High throughput single cell/nuclei RNA sequencing (scRNA-seq/snRNA-seq) has been used to characterize the gene expression at the cellular level in disease control studies. Differentially gene expression analysis aims to emphasize the biological variation between samples without unwanted technical variation. Batch effect correction is occasionally performed where cells from each individual sample are treated as being generated from a batch. We analyzed snRNA-seq data from ApoE neutral and detrimental mouse models of Alzheimer’s disease (AD) to test whether batch correcting the data using all cells from each individual biological sample as representing a batch would result in 1) loss of disease relevant associations, 2) loss of biologically relevant cell types, and 3) reduced association between cell types and phenotypes. We performed scRNA-seq analysis of seven samples from two ApoE genotypes using Seurat workflow applying Harmony batch correction, using each sample as a batch, and without correction. Since we measured two disease-related phenotypes in mice from the two genotypes, we asked whether cell cluster membership associated with genotypes are also associated with unit changes in brain-diseases related phenotypes. After applying individual sample batch correction, we found differences in number of cell types (clusters) before and after batch correction, we showed loss of cell types/clusters specifically from the detrimental genotype, shrinkage of the differences in cell cluster membership across samples and reduced association between cell type membership, genotypes and phenotypes. In conclusion, batch effect correction should be applied consciously based on the experimental design to avoid over correction of biological variability and an appropriate design should help to avoid unwanted technical variation.

Circadian rhythms are adaptive biological processes that govern the synchronous timing of biological functions and behaviors with the daily light-dark cycle. These rhythms are crucial for the timed regulation of sleep-wake cycles, metabolism, and hormone release, as well as for maintaining harmonious physiological functioning within the body. Within the mammalian brain, a central pacemaker, the Suprachiasmatic Nucleus (SCN), governs the timing of these circadian rhythms. This area contains a subpopulation of neurons that express and release the neurotransmitter Vasoactive intestinal peptide (VIP). These neurons play a role in synchronizing activity across the entire SCN network. Thus, this project is aimed at better understanding how VIP-neurons change shape throughout the day. Preliminary research in the de la Iglesia lab suggests that these neurons change shape across the day, such that VIP neuron fibers-axons and dendrites- are more branched during the day than the night. These results were obtained through the use of mouse models expressing a red fluorescent protein in the VIP neurons (VIP-TdTomato). The mice were perfused at a range of timepoints, and the samples underwent a tissue clearing protocol so that the entire SCN can be captured in one image. Then the software QuPath was used to train a machine learning algorithm to aid in the identification of VIP neuron fibers based on fluorescence expression. However, the performance of the machine learning algorithm has not been validated. To address this issue, I compared the algorithm-generated segmentations with manual annotations from humans, finding agreement 75.3% of the time in terms of fiber location and 77.4% of the time regarding background. These rather promising results demonstrate the usefulness of the algorithm in aiding the investigation of the entire dataset. This research provides a step towards better understanding the structural organization of the SCN, and thus circadian control of essential physiological processes.

Changes within the body that repeat approximately every 24 hours, called circadian rhythms, are controlled by a central pacemaker in the mammalian brain, the suprachiasmatic nucleus (SCN). Circadian rhythms can synchronize to cues like the light-dark (LD) cycle, allowing them to predict the 24-hour environment. SCN neurons are interconnected and connect to other regions within the brain. Our hypothesis is that SCN neurons have the ability to physically change connections throughout the day and that these changes are essential for it to act as a master clock. I explored this plasticity through the study of vasoactive intestinal peptide (VIP) and polysialylated neural cell adhesion molecules (PSA-NCAM). VIP is a neurotransmitter expressed in a subset of SCN neurons and plays a role in the SCN's ability to respond to light. PSA-NCAM is involved in decreasing cell interactions through facilitating events like cell migration and axon guidance; it is only expressed in areas of the adult brain in which neurons display plasticity in their fiber connectivity. Mice house in a 12 hour:12 hour LD cycle were sacrificed at two times, 12 hours apart. I used immunohistochemistry against VIP and PSA-NCAM to determine the levels of these molecules in the SCN. I found that the expression of VIP is higher 9 hours after lights were turned off (ZT 21) compared to 9 hours after lights were turned on (ZT 9). I found that PSA-NCAM has a higher trend of expression levels at ZT 9 than ZT 21. Although these results are preliminary, we find the implication of the results promising. VIP and PSA-NCAM express in anti-phase; as a negative regulator of cell adhesion, higher levels of PSA-NCAM should correlate with lower levels of VIP. Understanding how mammals keep time is important because circadian rhythms are essential for virtually every aspect of an organism's behavior.

