Phase separation in phospholipid membranes occurs in living systems like yeast vacuole membranes and consists of domains enriched in specific lipid components. Phase separated domains coalesce and merge together into a singular phase as membranes are heated above the phase transition temperature, T_mix. The phase transition temperature depends on the lipid composition of a membrane. In the lab, it is useful to produce simple, model membranes to isolate phenomena like phase separation from the complexity of biological systems. Emulsion phase transfer is one such technique used to generate giant unilamellar vesicles (GUVs) by using a centrifuge to drive emulsion droplets coated in lipids through a lipid-oil solution and water interface. However, there are several specific challenges for emulsion phase transfer that require optimization: drying the lipids down with nitrogen gas into a lipid film, the time sensitive creation and layering of the lipid-oil emulsion, and finding the optimal centrifugation parameters. Here, we optimize emulsion phase transfer in three ways: 1) evenly coating lipids films via swirling, 2) creating the emulsion and depositing it as quickly as possible, and 3) tuning centrifugation to maximize vescile formation and minimize vesicle aggregation. Further, we measure the phase transition temperature for GUVs made of a ternary lipid mixture consisting of DPPC (16:0 PC), DOPC (18:1 PC), and Cholesterol in a 1:1:3 ratio. To visualize membrane phase separation, a fluorescent lipid that partitions preferentially to only one phase was added to lipid mixtures used to prepare GUVs. Due to the crucial role of cholesterol in membrane phase behavior, the phase transition temperature of GUVs generated through this technique will vary from those produced by other techniques due to poor cholesterol incorporation.

A keystone bacterium involved in the pathogenesis of chronic periodontitis, commonly known as gum disease, is Porphyromonas gingivalis. This pathogen produces multiple structures on the surface of its cells categorized as virulence factors, including lipopolysaccharides (LPS) and outer membrane vesicles (OMV). Lipopolysaccharides are anchored to the outer membrane of P. gingivalis by Lipid A structures. Previous studies in our lab have found that the abundance and cargo selection of OMVs released from the cell’s surface can be modulated by the structure of Lipid A on P. gingivalis. The virulence factors investigated assist P. gingivalis in the colonization of the host at a cellular level – such as the formation of biofilm, or aggregated bacteria on a surface. OMVs released from the cell's surface operate as a delivery system for the various structures found on the outer membrane of the bacterial cell. We hypothesize that the biofilm density and morphology formed by P. gingivalis are influenced by the changes in OMV abundance and content derived from the modulations in the Lipid A structure. To test this, we utilize biofilm assays, where live cultures from various strains with differing Lipid A structures can grow and aggregate on a glass coverslip for 48-72 hours. Morphology differences are revealed from the analysis of the biofilms and pixel intensity is quantified and compared among strains. Various assays are used to compare the activity and concentrations of protein and lipid cargoes within the OMVs to understand how they are connected to biofilm morphology. The biofilms formed by P. gingivalis contribute to its pathogenesis, therefore it is important to understand the impact that secreted outer membrane vesicles have on its structure and integrity.

Clearance of wound infections can be hindered by a bacterial biofilm; a complex extracellular matrix (EM) secreted by adherent bacteria that allows them to evade the host immune system and obviate antibiotics. A novel, synthetic peptide—known as an anti-α-sheet inhibitor—can disrupt biofilm stability by inhibiting the formation of amyloid fibrils, which contribute to the biofilm EM. This project aims to design and characterize alginate porous scaffolds that elute these synthetic peptides, for use as anti-biofilm wound dressings. The physical properties and peptide release kinetics of the scaffolds will be optimized for clinical applications, supported by in vitro efficacy studies with live bacteria. This project draws upon past work from the Bryers Research Group on engineering infection immunity and tissue scaffolds, in which biofilms are prevalent. Results of this project will provide an alternative approach to biofilm prevention, thus reducing the burden of biofilm-related infection complications.

