In trying to understand biology’s dynamic heterogeneity, scientists have sought to recapitulate the spatial complexity and temporal presentation in which proteins are naturally presented to cells. Currently, the most promising strategies in this regard exploit sequential ligation/cleavage reactions, each controlled in time and space using light so as to reversibly immobilize proteins within synthetic biomaterials. Though our lab has utilized these approaches to spatially control complex biological fates with micron-scale resolutions, previous methods suffer from complex syntheses, as well as requirements for specialized equipment and skillsets rarely available in bio-based laboratories. Improving upon these fundamental limitations, our group has developed a scalable system wherein proteins can be bound/released from hydrogels using light, without the need for such expertise/equipment, by being fully genetically encodable. In this approach, biology performs all the modifications necessary to photopattern protein binding to gels, as well as install the reactive species requisite for the protein’s photo-mediated release. We have accomplished this using a photoactivatable protein-peptide ligation reaction developed by our lab, wherein UV irradiation “activates” the protein to ligate specifically with the peptide tag. Additionally, we exploit co-translational chemoenzymatic modification strategies to install a functional handle for tethering the protein into polymeric hydrogels during protein expression. To the peptide tag, we append a photocleavable protein that cleaves when irradiated by visible light, fused to a protein of interest (POI) to be tethered to the hydrogel. Expressing these proteins in E. coli yields the first-ever fully genetically encodable system which can reversibly pattern proteins into hydrogels, by first shining UV light to tether POIs into biomaterials, then subsequently shining visible light to photocleave the protein and trigger POI release. Highlighting the system’s versatility, we demonstrate that the approach is compatible with fluorescent proteins and bioactive growth factors to direct 4D cell fate.

Water-swollen polymeric networks (i.e., hydrogels) provide a structural platform for the manipulation of chemical and mechanical signals that mimics the complex heterogeneous environment experienced by cells in vivo. Photoresponsive chemistries have been of particular interest to this end, as they allow for precise spatiotemporal control of physiochemical properties and, thus, cell behavior. Here, we present a novel protein-based network that will allow for the photo-mediated stiffening of genetically-encoded hydrogels. In this system, we exploit a biochemical technology recently pioneered by our lab in which two pairs of proteins undergo irreversible, covalent heterodimerization after photoactivation. Through the incorporation of an inert, unstructured polypeptide backbone, we have exploited the aforementioned reaction to induce gelation in response to light through the formation of four-arm protein crosslinks. Unlike previous synthetic polymer-based hydrogel systems, this system is entirely genetically encoded, which provides significant advantages in terms of cost, time, and production simplicity. As we intend to demonstrate through photorheometry, this reaction proceeds in a dose-dependent manner, providing step-wise control of both where and when gel stiffening occurs. Such 4D control of a gel’s mechanical properties can be used to influence cell migration, growth, and differentiation, and, thus, could have applications in tissue engineering. Furthermore, we anticipate our system could be utilized to model the stiffening of the extracellular matrix, which is commonly associated with pathologies such as cancer, fibrosis, and cardiovascular disease.

The growing field of disaster resilience research deals with the complex and essential question of how we can prepare for and recover from disruption. Food assistance organizations have the potential to play an essential role in supporting communities after disaster events, especially for low-income residents. The goal of this study is to explore the factors that contribute to the resilience of Seattle’s food assistance organizations. My research will address how these organizations maintain food availability, acceptability, accessibility and stability before, during and after a disaster. I will conduct key informant interviews with representatives from food-assistance organizations, in-person and over the phone. I will sample all food assistance organizations from the Seattle areas of Cedar Park/Meadowbrook, University District and the Duwamish Waterway (South Park, Delridge and High Point). These include food banks and food pantries, meal centers, government assistance programs and school meal programs. To contribute to qualitative trustworthiness, a summary of key points gleaned from the interviews will be sent to all participants after their interview and they will be asked to verify that it represents what they wished to convey. I will analyze the interviews by coding for and synthesizing common themes in the transcripts using Nvivo software. I hypothesize that many of the organizations will cite strategies for maintaining day-to-day food security for their clients with less specific focus on disaster preparedness. I also hypothesize that infrastructure, funding, and food donations will be barriers to resilience while community partnerships, staff and volunteers will be factors contributing to resilience. Results from this project may inform Seattle's disaster planning efforts. 

Group B Streptococcus (GBS) is a gram-positive bacterium which causes nearly 150,000 stillbirths and infant deaths per year globally. About one in five pregnant women carry GBS, a major cause of infant disease. GBS encodes an extracellular enzyme known as Hyaluronidase, which increases the severity of GBS infection and is associated with adverse outcomes such as preterm labor. In this project, we investigated how the GBS Hyaluronidase enzyme suppresses the host pro-inflammatory response in the presence or absence of two crucial extracellular immune response receptors (Toll-Like Receptor(TLR)-2 and TLR4) . While both TLR2 and TLR4 receptors bind to pathogens and pathogen associated molecular patterns (PAMPs), TLR2 is specific to recognizing gram-positive bacteria such as GBS and plays a critical role in initiating the gram-positive pro-inflammatory response. Dendritic cells and macrophages - key components of innate immune defense - were generated from mice that included the wild type (WT) and those lacking either TLR2 or TLR4 or both TLR2/TLR4 . These immune cells were then treated with GBS (GBS WT) or a mutant strain lacking Hyaluronidase enzyme (GBS DhylB). Post GBS infection, host cell supernatants were analyzed for the production of pro- and anti-inflammatory cytokines using multiplex cytokine arrays. Fluorescence microscopy and quantification of bacterial colony forming units were used to determine the extent of bacterial uptake and immune cell death. We expect that GBS Hyaluronidase may suppress pro-inflammatory cytokine production in a TLR2/4 dependent manner in dendritic cells, similar to previous observations with macrophages. Data from this project can elucidate details about the GBS immune response pathway, and may inform the development of therapeutic strategies to prevent Group B Streptococcal infections, stillbirths, preterm births and early onset neonatal sepsis.

In a given year, a combined surplus of 20,000 transplants are performed in the US for patients requiring a new kidney, liver, or heart, and the need for these organs continues to increase rapidly with changes in societal and cultural outlooks on personal behavior. Recent regenerative medicine techniques have been implemented in attempts to create engineered tissues that support solutions to these problems, yet they are limited to thin or avascular tissues. In order to create thicker tissue constructs for implantation, vascular networks must be introduced to supply nutrients and oxygen to highly metabolic tissues. Yet, current methods can be expensive or require high-tech equiptment. To address this issue, this project aims to design a construct that integrates two vascularization techniques into a multilayered tissue. This new design of a thicker tissue will benefit from the advantages of both independent systems, endothelial cords and perfusable, patterened microvessels, advancing the tissue engineering field.