Tornadoes are rare events in the Pacific Northwest and are extremely difficult to predict along the coast, causing much surprise when they do form. After witnessing the destruction and rebuilding of a Port Orchard neighborhood that was ravaged by a F2 tornado in December 2018, I was inspired to explore these uncommon occurrences. For this project, I am investigating the near storm environments of tornadic systems that are generated from cold air outbreaks during the winter months along the coastal Pacific Northwest. This is done by analyzing data from massive tornado datasets, METAR (Aviation Routine Air Report) observations of convective precipitation such as graupel, and piecing together upper air reanalysis data to compare them to weather indexes that have been defined to determine atmospheric instability and can be used to predict extreme weather. The goal is to find patterns that are associated with these tornadic events to create more accurate forecasts and to paint a detailed picture of tornado climatology in the Pacific Northwest. My hope for this project is to shed light onto the factors that are at play for tornadoes, hail, and other severe weather that could potentially save lives.

Trauma-induced coagulopathy is a severe complication of trauma that alters the normal mechanism of blood clotting through a number of complex factors. If clots are hypercoagulable, there is risk for dangerous vascular blockages. Conversely, if the clotting is hypocoagulable, it can lead to fatal hemorrhaging. Prior research indicates that actin has a major impact on platelet activity and blood clot formation. Actin is a highly abundant cytoskeletal protein that forms long, insoluble filaments. When released into the blood during cellular death, these filaments have complex effects on blood clot formation. Actin filaments can be integrated into the scaffolding of the clot, increasing strength. My experiment aims to investigate the roles of actin and on human blood clotting. Healthy donor whole blood in 3.2% sodium citrate was spiked with either a saline control or recombinant human skeletal muscle-derived actin (final concentration 200 nM) and allowed to incubate for 5 min. Samples were then activated with either 10 mM adenosine diphosphate (ADP) or 2 mg/mL collagen. The platelet aggregation response was then measured by impedance aggregometry. Each pair of control and actin conditions was run simultaneously. The impedance area under the curve (AUC) was compared between control and actin groups under each activation condition using a paired t-test with significance at p<0.05. Preliminary results show ADP was no different between the control and actin groups (p=0.400, n=5). The AUC in response to collagen was significantly higher in the presence of actin compared to control (p=0.005, n=7). Exogenous muscle actin appears to increase platelet aggregation through the collagen but not the ADP activation pathway. Further investigation is required to better characterize this interaction. A better understanding of the mechanisms of actin on hemostasis could direct research into pharmaceuticals and therapies that could yield better outcomes for trauma patients.
 

Corepressors are proteins recruited by partner proteins to negatively influence transcription of genes. TPL is a corepressor from the model plant Arabidopsis thaliana, and while we understand a lot about how TPL works, there are still many mysteries remaining. My project aims to identify other proteins that work with TPL to form a transcriptional repression complex at a single-engineered promoter site. First, we created a synthetic repressor called dCas9-TPL that binds and represses the transcription of the RUBY reporter. The RUBY reporter is a visual marker designed to express throughout the entire plant, turning the green plant a bright purple. Our engineered RUBY line also carries two guide RNAs in its promoter with sequences not found anywhere else in the Arabidopsis genome. This allows dCas9-TPL to bind to and repress this particular gene and not affect the transcription of other genes. Visual screening of the repressed RUBY line showed these plants turn a faint whitish-pink instead of bright purple, signifying that the repression by TPL is working. I have identified the promising repressed RUBY homozygous line and have generated three mutagenized populations of 40,000 individuals using the chemical Ethyl methanesulfonate (EMS). The EMS protocol creates new point mutations allowing us to identify genes involved in repression that we can map through DNA sequencing. I will use visual screening to search for plants with bright purple organs, meaning that the repression by TPL is broken and that a putative TPL interactor may be mutated. By identifying regulators of corepressor function in plant biology, I hope to learn principles that can inform cellular engineering across many organisms and better understand why certain mutations associated with transcriptional repression cause developmental defects or diseases like cancer in humans. 

