Enzymes have recently been incorporated into multiple high-value industrial syntheses, demonstrating the utility of enzymes as highly selective catalysts for practical industrial processes. However, the current scope of non-biological enzymatic reactions is narrow and new reactions and reaction pathways need to be engineered. The goal of our work in the Zalatan lab is engineering enzymes as catalysts in carbon-hydrogen bond functionalization reactions, a transformation critical for practical industrial synthesis where selective catalysis is still a major challenge. Importantly, we are interested in exploring more efficient and informed engineering approaches by establishing structure-function relationships with the enzymes that we work with. Our model system for this work is the non-heme iron(II) 2-oxoglutarate dependent oxygenase superfamily (Fe(II)-2OGs). We are using a high-throughput microfluidics based kinetic assay to determine key sites that we can target for mutagenesis and directed evolution in a candidate Fe(II)-2OG found to catalyze a new reaction. Overall, we expect that this work will enable new directions and principles for engineering Fe(II)-2OGs, and that lessons learned here can then be extended to additional industrially-relevant enzyme families.

Irritable bowel syndrome (IBS) is a disease that affects many people worldwide. Clinical trials using probiotic bacteria to treat IBS show promising outcomes, although the molecular mechanisms of probiotics and their effect on gut health is not completely understood. Multiple studies comparing different strains of probiotics show Lactobacillus plantarum and Bifidobacterium longum as the most effective strains in inhibiting the release of proinflammatory factors in the gut and promoting proliferation of healthy gut bacteria. The purpose of this study is to evaluate the protein profile of probiotic bacteria Lactobacillus plantarum and Bifidobacterium longum in both healthy gut and IBS gut conditions. To observe how Lactobacillus plantarum and Bifidobacterium longum respond to different simulated gut conditions, samples of the bacteria will be isolated from probiotic supplements. These samples will be grown in aliquots representing different simulated anaerobic gut conditions for 12-16 hours at 37°C. Lactobacillus plantarum and Bifidobacterium longum will be grown separately and grown combined within these conditions. To enrich secreted proteins, the supernatant from each aliquot sample will be clarified using centrifugation. Secreted proteins will be solubilized and denatured with sodium deoxycholate. The denatured proteins will be reduced with dithiothreitol and alkylated with iodoacetamide. The sample will be diluted using ammonium bicarbonate and digested by trypsin. Tryptic peptides will be analyzed using liquid chromatography and mass spectrometry. Tryptic peptides will be identified using the SEQUEST search engine with a reference database. I anticipate the probiotic strains will demonstrate differential proteomics when isolated and combined in the healthy gut and IBS gut conditions. The secreted protein analysis completed in this study will further the understanding of the probiotic molecular mechanism of promoting gut health.

Standard diagnostic tests for common bacterial infections such as strep throat are invasive and uncomfortable, especially for children. Difficulty performing these tests may delay or prevent diagnosis, which leads to more severe consequences, such as secondary conditions or organ damage. To address these barriers to care, we developed the CandyCollect, a novel saliva collection platform that is child-friendly, effective at pathogen collection, and suitable in at-home and clinical settings.The purpose of this study is to test the feasibility of our device’s bacteria capture for home use in healthy people. Staphylococcus aureus and Streptococcus mutans are common oral bacteria that are targeted in this study to assess our device’s ability to capture and detect bacteria. The findings in this study will inform our ability to use the CandyCollect for the bacterial pathogen responsible for strep throat, Streptococcus pyogenes, in future studies. We initally recruited 5 participants (>18 years) nationwide to test our CandyCollect device against oral swabs and spit tubes to compare the efficacy and user feedback of CandyCollect with traditional methods of saliva collection. The study is continuing with additional participants. qPCR analysis and fluorescent imaging were used to detect the target oral bacteria in collected samples. Responses from the user feedback survey indicated that the majority of the participants were satisfied with the design and were optimistic about implementing this device for children. Future applications include quantitative determination of bacteria concentration as well as targeting S. pyogenes. We aim to expand this human subjects study to recruit younger participants, especially children (> 3 years), which will help us achieve the ultimate goal of delivering a comfortable saliva collection platform to pediatric patients.

