Non-tuberculous mycobacteria (NTM) are ubiquitous in the environment and survive in various settings such as soil and water. The purpose of this project is to evaluate the ability of humoral responses produced from NTM pre-exposure on the prevention of Mycobacterium tuberculosis (M.tb) disease progression in mice. Additionally, we want to determine if antibody responses from immunization with bacille Calmette-Guerin (BCG) and/or candidate tuberculosis subunit vaccines contribute to protection against M.tb. To test this, we collected serum samples from mice that received various combinations of treatments before and after infection with M.tb. Groups either received NTM in the drinking water or normal drinking water, with or without BCG vaccine, and with or without a candidate vaccine or a combination of the treatments. M. avium ATCC 25291 NTM-lysate was used as an antigen for ELISAs and it was created by growing the mycobacteria and lysing the bacterial cells by multiple freeze-thaws. NTM-specific total IgG ELISAs were performed on serum samples following different vaccine strategies. Before M.tb infection, mice vaccinated with a candidate subunit vaccine regardless of NTM exposure or BCG vaccination had a significantly higher total IgG response. After M.tb infection, mice that received immunization with BCG followed by the candidate vaccine had significantly higher NTM lysate-specific total IgG responses compared to the saline group, however, this was observed only in NTM unexposed mice. NTM-exposed mice had equivalent levels of NTM-specific total IgG in the saline group when compared with the different vaccine regimens. This suggests that NTM exposure is able to produce potent NTM-lysate total IgG antibody responses in unimmunized ‘saline’ control mice, which suggests that the mice were likely boosted following M.tb infection. Given these findings, we plan to investigate whether the NTM-specific antibody response is reducing vaccine-mediated protective responses against M.tb.
 

The COVID-19 pandemic spotlighted Asian American (AA) mental health and their foreign position, underscored by anti-Chinese rhetoric and policies and the concurrent surge in hate incidents, crimes and xenophobia against AAs. However, framing COVID-19 as the sudden cause of anti-Asian hate facilitates misconceptions about AAs: it undermines a storied history in which AAs have long survived and challenged their treatment as “forever foreigners.” To understand the extent to which anti-Asian hate and mental health challenges among AAs during COVID-19 was a unique event in AA racialization, it is paramount to understand AA marginalization and its implications for mental health prior to COVID-19. To do so, I use the 2016 Collaborative Multiracial Postelection Survey (CMPS). The survey gathers data on respondent attitudes across a diverse set of political, psychological and socio-demographic measures and is ideal for Asian ethnic subgroups and intersectional analyses of gender, age and immigration generation. My analysis includes bivariate and multivariate regression analyses using the sample of AAs (N= 3006). Thus far, I have found that a substantial proportion of AAs indicate low feelings of belonging in the U.S., personal experiences of racial discrimination, and depressive symptoms. For the project’s next step, I will examine the extent to which AAs share a common foreign experience prior to COVID-19 and how this contributes to depressive symptoms. I expect that there will be significant subgroup differences at the intersections of ethnicity, gender, and immigration generation in the relationship between experiences of racial discrimination, feelings of belonging, and depressive symptoms when community involvement and local contextual factors are considered. This work helps situate anti-Asian hate during the pandemic as part of a more extensive history of racism in the U.S. and broadly, contributes to more informed mental health services by highlighting the implications of racism for mental health.

This study explores the adaptive dynamics of Pseudomonas aeruginosa (P. aeruginosa), a bacterium that often plagues cystic fibrosis patients. In P. aeruginosa, many genes are regulated through quorum sensing (QS), a form of cell-to-cell communication. In wild-type P. aeruginosa, strain PAO1, LasR acts as a QS regulator, controlling several genes, including those encoding proteases, as well as two other QS regulators, RhlR and PqsR. QS-regulated proteases are secreted by P. aeruginosa into the environment, benefiting the whole population– a cooperative process. When grown in conditions requiring proteases for growth, such as on casein, mutated P. aeruginosa emerge that are capable of using proteases without producing any themselves. While disadvantageous to the community, these “cheaters” experience a fitness advantage. Over time, the proportion of cheaters increase until the population cannot support itself and collapses. Related to QS is the transcription factor MexT, that, when active, down-regulates RhlR. Another transcription factor, MexS, down-regulates MexT expression. In PAO1, MexS is inactive, resulting in constitutively expressed MexT. I was interested in how variants of MexT and MexS that alter expression of QS regulators might influence cooperative behaviors in P. aeruginosa. I compared survival and function of wild type PAO1 to two engineered strains, one with restored MexS function (MexS+) and one with MexT deleted (ΔMexT). I passaged these strains in casein broth for 30 days, evaluating every five days for the emergence of protease-negative mutations. I am also analyzing the genetic composition of each population, focusing on categorizing lasR and a gene involved in peptide transport, psdR. Preliminary results show fewer mutations in psdR than expected in all strains, and fewer mutations in lasR in the ∆mexT strain. Continued exploration of the complex QS and gene regulatory pathways in P. aeruginosa may point to mechanisms of virulence and to potential targets for antibiotic therapies.