Tropospheric reactive halogens are a sink for ozone and influence the oxidizing capacity of the troposphere. Daily measurements of chloride enrichment, bromide enrichment, and iodine in sub- and super-micron aerosol were collected in June 2022 and January-February 2023 in Bermuda during the Bermuda boundary Layer Experiment on the Atmospheric Chemistry of Halogens (BLEACH) campaign. Gas-phase halogen radicals originate from heterogeneous reactions (chemical reactions involving more than one phase of matter) on the surface of halide-containing aerosol. These reactions lead to enrichment or depletion of aerosol halides (e.g., particle phase chloride, bromide, or iodide) relative to their ratios with sodium in sea water. We compare these measurements with simultaneous observations of ozone and gas-phase halogen concentrations in order to understand the relationship between aerosol and gas-phase halogens and their impact on tropospheric ozone abundance. Observations from these campaigns will aid in improving atmospheric model outputs and environmental policy. I extracted the ions from more than 60 quartz air-filter samples and aided in method development with Ion Chromatography (IC). Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was primarily used for iodine measurements. Data analysis and lab work is still ongoing, therefore only preliminary observations are available. We’ve observed that sodium, iodine, chloride, and bromide agree well with previous observations at Tudor Hill (Arimoto et al., 1995; Sander et al., 2013). Higher depletions of chlorine excess and bromine excess and higher iodine and non-sea salt sulfate concentrations are observed during the first half of the summer campaign.

Without proper genetic regulation, the creation and maintenance of cells within eukaryotic organisms such as yeast, plants, and humans is doomed to fail before it even begins. Protein corepressors are key to the genetic repression in all eukaryotic organisms and are vital for an organism to be able to properly coordinate their development and respond to environmental stimuli. In Arabidopsis thaliana, a model plant for genetic studies, the corepressor TOPLESS (TPL) is one of the main proteins that is used to repress the auxin pathway, which is essential to development and organ creation. Recently, the active domain of TPL has been pinpointed to an 18-amino acid long region named LIS1 homology (LisH) that is sufficient for activity. Previously, we found that the helix H1 of LisH in a plant corepressor functioned as a transcriptional repression domain in yeast. These observations suggest a broad conservation of mechanisms across kingdoms, suggesting this motif could be engineered to be a potent, short, and adaptable protein domain suitable for synthetic biology and therapeutics. My project aims to test the ability of the LisH protein domain to repress gene transcription in metazoans using mammalian cell culture. We will transfect human cancer cell lines with DNA encoding a dCas9-TPL fusion protein, which can be targeted to promoters of endogenous genes such as the cell surface antigen CD4, or synthetic constructs such as fluorescent reporter genes to detect differences in protein levels. Results of the project are expected to show that TPL and other foreign corepressors can function within the human cell just as efficiently if not more than human corepressors. Research into LisH's abilities will provide knowledge of its active domains and mechanisms in mammalian cells while also having the possibility to aid the scientific community by developing TPL as a rapidly deployable synthetic biology tool.