Test questions are commonly written with the intent to assess a student's higher level thinking skills, but the phrasing of a question may not align with this intention. One way to remedy this is to assess the cognitive complexity of existing test questions, and use these findings to reform instructional materials and subsequent examinations. We used Marzano’s Taxonomy to characterize a bank of multiple-choice exam questions from general chemistry by the level of cognitive complexity required of students' thinking. Marzano’s Taxonomy has been applied to introductory physics questions to characterize quantitative thinking and problem-solving skills (Teodorescu et al., 2013), and to general chemistry curricula to guide course and assessment development (Toledo & Dubas, 2016). Here we will describe our implementation of Marzano's Taxonomy in the context of large-lecture general chemistry, and our development of a rubric that chemistry instructors can utilize to evaluate their own exam questions. The development team included an undergraduate biology major and former general chemistry peer mentor; a chemistry graduate student and experienced general chemistry teaching assistant; and a chemistry faculty member. When applied to multiple choice questions, we found that the cognitive level of most questions was lower than expected, even for lengthy questions that are normally considered challenging by students and instructors alike. We will discuss ways to increase the cognitive complexity of problems to allow instructors to elicit the intended level of thinking from the student, and to align the cognitive levels of assessment and instruction. This can also improve the clarity of expectations of the cognitive complexity that is required of the student and promote higher-order thinking.

Metal–organic frameworks (MOFs) are crystalline, porous extended solids that are formed through coordination between metal cations and bridging organic ligands. Since their discovery in the late 1990s, MOFs have been a topic of acute interest in the scientific community due to their intrinsic porosity, high surface area, and precise tunability. However, MOFs are typically insulating, which limits the scope of their applications. The recent development of electrically conductive MOFs has opened the door to exciting multifunctional applications in electrocatalysis, advanced electrochemical energy storage, chemical sensing, and much more. However, a molecular-level understanding of charge transport in MOFs remains lacking. My research aims to address this knowledge gap through the investigation of one-dimensional (1D) metal organic chains. These 1D chains can be thought of as the primary subunit of higher-dimensional MOFs; they allow for high synthetic and electronic tunability, making them ideal model materials for studying the genesis and tuning of electronic properties in conductive MOFs. Here, I will present the synthesis of a series of highly-tunable 1D metal–organic chains that exhibit delocalized π systems and high electrical conductivity along with our studies of how structural parameters such as metal identity, chain structure (linear vs. zig-zag), and metal/ligand oxidation state can influence the overall electrical and magnetic properties of the resulting chain.

Microfluidics is the technology of systems in which microscale channels are used to manipulate small quantities of fluids (microliter to picoliter or less). Open microfluidics provides a platform to control the movement of microscale volumes of liquid in open space, making every position more accessible than conventional closed microfluidics. In many applications–including cell culture, chemical synthesis, and high throughput screening–merging droplets is essential for the experimental workflow. For example, merging droplets containing reagents can initiate a chemical reaction, or adding a drug to a cell culture can stimulate cells. In conventional closed droplet-based microfluidics, merging techniques often rely on external components such as merging by electrofusion using electrodes. In other cases, like merging using hydrophilic strips in a channel, the merging method is built into the device and the location of merging must be determined in advance. Therefore, the ability to easily initiate droplet merging in an open microfluidic system would be beneficial to researchers and offer flexibility in experimental design and applications. In this study, I developed an easily accessible droplet fusion technique with the prick of a needle at the liquid interface; my method fuses multiple droplets simultaneously (2-15 droplets). Importantly, this fusion method can be used on-demand at any point in the open microfluidic device and does not require external equipment nor features built into the device. This presentation characterizes the experimental parameters required for successful and controlled droplet fusion and explores the physics behind this phenomenon.

The Wnt signaling pathway plays a critical role in mammalian cell development and regulates cell growth and differentiation. Two central proteins in this pathway are glycogen synthase kinase 3β (GSK3β) and β-catenin. Another protein, Axin, is responsible for holding these two proteins in close proximity in order to promote the reaction between them. The kinetic mechanism for the Axin-mediated reaction is well understood, but the relationship between this function and Axin’s structure is poorly characterized. I am currently generating a cryogenic electron microscopy (Cryo-EM) structure of Axin bound to GSK3β and β-catenin in order to investigate this mechanism. Cryo-EM is a technique for determining the structures of proteins that is especially suitable for large protein complexes, such as the one I have produced. Inital results indicate that the stoichiometry of the complex is much more complicated than initially assumed and does not follow the predicted 1:1:1 complex. Elucidating this structure will provide insight into how the structure of Axin promotes the reaction between GSK3β and β-catenin as well as how it provides Wnt signaling specificity. This structure will also be important for understanding and intervening in diseases such as cancer where Wnt signaling is dysregulated.

