My research project involves the AP-3 (adaptor protein) complex, which plays a key role in membrane trafficking within cells. Vesicles (small, membrane bound structures) mediate the transport of proteins and lipids among cellular organelles. These vesicles are created in various ways by proteins throughout the cell, with some including a protein "coat" around the vesicle as it travels to its destination. AP-3 is a coat protein complex that mediates vesicular transport from the trans-Golgi network to the lysosome. We have utilized a gene reporter system called GNSI to analyze AP-3 function in different genetic backgrounds of Saccharomyces cerevisiae (baker’s yeast). One output of the reporter system is a colorimetric assay that measures the intensity of colored halos around yeast colonies grown on an agar plate. We found that targeted truncations of proteins of the AP-3 subunits Apl6 and Apl5 at the C-terminus of AP-3 (specifically the “ear domains”) resulted in increasing defects in AP-3 trafficking ability. However, it is unknown whether the defects are in membrane recruitment to the trans-Golgi network, or whether recruitment occurs but vesicle budding defects arise. Therefore, the current aim of the project is to begin analysis of the truncation defects in AP-3 by fusing Apl5 trunctions with a C-terminal mNeonGreen fluorescent protein and use live cell fluorescence microscopy to further examine AP-3 localization These basic studies will further our understanding of membrane trafficking and may provide insight into diseases linked to AP-3 function, including HIV-1 particle assembly and human genetic disorders.

Newly developed, rational syntheses of the phosphaethynolate anion have taken it from an academic curiosity to a useful synthetic reagent. Since the new state-of-the-art synthetic method was published by Grützmacher et al. in 2014, many studies have emerged investigating the phosphaethynolate anion’s possibilities as a synthetic building block, cycloaddition reagent, and P atom transfer agent. I have performed very preliminary work (cut short by COVID-19) on using this anion to generate phosphide thin film materials. Metal phosphide thin films have many applications in advanced electronics and thus are a valuable manufacturing target. The solution-processed, electrochemical method proposed would have marked advantages over current methods to generate phosphide thin films such as MOCVD and MBE. If realized, this unique method of producing metal phosphide thin films would be entirely unprecedented.

Natural proteins often assemble into various complex geometric structures based on their interactions with each other. The King Lab at the University of Washington's Institute for Protein Design uses the way these proteins behave to develop computational models that enable the design of novel self-assembling protein cages, or nanoparticles. The designed particles are capable of holding and transporting molecules or displaying antigens on their surface, making them effective vaccine candidates. My project involves recovering the solubility of one of these protein cages known as T33_dn2. T33_dn2 is a tetrahedral protein cage comprised of four copies each of two trimeric components known as T33_dn2A and T33_dn2B. While both components can be expressed individually through E.coli before being assembled in vitro, they can also be expressed bicistronically and assemble in vivo. Currently, the use of T33_dn2 as a vaccine scaffold is limited because T33_dn2B is insoluble, and only seems to be stabilized in solution when associating with T33_dn2A. When expressed bicistronically, however, the cage has an extremely low yield. For a protein to be developed into a vaccine, it must be soluble. To recover the solubility and yield of T33_dn2B, I am testing ten plasmid variants of bicistronic T33_dn2. The “original” plasmid consists of one gene coding for a high-expressing cleavable SUMO protein attached to T33_dn2A and another coding for T33_dn2B. The additional nine variants have single point mutations at specific locations on the T33_dn2A gene intended to affect binding strength. After expression, introducing wildtype T33_dn2A in vitro will allow for the formation of T33_dn2. I will be presenting the results of these expression, purification, and assembly tests.

Although biosensors are commonly used to detect many different molecules of interest, they cannot effectively detect small hydrophobic molecules in biological systems. We propose combining chemically induced dimerization (CID), in which two proteins dimerize only in the presence of a ligand, with a traditional luciferase assay to create a biosensor that luminesces when the desired molecule is introduced. Using molecule-specific nanobodies, we can design the two CID binders to attach to a wide variety of small molecules, even those that are challenging for conventional biosensors to detect. Through its specificity and ability to bind to small or hydrophobic molecules, the CID system overcomes problems that other biosensors face. As a proof-of-concept, we implemented an in vivo CID biosensor to detect the presence of cannabidiol. With the nanobody CID system, we hope to introduce a novel biosensor that can detect a variety of important small molecules across research, biotechnology, and medicine.