Giardia lamblia is a gastrointestinal parasite which causes diarrheal disease and hinders nutrient absorption. G. lamblia colonizes the small intestine by a two-phase life cycle: the reproducing trophozoite stage and the transmissive, infective cyst stage via a fecal-oral route. How G. lamblia detects encystation signals in the encystation process is unknown. Our laboratory discovered EncystR, a seven transmembrane protein which compartmentalizes after perceiving cholesterol depletion and increased pH. Knockdown of EncystR promoted encystation, indicating negative regulation. We hypothesize that EncystR is responsible for perceiving encystation stimuli and de-repressing cAMP signaling to promote encystation. However, the mechanism responsible for triggering cAMP signaling is uncharacterized. This project hopes to identify EncystR transient interactions using proximity labeling and mass spectroscopy (LC-MS/MS). Proximity labeling of EncystR over an early encystation time course will identify proteins involved in downstream signaling. To determine time points of interest, EncystR was endogenously labeled with mNeonGreen (mNG), and imaging of non-encysting cells displayed EncystR-mNG at the plasma membrane. To delineate the EncystR trafficking pattern, we induced encystation and followed EnystR localization for several hours using fluorescent microscopy. Individual cells were categorized by localization to peripheral vesicles, a novel acidic compartment, or non-responsive cells where localization remained on the plasma membrane. Percent peripheral vesicle localization peaked at 1h and percent compartmentalization stabilized after 3 hours. Proximity labeling proteomics at these time points can identify connections to cell trafficking to the novel acidic compartment. While EncystR does not tolerate TurboID proximity labeling, miniTurbo produced significant biotin labeling after 30 minutes in 50 mM biotin. Biotin-tagged proteins are sequestered using Streptavidin-coated columns through liquid chromatography, then cataloged using mass spectroscopy (LC/MS-MS). Proximity-tagged proteins using miniTurbo can inspire drug inhibition candidates of Giardiasis. Due to Giardia’s model nature to other parasites, such as reliance on cholesterol, parallel drugs may be found as well.

The dynamic expression of genes in an organism creates the biological complexity of life. Many are unique to a given lineage, while other genes are conserved and carry out the essential functions of life. Unsurprisingly, these essential genes are complicated to study, as interfering with their function often leads to death. One critical component of transcription is the multi-protein Mediator complex, which is found at every eukaryotic promoter where it helps coordinate the activation of gene expression. My project focuses on a core component of the Mediator complex, Mediator 21 (MED21). While MED21 is required for gene activation, my lab found that it also plays a role in the repression of gene expression, suggesting a complicated interplay between these two states. This can be challenging as many mutations in MED21 lead to lethal phenotypes. As an alternative, I hypothesize that using an integrase-based molecular switch to create a switchable MED21 will then allow me to differientate the role that MED21 plays in activation through the Mediator complex versus repression through the corepressor protein (TPL) in the model plant Arabidopsis. Integrases are capable of inverting DNA sequences flanked by unique sites, and I am engineering a switch that will turn off MED21 in certain tissues or in response to the addition of a chemical. By expressing an integrase protein from a lateral root-specific promoter, we can engineer a MED21 loss of function only in those specific cells while the rest of the plant is wild type and healthy. Future experiments include a switch from wild-type MED21 to a mutant form incapable of binding to corepessor TPL. This study will help us better understand the role MED21 has in repression versus activation, and also how state switching contributes to organogenesis.

The protozoan parasite Giardia lamblia infects hosts via the ingestion of cyst-contaminated water. We recently identified EncystR, a 7-trans membrane protein at the cell surface that acts as a negative regulator of encystation and responds to encystation cues by internalizing. By following the localization of EncystR through an encystation timecourse we discovered a novel compartment of unknown function. The Giardia endocytic pathway was only believed to include hybrid endosome/lysosome compartments statically positioned beneath the plasma membrane. We suspect this newly identified compartment may be a lysosome-like compartment. Using EncystR as a marker for this compartment we found that the compartment is highly acidified based on the florescent reporter pHluoren2. Canonically endomembrane compartments are marked by specific phosphatidylinositol phosphates (PIP). Relevant to endomembrane compartments, PI(3)P marks endosomes and PI(3,5)P2 marks multi-vesicular bodies and lysosomes. To test if this newly identified compartment is evolutionarily related to lysosomes, we will generate reporters for these phosphoinositides. Namely, we utilized the FYVE protein’s PIP binding domain as a sensor for PI(3)P which is located on endocytic membranes, the pH domain of PLC delta as a sensor for PI(4,5)P2 or PIP2 which is necessary for endocytosis and membrane-based cytoskeletal protein regulation, and ML1N for PI(3,5)P2 which is localized to lysosomes. Additionally, a mutant variant of the ML1N protein which is incapable of binding to PIPs is utilized as a negative control. These protein sensors were fused to the fluorescent protein mNeonGreen and imaged via fluorescent microscopy. While the experiment is currently in progress, we hypothesize that the localization of PIPs to the novel compartment will likely feature PI(3,5)P2 due to the previously found acidic nature of the compartment, implying a lysosome-like functionality. This possibly novel or conserved compartment could give insight into the evolution of eukaryotic cellular organisms and could potentially offer treatment routes for Giardia.