Since the beginning of the COVID-19 outbreak in 2020, many students have participated in online learning. Online students spend many hours every day studying at home in indoor spaces that lack ventilation and may have hazardous air quality. Prior research conducted by Tyler Jacobsen et al, (2019) reveals that CO₂ concentrations over 1,000 parts per million (ppm) can increase physiological stress and lower cognitive abilities. The goal of this project was to determine if the CO₂ levels in the at-home study environment of a student conducting online learning are higher than those of a student conducting in-person learning during study times. I predicted that if a student participates in online learning, then the CO₂ levels in their study environment will be higher than those in the study environment of a student conducting in-person learning during school hours (8 am-3 pm), but the levels will be similar during homework hours (4 pm-5 pm). A CO₂ air monitor collected data in the study rooms of an online learning student and an in-person learning student. Over several days, I recorded data every hour from 8 am to 5 pm, resulting in a time series showing CO₂ concentrations throughout a school day. Preliminary data reveals that the online student’s room consistently exceeds 1,000 ppm and varies greatly throughout the day, while the in-person student’s room remains below 1,000 ppm until they arrive home at 4 pm, at which point levels increase significantly. I only observed two locations during this quarter-long research study in General Chemistry and I need to collect data at more locations to determine if this study’s findings are representative of online learning versus in-person learning as a whole. Findings may be useful to educators when they are deciding if online education is a viable option for students in the future.

The construction sector has always had a significant influence on CO₂ footprints in a given area (Pomponi, 2021). However, there are no recent studies quantifying the effect of construction activities on localized CO₂ concentrations. Previous research conducted in the early 1990s has shown that construction increases CO₂ concentrations by over 11% (Mazria, 2018). However, this research was done over three decades ago, and since then, CO₂ concentrations have increased globally over 40%, necessitating updated measurements. My research group and I will investigate construction sites of various degrees in the Puget Sound region to determine the effect of construction activities on local CO₂ concentrations. Preliminary data has shown an increase of 140 ppm (±8 ppm) in CO₂ concentration when implementing a method of measuring CO₂ concentrations in regions of high and low construction activity within urban, suburban, and rural areas. This research is key to understanding the health implications of the increased construction within the Puget Sound region and its effects on our ecosystems.

Chronic inflammation in the lung often leads to airway remodeling, which can worsen symptoms in inflammatory diseases such as asthma. Airway remodeling is attributed to the excessive deposition of the extracellular matrix (ECM) by myofibroblasts, which are a differentiated form of fibroblasts. Eosinophils, when activated by interleukin-3 (IL-3), release certain soluble factors that were found to be associated with inflammation in asthmatic tissues. Hence, it is crucial to study cellular communication in airway remodeling to facilitate the development of treatments. The aim of this project is to establish an in vitro model of asthma by coculturing primary human lung fibroblasts and eosinophils to study the soluble factors that trigger airway remodeling. We hypothesize that IL-3 activated eosinophils, when immunoglobulin (IgG) is added, release soluble factors that trigger the gene expression and phenotypic changes in fibroblasts. The coculture device has two chambers, in which two types of cells can be cocultured in the shared media while being physically separated by a half wall. Eosinophils are seeded in the outer chamber of the devices and degranulated. The differentiation of fibroblasts would then be quantified by utilizing immunocytochemistry to see the differences in expression levels of alpha smooth muscle actin (É‘SMA) in fibroblasts, in addition to quantitative polymerase chain reaction (qPCR) to detect messenger RNA (mRNA) level associated with inflammation and tissue remodeling. The initial experiments were focused on the monoculture of fibroblasts to ensure that reliable readouts can be obtained from fibroblasts before initiating the coculture. The preliminary data suggest that the fibroblasts treated with transforming growth factor beta 1 (TGF-β1), which promote differentiation, result in significantly higher expression of É‘SMA. Our future experiments include initiating the coculture of eosinophils and fibroblasts to fully illustrate this crucial cellular communication in airway remodeling.

 Quantum dots are semiconducting nanomaterials that are useful in converting solar energy because of their high absorption and tunable photophysical and chemical properties. They can have various organic ligands bound to their inorganic surface, creating quantum dot-ligand systems useful for charge and energy transfers. When a quantum do is excited by external radiation, an electron inside the dot is promoted to a higher energy level and can be extracted form the quantum dot through the ligands surrounding the dot. We hypothesize that by binding the charge accepting ligand directly to the quantum dot, charge extraction from the quantum dot will be promoted. In my experiments, I am trying to determine the extent of ligand binding to the quantum dot and how this binding is affected by the identity of the binding moiety. To do this, I am synthesizing cadmium sulfiide quantum dots and attaching various ferrocene derivatives through a titration experiment. I analyze each step of the titration experiment for each quantum dot-ligand system with cyclic voltammetry (CV), an electrochemical technique that is sensitive to the diffusivity of the charge acceptor. The resulting CV curve can then be modeled with DigiElch to extract the favorability of the binding of the ligand to the quantum dot. Based on the magnitude of the equilibrium coefficients, we can determine which ligand binds most favorable to the quantum dot and compare this to the efficiency of charge extraction. This research has many applications, from solar energy to medical imaging, and the ability to extract charge outt of the quantum dot in an efficient and quick manner is one worth researching. This research is supported and overseen by Prof. Brandi Cossairt, and graduate students, Micaela Homer and Florence Dou. 