Current technology to control 3D cell function takes advantage of bioorthogonal photochemistry to immobilize proteins into materials by using photocages—photoremovable molecular groups that block protein activity. However, diffusion limitations necessitate patterning times ranging from hours to weeks, far longer than the timescales of many biological processes. Protein activation aids signaling events within the extracellular matrix (ECM), leading to downstream changes in cell fate and physiological responses in our bodies. In order to probe and investigate biological systems, hydrogel materials provide an ideal synthetic platform, due to their polymeric, water-swollen characteristics that mimic the native ECM. In this project, I will use light to control the spatial and temporal presentation of biochemical cues through the photoactivation of proteins within hydrogels. We hypothesize that the kinetics of the protein activity between solution and biomaterial studies should correlate, given their dose-dependent response to light exposure—by varying the intensities of light and time intervals of exposure. By characterizing the photoactivatable protein system and controlling protein activity, we intend to use this platform to photoactivate biologically relevant proteins to control signaling that occurs on shorter time scales, applicable to biochemical processes.

 Injectable biomaterials can be used for targeted, local delivery of drugs and small protein factors. However, designing a biomaterial that is self-healing, bioresorbable, highly-defined, and allows for the independent release of multiple protein drugs remains a challenge. Interpenetrating network (IPN) hydrogels formed by physical crosslinks provide a solution as they combine the properties of two networks into one material, as well as form gels that are injectable and self-healing. Our project aims to produce the first ever reported fully recombinant protein-based IPN hydrogel that can be used to deliver multiple drugs simultaneously. Recombinant protein-based polymers are advantageous for forming biomaterials due to their bioactivity, biocompatibility, and tunability. Each biopolymer contains coiled-coil proteins linked by a flexible hydrophilic linker that is bisected by a click-like chemistry protein system, known as a polyproteam. These polyproteams allow drugs or protein factors to be covalently ligated to the networks, so that drug delivery is dependent on material erosion instead of diffusion. We characterized the material’s properties using rheometry to measure material stiffness, percent strain at which the material liquifies, and self-healing behavior. Then, we tested the ability of our hydrogel to deliver drugs by ligating epidermal growth factor and insulin growth factor to the hydrogel and measured the release of protein over time through a Western Blot. The bioactivity of the growth factors was confirmed by culturing with human mesenchymal stem cells (hMSCs) and using a Western Blot to measure activation of the ERK signaling pathway. If successful, the IPN hydrogel can be used for clinical applications, as a tool for drug delivery.

Bioconjugation, or the covalent linkage between a biomolecule and another chemical group, creates hybrid "conjugates" that exhibit the properties of both biomolecules and exogenous moieties. The N-termini of proteins often fall outside of their final fold, making the N-terminus an optimal site for conjugation while preserving a protein’s native folding and bioactivity. Consequently, N-terminal modification of proteins and peptides has been a long-standing goal in fields like drug delivery, biotherapeutics, and cellular imaging. However, the current techniques for N-terminal protein conjugation are limited by either the introduction of bulky protein assemblies at the conjugation site, the need for multiple costly and complicated steps, or low site selectivity. In this project, we aimed to develop an improved route for N-terminal bioconjugation. We created a generalizable platform for single-step purification and near-scarless N-terminal bioconjugation of proteins by leveraging the chemistry of the atypically split intein VidaL. To evaluate the effectiveness of our platform, we first examined the kinetics and reaction conditions of VidaL bioconjugation, confirming its ability to modify the N-termini of proteins successfully and selectively. Then, we used our platform to conjugate an alkyne, biotin, or FAM-biotin moiety to the N-termini of fluorescent proteins (EGFP and mCherry), a model enzyme (beta-lactamase), and a model growth factor (EGF). Through measuring fluorescence and conducting nitrocefin and proliferation assays, I found that, regardless of the moiety added, bioconjugation did not impact the native function or activity of these proteins. In the future, we expect that this platform's ability to easily N-terminally bioconjugate proteins with minimal impact on their functionality will find use across the growing fields of applied chemical biology.