Giardia Lamblia is an intestinal parasite known for causing the diarrheal disease giardiasis in host organisms. It commonly infects humans and companion animals such as cats and dogs. Giardia’s life cycle is defined by its infectious cyst stage and proliferative trophozoite stage. GlRac is a small Rho-family GTPase which we recently determined to play a role in regulating encystation in Giardia, the process of transition from a trophozoite to a cyst. Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) are key regulatory proteins of GTPase activity. The GEFs and GAPs that regulate GlRac activity are currently unknown, but four candidates for GAPs and three candidates for GEFs have been identified. We are testing these GAP and GEF candidates in Giardia with a two-phase approach. First, we are using the protein-protein interaction reporter NanoBit to determine if the candidate GAPs and GEFs interact with GlRac. We are verifying results for NanoBit assays, a split NanoLuciferase reporter that indicates protein-protein interaction. Nanobit can reveal interaction but not the localization of the interaction, so we are performing co-localization with deconvolution microscopy. Candidate GAP and GEF proteins are being visualized using a mNeonGreen fluorescent tag while GlRac is being followed using the halogenase (HALO) tag labeled with Janeliafluor 646. The microscope assays allow us to determine if and where GlRac co-localizes with candidate proteins. Subsequently we will test the role of the identified GAP and GEF proteins in encystation through transcriptional repression using CRISPRi and translational repression using morpholino-modified antisense oligonucleotides (MOs.) If the candidate proteins are truly GAPs and GEFs, the knockdown cells should not progress through encystation as normal. GAPs and GEFs are potential drug targets for treatment of Giardiasis, as well as important in understanding the process of encystation in Giardia.

Iron centers which feature metal ligand multiple bonds can be powerful group transfer agents, for example, terminal Fe-oxo intermediates in soluble methane monooxygenase can perform oxo-atom transfer for the selective oxidation of methane to methanol. Abiologically, ligand constructs which enforce desirable electronic and structural configurations have been shown to enhance group transfer to a range of organic substrates. We developed and studied an iron (Fe) molecular complex with two aminophosphine selenide ligands (Se=PPh₂NTol; Ph=Phenyl, Tol=4-Tolyl) that chelate the metal center via the selenium and nitrogen. The iron complex (FeL₂) was synthesized by a reaction between Fe(HMDS)₂ (HMDS = bis(trimethylsilyl)amide) and the aminophosphine selenide. Characterization shows a tetrahedral, high spin, symmetric compound. We hypothesized that FeL₂ can activate and transfer heteroatoms and explored the reactivity of FeL₂ with oxidants, oxo atom donors, and organic azides. Treatment with iodine (I₂) results in oxidation of the iron center (Fe(II) to Fe(III)) and coordination of the iodide counterion results in structural reorganization to a five-coordinate square pyramidal complex. Reactivity with oxo atom donors shows that either the ligand or Fe center are oxidized, and we identified a µ₂-oxo dimer, which is the first Fe-O-Fe dimer to have selenium in its first coordination sphere. We found that FeL₂ forms Fe-nitrenoid intermediates and can perform nitrene transfer to form diazos or do C-H amination, when treated with aromatic and aliphatic azides, respectively. The presented complexes are characterized by single crystal X-ray diffraction (XRD), Evan’s method, nuclear magnetic resonance (NMR), and Ultraviolet-Visible Spectroscopy (UV-Vis). This research builds upon the knowledge of transition metal complexes for heteroatom transformations.