Humans are incapable of regenerating a majority of their major tissues following traumatic injury. Tadpoles from the frog species Xenopus tropicalis have the ability to regenerate lost spinal cord, vasculature, muscle, and cartilage within a few days following injury. The regulatory mechanisms of gene expression necessary for regeneration have not yet been well defined. My primary interest is in understanding the relationship between stress signaling and gene expression during regeneration. The lab has shown that the stress responsive transcription factor Hypoxia Inducible Factor 1α (Hif1α) is necessary for the expression of Wnt target genes, one of the primary signaling processes necessary for regeneration. While we have found that Hif1α is necessary for Wnt target gene expression, we do not know the epistatic relationship between Hif1α and Wnt. In order to test this relationship, I utilized the drug IWR to antagonize Wnt and found that tadpoles treated with IWR have reduced tail regeneration 72 hours post amputation (hpa). I then supplemented these tadpoles with DMOG to stabilize Hif1α and found that DMOG is sufficient to rescue tail regeneration, suggesting that Hif1α is downstream of Wnt. In order to determine if Hif1α is sufficient for Wnt target gene expression, I extracted RNA from regenerating tails 24 hPa and used quantitative PCR (qPCR) to determine relative gene expression. I also utilized in situ hybridization to see if expression of these genes is restricted to regenerating tissues. As Wnt is a known regulator of neural and muscle development, I investigated how inhibiting Hif1α would impact complex tissue regeneration and found that Hif1α is necessary for regeneration of axons and muscle specifically. By determining the epistatic relationship between Hif1α and Wnt through the analysis of specific gene expression, we continue to improve our understanding of how regenerative organisms convert stress signals to cell fate signals.

Wildfires are known for their destructive capacity towards ecosystems and devastating impacts to human life and property. Regions in the western United States are particularly prone to such events, including large crown fires. However, there is limited research on the potential for smoke from these wildfires to carry and redistribute nutrients in the form of aerosols. Here I show that higher levels of airborne nutrients, specifically iron, phosphorus, and potassium, can be associated both with the timing of active wildfires and a known smoke tracer, elemental carbon. Data from the Interagency Monitoring of Protected Visual Environments (IMPROVE) show a strong correlation with fire presence and local peaks in levels of potassium, with less strong associations with phosphorus and iron. Across a multi-year period, there is also a correlation between the number of acres burned in a particular year and the average concentration of iron and potassium in that region. Using data from the NASA DC-8 airborne laboratory corroborates this information, with calculated potassium concentrations matching those on the ground. These findings indicate the wildfire plumes have a potential to be significant sources of nutrients in the short term. This is especially relevant for areas impacted by wildfires, as these nutrients are key for plant growth and development. These elements may also be carried into the ocean and affect the aquatic biosphere. All of the information gathered will help improve our understanding of the complex networks that make up Earth’s biogeochemical cycles.

Viral pathogens, especially those that undergo rapid mutagenesis, pose a significant threat to public health. Viruses that exemplify this issue include influenza, HIV, and Ebola. Given the low efficacy of seasonal vaccines for influenza, our project focuses on improving existing influenza vaccinations. Instead of using conventional methods, such as injecting inactivated pathogens or viral subunits, mRNA sequences encoding the viral hemagglutinin (HA) fused to our recently-developed self-assembling I53-dn5 nanoparticle platform will be administered in vivo. The organized array of the protein platform can lead to stronger B-cell crosslinking and a robust immune response. However, characterization of I53-dn5 in vitro is critical before use in vaccination studies. My work focused on optimizing the expression, secretion, and assembly of the I53-dn5 protein platform. To mimic in vivo conditions, I transfected DNA encoding HA-fused I53-dn5 into HEK293F cells. Past experiments have shown that when dn5A and dn5B are transfected separately, they express at disproportional concentrations. To resolve this, we encoded both components onto one DNA plasmid for transfection. However, with this new approach, we also needed to cleave the two components after expression. To do so, we incorporated different cleaving peptides, such as T2A and Furin cleavage sites. Through western blots, SDS page electrophoresis, SCC protein purification, and electron microscopy, I analyzed how these cleaving peptides impacted assembly and secretion of the protein platform. Once we are able to consolidate an effective model, we will be able to start in vivo studies. Furthermore, if effective, our model can be used to create vaccinations against other viral illnesses, including HIV and coronavirus.