogenic activity regarding greenhouse gases, the most recent major global warming event was the Middle Miocene Climatic Optimum (MMCO). 17 14 million years ago, this event was a short aberration to a long-term cooling trend that lasted over the last 53 million years, with global temperature averages during the MMCO estimated to be about 8°C warmer than preindustrial averages. Through statistical analysis of leaf functional trait measurements such as leaf area, leaf perimeter, tooth count, and petiole width, we seek to help document an example of how global warming affected vegetation in Earth’s past by comparing changes in leaf traits across the MMCO to modern leaves. While studying Miocene fossil leaves, we are creating a modern leaf database to better interpret trait trends analyzed from samples. This is building off of a previous study’s global dataset to create a model that can be used as an analog for categorizing fossil plant assemblages into different vegetation types using functional trait distributions, as well as assist in trait trend interpretation. At this point in time, we have analyzed enough modern samples to be prepared to interpret trait trends preserved in MMCO leaf fossils. Our objective during the 2021-2022 school year will be to collect enough MMCO trait trend data to make stronger predictions about how modern plant communities will be affected by climate change.

 The methionine salvage pathway (MSP) is a set of metabolic reactions that is highly conserved among species. The SPE3 gene of the budding yeast Saccharomyces cerevisiae, characterized as essential for growth, encodes spermidine synthase, which catalyzes the 3rd enzymatic step of the MSP and is involved in biosynthesis of spermidine. We investigated the effects of mutations in SPE3 and other genes of the MSP by comparing strains containing mutations in genes encoding for different MSP enzymes. Diploid heterozygotes were created via mating, and double mutants were created via knockout PCR, transformation, and recombination. Cell growth rate, viability, vacuolar morphology, and genetic relationships were analyzed via growth curves, viability tests, microscopy, and spot test assays. We found that SPE3 knockout haploid (spe3Δ) mutants are viable with a generation time unaffected by growth in minimal media. We also show that SPE3 mutations result in a hindered ability to respond to stressors. Characterization of these mutant strains and their responses to stressors will lead to better understanding of spermidine biosynthesis and the functions of the Spe3p enzyme and other MSP enzymes in S. cerevisiae.

Understanding the major patterns of phenotypic variation is a central goal of evolutionary biology. In vertebrates, body shape is one of the most prominent features of trait variation. Body shape diversity can be influenced by the locomotor modes required by different niches, which is what we explored within the Sciuridae (squirrel) family. Squirrels can be sorted into three distinct ecotypes—ground, tree, and gliding—that invite many questions about the adaptive significance of different body shapes for these different niches. We hypothesized that there is a relationship between the morphology of the axial skeleton (the spinal column) and the appendicular skeleton (the limbs) that may then correspond to ecological and functional adaptations. To quantify squirrel body shape, we took measurements of osteological specimens held at the Burke Museum and conducted phylogenetic comparative methods on these data. Our findings include how the axial and appendicular components of the different squirrel ecotypes contribute to their overall body shape. Our results also indicate whether any of the measured squirrel species can be considered elongate—having a high ratio of body length to body depth. This research marks the first time the axial skeleton of Sciuridae has been explicitly measured and aims to help advance the field by elucidating the trends in convergent evolution of elongate body shape across vertebrates. Future research could include investigating the relationship between the axial and appendicular skeletal components of different clades to diversify the data collected. Additionally, to explore the effect of dietary ecology in addition to locomotion, measurements of the jaw, forefoot, and hindfoot could be collected and analyzed.

Thirteen months after the first confirmed case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the United States (U.S.), over 500,000 people have died. However, the pandemic in the U.S. has not affected all populations equally; there are vast differences in morbidity and mortality in areas of differing population densities. We hypothesized that the number of SARS-CoV-2 mutations would be higher in densely populated areas due to closer proximity among inhabitants, which would lead to increased viral spread from person to person, and thus a greater number of mutations. These mutations can impact and reduce vaccine efficacy, as well as morbidity and mortality, depending on where they occur in the virus’s genome. The complete genomes of SARS-CoV-2 cases from March 1^st, 2020 to March 1^st, 2021 were collected from the Global Initiative on Sharing Avian Influenza Data (GISAID) for counties of varying population density. These genomes were analyzed to identify geographic areas where problematic mutations had the potential to occur. Demographic data was collected at the county level from Integrated Public Use Microdata Series (IPUMS) for additional analysis. Mapping the incidence of mutations in the SARS-CoV-2 genome and the correlation of these mutations with population density and other demographic indicators may help decrease disease spread and ensure the vaccines will remain effective.

Alzheimer’s disease, the most common human neurodegenerative disorder, is characterized by hyperphosphorylation of the protein tau, leading to the protein’s aggregation and the formation of neurofibrillary tangles. The subsequent neurodegenerative consequences of these tangles may influence the biological response and sensitivity to neurological stressors, such as a traumatic brain injury (TBI). A TBI usually results from a strong blow to the head that leads to damaged brain cells and neurodegeneration. In the Promislow Lab, we are currently examining how neuronal tau affects the mortality response to a TBI-like trauma in the fruit fly, Drosophila melanogaster. Using the UAS-GAL4 gene expression system, we are able to induce the expression of the tau gene in fly neurons from eclosion. From both an experimental fly genotype (tau expression) and a control fly genotype (no tau expression), we are currently sampling flies at various time points during their lifespan and administering a TBI-like trauma on the flies using a high-impact trauma device. To quantify the impact of tau on the flies’ response to the TBI-like trauma, we are recording the percentage of flies dead 24 hours later (24 hour mortality index). We hypothesized that inflicting a TBI-like trauma would lead to a significantly increased 24-hour mortality index in the experimental genotype compared to the control genotype due to increased sensitivity in the former. Thus far, we have observed significantly increased mortality in response to a TBI-like trauma in the control genotype compared to the experimental genotype. The decreased mortality in the experimental genotype suggests a novel positive role of tau in the response to a TBI, which holds significant implications for targeting clinical TBI treatments and therapies. Further analysis and follow-up experiments will provide useful insight into understanding the mechanisms of tau’s role and the pathways of both Alzheimer’s and TBIs.