The geologic record provides opportunity to provide actual examples of how plant communities have responded to climatic changes, providing important perspective for modern anthropogenic-driven climate change. Two important climatic events in the Miocene offer such an opportunity, including a global warming event, the Miocene Climatic Optimum (MCO; 17-14 million years ago), and a global cooling event, the Middle Miocene Climatic Transition (MMCT; 14-12 million years ago). This study is assessing how the diversity and prevalence of ecological strategies within Pacific Northwest (PNW) plant communities changed in response to these events, by analyzing ~6 PNW fossil plant sites that span these events in time. At each site I characterize ecological strategies of taxa comprising these ancient communities by measuring leaf vein density (LVD) of fossil angiosperm leaves, which relates strongly to the maximum photosynthetic rates of the plant. Photosynthetic rates influence ecological strategy by placing plants along a spectrum with fast growth but low tolerance to resource scarcity at one end, and slow growth and high tolerance at the other. I am digitally measuring leaf vein density using microscope images of fossil leaves previously taken at several museums where these fossils are housed. I expect that during the MCO, evergreen plants with slower growth rates become more dominant and the diversity of ecological strategies increased (lower mean and higher variance of LVD). Across the MMCT, I expect that deciduous plants with high growth rates became more dominant and stronger abiotic filtering caused a decrease in the diversity of ecological strategies present (higher mean and lower variance of LVD). This study provides a real-life example of how climatic events reshaped the assembly of plant communities and provide an important perspective for present and future climate change.

The Miocene Climatic Optimum (MCO) was a period of global warming 17-14 million years ago, where temperatures increased 2-4°C and CO₂ levels increased to ~400-600 ppm. Overlapping with the MCO were the Columbia River Basalt eruptions (CRB: 6.6-15.9 Ma), where extensive lava flows spread across the Pacific Northwest, resulting in primary succession. My study is focused on reconstructing the vegetation across the MCO and during CRB eruptions using epidermal phytoliths (i.e., Microscopic Biosilica) to understand how these conditions impacted plant communities. Epidermal phytoliths are formed within living plant matter reflecting the current environmental conditions in their size and undulation. The plant matter then falls to the forest floor and decays leaving behind the resilient Microscopic Biosilica, which is preserved within that sediment. Leaves formed in ecosystems with an abundance of sunlight reflect open-canopy vegetation, with small circular phytoliths; while large-undulated phytoliths come from closed-canopy, shady environments. Previous work has shown a correlation between the average size and undulation of epidermal phytoliths with leaf area index (LAI; i.e., a measure of canopy openness). I am using this process with sediment samples collected across four sites in Central Oregon to calculate ancient reconstructed LAI (rLAI), and thus reconstruct the canopy cover. Each site was chosen due to the time period it represents, with different exposure to increasing variations of CO₂ and CRB impacts. I hypothesize increased temperature and atmospheric CO₂ concentrations during the MCO created favorable conditions for plant communities, which promoted a productive closed-canopy forest structure. Additionally I hypothesize, the primary succession induced by CRB volcanism prevented the re-establishment of forests, leading to open-canopy vegetation structure. As modern day anthropogenic-driven climate change invokes alterations in our planet's ecosystems, we need to better predict and anticipate future responses of plant communities to these environmental perturbations. 

As the planet progressively experiences the effects of climate change, the US government is increasingly more interested in limiting the country’s resource consumption with a focus on limiting the footprint of corporations and manufacturers. The US Department of Energy (DoE) created a national network of Industrial Assessment Centers (IAC) within colleges, including the University of Washington, to provide recommendations that will be backed by DOE matching funds to facilities. The mission of the UW IAC is to reduce the energy consumption and emissions, improve overall efficiency, and implement cutting-edge technologies in a minimum of 20 facilities across Washington state and the Pacific Northwest, annually. We hypothesize that, by giving corporations free energy audits with financial incentives and training students to be energy savvy engineers, Washington state will reduce its overall energy consumption and be a leader nationally in resource management. The main methods to my research include initializing our energy audits with companies, preparing preliminary recommendations based on the company’s industry classification, touring the facility while collecting machinery and energy data, and writing detailed DOE-accredited reports entailing the best potential savings for the facility’s overall energy reduction. To date, the UW IAC has provided 250+ recommendations and 50+ assessments to industry, saving 1.49+ TBtu of energy. We have and continue to analyze a breadth of technologies ranging from air compressors, refrigeration, HVAC, lighting, automation, motors, and overhead operations in corporations across a variety of industries including aerospace manufacturing, food production, wastewater treatment, and paper mills. The significance of this pursuit is providing corporations an incentive to reduce their environmental footprint through an increase of revenue and modernization of their practices. We ultimately strive to bring Washington state to the forefront of efficient and clean energy practices.