Polymers are important in all areas of life from commodity plastics to vaccine delivery. Currently, plasticizers are small molecules used to make rigid polymers into the flexible plastics we need. However, the problem with these plasticizers is that by disrupting intermolecular polymer chain interactions, they can degrade the polymer over time and leach out into the environment. When plasticizers escape, they can be toxic to human health and harmful to the environment. By inserting a plasticizer into the backbone of the polymer rather than into the polymer solution, we reduce the risk of toxic molecules escaping the polymer. To solve this problem, a fluxional molecule such as bullvalene can be inserted into the backbone of the polymer. Bullvalene is a small molecule and due to its fluxional property, it will induce flexibility into the polymer as an internal plasticizer. Bullvalene is considered fluxional because it can undergo Cope rearrangements at room temperature. The purpose of this research is to develop the methods for synthesizing a novel polymer and to analyze its chemical and mechanical properties such as glass transition temperature and tensile strength. My project focuses on polycarbonates, a specific class of polymers. Polycarbonates are in many plastic materials such as water bottles and are increasingly important for consumer electronics. The results will have positive implications for human and environmental health by providing methods to develop safer plastic material.

The rapid rise of bioanalytical testing has renewed discussions in improving the collection of bacteria through oral sampling. Current diagnostic tests for common respiratory illnesses such as strep throat are invasive and uncomfortable, especially for children. Undiagnosed, these treatable diseases can cause serious damage. The Theberge lab has created the CandyCollect, a saliva sampling device intended to be a child-friendly alternative to current sampling techniques for home and clinical settings. The CandyCollect utilizes candy in the design to appeal to children, a unique component that requires additional testing and standardization to be appropriately implemented. This study explores the intersectionality of safety, design, effectiveness, and ease of application by using candy to increase children's compliance and act as a built-in timer. Our goal is to modify the existing device with these objectives in mind. Several types of candy were made using different ingredients, flavors, and textures, and the interaction of these candies with oral bacteria and saliva was investigated. We engineered the device using rapid prototyping, computer-numerical-control (CNC) milling, and silicone mold development. These methods allow for the flexibility to modify the design of the candy and sampling device. We then designed a survey for a human subjects study for adults (>18 years) to receive useability feedback and adapt the device components. Our analysis of these results determines the variety of candy that is most suitable for large scale use. The findings of this study will offer an improved method for child diagnostics and encourage other industries to redesign traditional sampling procedures. 

T cell activation is an essential element of the adaptive immune response. When the T-cell antigen receptor (TCR) interacts with a peptide antigen bound to a major histocompatibility complex (MHC), lymphocyte-specific protein tyrosine kinase (Lck) phosphorylates the CD3ζ chains of the receptor complex, initiating an intracellular signaling cascade. Therefore, Lck plays a crucial role in TCR signal transduction and thus Lck’s kinase activity and scaffolding function are tightly regulated by intramolecular protein-protein interactions (PPIs). Regulation is conferred by phosphorylation of two tyrosine residues on Lck, leading to conformational changes. When the C-terminal tail tyrosine is phosphorylated, the alignment of catalytically necessary residues is perturbed, rendering an inactive closed conformation. However, when the C-terminal tail is dephosphorylated, and the activation loop tyrosine is phosphorylated, Lck adopts a catalytically active open conformation. Using its regulatory domains, Lck also functions as a scaffold. First, Lck uses its tyrosine kinase domain to phosphorylate the kinase ZAP-70. Then, Lck bridges ZAP-70 to LAT by binding both proteins using its regulatory SH2 and SH3 domains. To study the intramolecular regulation of Lck’s kinase activity, we performed a Deep Mutational Scan (DMS) in yeast to measure the activity of ~5,000 single amino acid variants of Lck. Because Lck’s kinase activity is toxic to yeast, we can infer information about Lck’s kinase activity from changes in abundance of yeast expressing individual Lck variants, relative to wild type, over time. Specifically, we analyze the mutations along the SH2-SH3/kinase domain PPI which we hypothesize perturb the accessibility of Lck’s SH2/SH3 domains. In Lck variant expressing T cells, we expect to see differences in Lck’s binding partners due to differences in Lck’s SH2 and SH3 domain accessibility. This research will help us better understand Lck’s role in T-cell activation, with the long-term vision of leveraging Lck variants in next-generation T-cell therapies.