As anthropogenic climate change progresses it is drastically altering the health of watersheds globally. In efforts to mitigate changes to marine ecosystems, many studies are using physical and chemical measurements to inform plans and create legislation. The impacts of climate change on local ecological communities are harder to track and take longer to show themselves which is why it is vital that we develop accurate techniques for measuring this kind of change quickly. This project, completed as part of Puget Sound Foraminifera Research Project at the Burke Museum, uses calcareous benthic foraminifera recovered and identified from sediment samples collected by the Washington State Department of Ecology between 2017 and 2018. Benthic foraminifera are marine protists that live on or within sediment and form shells of calcium carbonate or agglutinated sand grains. Foraminifera used in this project were stained with Rose Bengal to ascertain whether they were alive at the time of collection and grouped according to World Registry of Marine Species protocol. Stained and unstained individuals were counted to create living and dead assemblages. The goal of this study is to determine the validity of using total assemblages that include both living and dead foraminifera as a proxy for quantifying the living assemblages in Puget Sound. This is important because previous research has found discordance between living and total assemblages of molluscs, pteropods and ostracods in embayments heavily impacted by anthropogenic activity. This study includes 5 embayments in Puget Sound. Results from Bellingham Bay and Sinclair Inlet suggest that the validity of using total assemblages as a proxy for living assemblages may vary across different areas of Puget Sound; while the total assemblage and living assemblage matched in Bellingham Bay, Sinclair Inlet has been found to have significantly different total and living assemblages.

Treatment of neurological disease has made little progress due to the inability of many therapeutics to access the brain environment. However, delivery vehicles like nanoparticles can allow therapeutics to overcome brain-specific biological barriers including the blood-brain barrier (BBB), the dense extracellular space (ECS), and cellular targeting. The ability of nanoparticles to overcome these barriers is influenced by surface properties which can be modified through the formulation process. One understudied parameter is the choice of surfactant, molecules which stabilize nanoparticle formation and likely form an interface between the nanoparticle and brain environment. First, we investigated the potential toxicity of several commonly used surfactants on brain cells and slices. We added surfactant solutions to mouse microglial cells (BV2) or cultured brain slices and assessed cell viability two days later with colorimetric assays. Our results showed that while surfactants cholic acid (CHA) and polysorbate 80 (P80) caused toxicity at high doses, they were nontoxic at the low doses involved with nanoparticle formulation. Other surfactants, including Pluronic® F127 (F127) and poly(vinyl alcohol) (PVA), were nontoxic throughout the tested dose range. Interestingly, although the F127 compound is nontoxic on its own, nanoparticles formulated with F127 reduced cell viability. This result was not observed with any other nanoparticle-surfactant combination. Confocal microscopy indicated higher intracellular accumulation of the nanoparticles formulated with F127 compared to all other formulations, suggesting that toxicity is mediated by nanoparticle internalization and surfactant choice. Finally, we used a live cell imaging technique to capture videos of the nanoparticle internalization process. Building off these results, ongoing experiments will evaluate several nanoparticle-surfactant formulations on their ability to accumulate within brain tissue after in vivo administration. Findings from this work will guide nanoparticle design for future clinical translation.

Brain extracellular matrix (ECM) structure mediates many aspects of neuronal function. When ECM structure becomes dysregulated in neurological disease, one resulting impact is impaired neuronal function. Therefore, probing changes in ECM structure could provide insights into disease mechanisms and expose potential therapeutic pathways. Previous work in our group determined that degrading neural ECM structures, including perineuronal nets (PNNs), leads to a significant increase in the diffusive ability of nanoparticles navigating the brain extracellular space. However, this diffusion-based analysis provides little insight into changes in PNN-specific morphology or structure; it only predicts whether or not they are present and the degree to which they may be altered from normal. With this project, we aim to quantify changes in PNN structure with high spatial resolution. PNNs are stained using a fluorescently labeled lectin (Wisteria floribunda agglutinin) and images are acquired via confocal microscopy. Using Python, a coding language, we developed an automated image processing workflow to characterize morphological and structural features associated with PNNs, including total number of branches, average branch length, average mesh size of the net, and the areal density of fluorescence. This approach was applied to brains that span a range of chronological ages, from 14 days old to adult. PNNs are known to increase in counts early on in life, so this age-based study served as a proof of concept for our methodology. This same approach can be applied to study the effect of various neurological diseases on PNN structure. Collectively, this work aims to enhance our understanding of neurological disease mechanisms and open new avenues of therapeutic intervention.

Extracellular Vesicles (EVs) are group of cell-derived structures including exosomes, microvesicles, and apoptotic bodies, which have been found to play a key role in intercellular communication, through the biological cargo that these EVs can carry. Their ability to deliver proteins and nucleic acids from donor cells to their target cells has led to growing interest in the potential of EVs being used as biomarkers for disease. But, a comprehensive understanding of EVs behavior is lacking, especially in neuroscience, which may hinder the development for further application of the EVs. Thus, we are interested in investigating the effect of brain-derived EVs (bEVs) on brain cells, especially microglia, the brain’s primary resident immune cells. To do this, we first extracted the bEVs from the rat brain through ultracentrifugation and purified them through size exclusion chromatography (SEC). We then applied the bEVs to cultured mouse BV-2 microglial cells and incubated for 24 hours before performing quantitative reverse transcription PCR (RT-qPCR) on the treated BV-2 cells to explore any bEV induced inflammation response. Preliminary results have shown that bEVs play a role in inducing both pro and anti-inflammatory responses in microglial cells, both to varying degrees in the cytokine markers expressed after incubation for 24 hrs. Furthermore, to better understand the interaction between bEVs and microglial cells, we labeled bEVs with fluorescent nano-sized semiconductor quantum dots (QDs). Through fluorescent confocal microscopy and time-lapse imaging, we were able to explore the time-dependent interaction of bEVs and BV-2 cells at high resolution. Our study can provide insights into bEV behavior, which can be used to better understand their potential use as biomarkers for specific brain disease models. 

The blood-brain barrier (BBB) is a layer of tight-junction endothelial cells that make up the capillaries in the brain and strictly regulate what molecules can pass from the blood into the brain. Many molecules, including insulin, cannot passively cross this barrier but require an active transport system at the surface of the BBB. Once in the brain, insulin plays a role in memory and cognition. Indeed, Alzheimer’s disease is characterized by decreased sensitivity to insulin, which could be explained by a malfunctioning insulin receptor (IR) or impaired transport at the BBB. However, before we can begin to investigate the IR under disease conditions, we must first understand its standard regulation and function in a healthy system. Specifically, we aim to determine what factors mediate the endocytosis of insulin into the endothelial cells of the BBB. To do this, we focused on clathrin and caveolin, two proteins involved in different endocytic pathways. We performed cardiac perfusions on mice, where we first administered a drug to inhibit either clathrin or caveolin, and then we perfused with radiolabeled insulin. Afterwards, brains were collected and dissected into regions. Radioactivity was measured in the hypothalamus, olfactory bulbs, and whole brain, and the data was graphed over time to determine if there were changes in insulin binding or transport rates. Our results help elucidate the molecular processes necessary for insulin transport and binding at the BBB, which can ultimately help us understand how IR uptake and insulin transport may go awry in Alzheimer’s disease.