Biological processes depend on the intricate functions of proteins, which serve as the driving force in vital reactions. Given the importance of proteins in biological processes, several methods have been developed to control their functions in biomaterial and in vitro. Current methods often rely on secondary fixatives and caustic chemicals, posing risks of functional impairment in target proteins. To address these limitations, my project focuses on modifying cells with non-endogenous machinery, enabling expression of photoactivatable proteins – those with photocaged amino acids installed at essential locations, and using them to optically stimulate protein function. This strategy enables the triggered activation of target proteins with light, mitigating the risks associated with chemical interventions. We predict that the degree of protein activation can be controlled in a dose-dependent manner by manipulating light exposure duration and intensity. Genetic code expansion is used to integrate noncanonical amino acids, site specifically, into proteins expressed in bacteria and mammalian cells. By incorporating these photoactivatable proteins into biomaterials and living cells, our research aims to gain insights into fundamental cell signaling processes through control of targeted proteins. By analyzing biological mechanisms at the molecular level, we can guide cell processes and make informed decisions regarding cellular behavior and fates.

Current trends indicate that while adolescents and young adults are starting to engage in sexual activity later in life and less frequently, there is a notable shift towards experiences characterized by low commitment and higher risk. Despite numerous studies on sex-related attitudes and practices among young people in the United States, broader insights are necessary to achieve a holistic understanding of the newly emerging landscape of youth sexuality. Against this background, our research examines sexual attitudes and reported behaviors in a sample of 1,200 undergraduate students at the University of Washington. Our study employs a comprehensive survey to identify patterns in sexual attitudes and behaviors among a representative sample. Administering online questionnaires is crucial in ensuring increased reliability and validity of responses on sensitive topics. These include but are not limited to, pornography viewership, kink and fetish interests, sexual self-concept, and more. Preliminary findings indicate a complex interplay between cultural, educational, and social factors in shaping sexual norms and practices among young adults. This study contributes to the broader discourse on sexual health education and highlights the need for nuanced approaches in addressing the dynamic and multifaceted nature of young people's sexual relationships. The results of this investigation aim to inform the foundation of modern sex education in order to address the evolving challenges of today's sexual landscape.

The 2021 Heat Dome represented a historic climate event for the Pacific Northwest, resulting in 159 excess deaths in Washington State alone. Within this region, public health departments and other government agencies regularly warn the communities they serve about upcoming extreme weather events such as extreme heat, to encourage people to take preventive actions that protect their health. Social media and online communications are some of the media commonly used to disseminate these advisory messages. From June 2023 to September 2023, we captured social media and web communications about extreme heat shared via city and county-wide government sources in Multnomah County, OR, King County, WA, Spokane County, WA, and Ada County, ID. Sources included Facebook, Twitter, Instagram, and official agency websites. We then used qualitative content analysis methods to assess the types of messages that were published, as well as differences across jurisdictions, message timing, and the frequency of reposting/sharing the same content, across organizations in the same jurisdiction. Entries were coded using NVivo qualitative analysis software. Some notable themes found in this content review included risk communication (e.g., identifying highly vulnerable populations); heat-related health symptoms and interventions for heat exhaustion and stroke; promoting formal and informal cooling centers, and communicating through languages other than English. Preliminary findings across Multnomah County, King County and Ada County indicate that Multnomah County’s messages frequently targeted high-risk populations through messages that promoted free transportation to/from cooling centers and by posting in four or more languages. These findings can be used to determine the feasibility of using social media and website communications to assess the implementation of public health extreme heat messaging on a large scale. Future research should explore the effectiveness of these communications in prompting behavioral changes and in reaching vulnerable subpopulations with targeted recommendations.