Diaryliodonium salts have recently been shown to facilitate metal-free mechanoredox free radical polymerizations. Prior literature reports focus on the role of diaryliodoniums as photoinitiators; these salts have well established fragmentation mechanisms and kinetic profiles. However, their use in mechanochemistry has not been extensively investigated. Mechanochemistry is an emerging field of chemistry that uses force as a stimulus for chemical reactions. Compared to traditional stimuli such as light, heat, and electricity, mechanical force avoids the use of transitional metal additives and often has a lesser environmental impact. This report looks to explore functionalized (e.g., electron-rich versus electron-deficient) diaryliodoniums and to determine the impact of reactivity in a mechanoredox polymerization setting. Herein we synthesized a library of salts of diverse electronic structures and tested them within an established mechanoredox ball mill system. We report data on their initiation based on radical trapping as well as changes in polymers molecular weight. The hypothesis is that salts with functionalities that withdraw electron density such as alkyl halogens or cyano groups will initiate faster than salts with electron donating functionalities due to their lower reduction potential as demonstrated in literature. Exploration of these functionalized salts will provide kinetic insight and open new avenues of synthesizing commodity polymers. This is particularly applicable in 3D printing, where having control over the rate of initiation could be used to tune downstream physical properties.

With increasing temperatures and changing ocean conditions, it is important to measure the effects felt on both a species specific and ecosystem level, to better understand the consequences of this change. To investigate this issue specifically off of the Northeast US Atlantic Coast, I worked collaboratively with the National Oceanic and Atmospheric Administration, using both bottom temperature and sea surface temperature as oceanographic variables to examine whether the changes observed have influenced fish consumption over time across seventeen prominent fish species. We calculated average annual fish consumption per species from 1993-2018, where I then compared this to both sea surface temperature and bottom temperature using generalized additive models. Additionally, we plotted the above variables independently using generalized linear models and linear models to analyze their respective trends. I also created a sea surface temperature model to compare the extreme temperature changes the ecosystem was experiencing. Overall, increasing trends in both sea surface temperature and bottom temperature were detected, and within species’ consumption trends, four species showed significant increases in consumption (buckler dory (Zenopsis conchifer), fourspot flounder (Hippoglossina oblonga), longhorn sculpin (Myoxocephalus octodecemspinosus), striped searobin (Prionotus evolans)) whereas two indicated significant decreases in consumption (Atlantic cod (Gadus morhua), thorny skate (Amblyraja radiata)). When compared to sea surface and bottom temperature, three species' consumption rates were found to be significantly influenced by these variables (longhorn sculpin, thorny skate, spiny dogfish (Squalus acanthias)). Given these results, it is likely that the adaptability of species and their respective mobility will influence the degree of impact by changing ocean conditions, constituting both winners and losers in this changing time period. Therefore, we recommend further analysis to better understand how various related biological factors influenced by climate change will be impacted in the future to develop a more thorough understanding of the consequences of this change.

The purpose of this study is to establish biomarker assays for physiological stress linked to arsenic exposure and microbiome perturbation in freshwater snails. The ASARCO smelter in Tacoma, Washington polluted soil and water around Puget Sound with arsenic for nearly 100 years. Previous studies have shown that environmental pollutants can alter the gut microbiome and modulate host-microbe interactions in mice. However, little work has been done to understand how chronic exposure to environmental pollutants can impact the microbiota or physiology of primary consumers in aquatic ecosystems. To address this gap in knowledge, we are studying Chinese Mystery Snails (CMS) collected from three lakes in the Puget Sound region: Lake Killarney (20 ppm As), Steel Lake (2 ppm As), and Pine Lake (trace As). We hypothesized that snails exposed to higher concentrations of As are subject to greater levels of physiological stress than those with lower levels of exposure, and that this physiological stress is impacted by the microbiota of snails living in each environment. We measured relative levels of HSP70 expression in snail gut tissues to determine usefulness as a biomarker in our study. We also used inductively coupled plasma mass spectroscopy to determine the amount of arsenic in the tissues of CMS harvested from each lake. Next, we are isolating DNA from snail guts, water, sediment, and plants from each lake. The purified DNA will be subjected to 16S rRNA amplicon sequencing, allowing us to determine the relative abundances of bacterial taxa in each environmental compartment and the overlap between the snail gut vs local environment. Our future research will focus on validating additional stress induced biomarkers, while assessing microbiome alteration linked to the biomarker(s). This work will lay the foundation to future studies focused on understanding the links between arsenic exposure, chronic physiological stress, and microbiome composition.