Long non-coding (lnc)RNAs have multiple biological functions, including recruitment of kinases to regulate signaling pathways involved in tumorigenesis and other human diseases. Of particular interest is the proposed interaction between Long Intergenic Noncoding RNA for Kinase Activation (LINK-A) and the membrane component phosphatidylinositol-3,4,5-triphosphate (PIP₃). The proposed interaction between PIP₃-LINK-A would be the first example of a direct interaction between a non-coding RNA and phospholipid. I investigated the proposed interaction between LINK-A and PIP₃ using Nuclear Magnetic Resonance (NMR) spectroscopy. Multiple NMR-based experiments were performed to assess the degree of binding on the basis of line-width broadening of NMR spectra. Wild-type and mutated RNA constructs were titrated into a 100 uM solution of PIP₃, but the NMR data showed no evidence of line-width broadening, indicating that no direct interaction occurs between wild-type or mutated RNA constructs. Presumably, the reported cellular interaction is not direct and might require an additional mediating factor. I determined the 3D structure of the LINK-A RNA hairpin, required to recruit PIP₃, using biophysical molecular modeling and NMR data. My results provide a biophysical foundation to elucidate the functional role of LINK-A in PIP₃ recruitment and kinase activation.

Growing reliance on antibiotics in livestock production, commercial agriculture, and human healthcare has created evolutionary pressures on bacteria. These pressures have given rise to a new threat to public health, drug-resistant microbes. In addition, antibiotics eliminate all microbes, including the beneficial ones, and are not advisable for long-term use. Recent advances in the understanding of bacterial virulence via quorum-sensing (QS) has presented the scientific community with a promising new approach for alleviating the ongoing overuse of broad-spectrum antibiotics. Quorum-sensing is a form of coordinated gene expression mediated through detection of specific population density. Bacteria use QS as a way to regulate behaviors including biofilm formation, virulence, and motility. Quorum-sensing inhibition (QSI), is the ability to prevent QS and is used by organisms across kingdoms. This prevalence suggests that disrupting prokaryotic communication is either a common defense tactic against infection or acts as a competitive advantage in resource acquisition. Fungi, a kingdom in direct competition with prokaryotes are prime candidates for broadening our understanding of the mechanisms behind quorum-sensing. With its abundance of endemic fungi, the Puget Sound region provides a unique opportunity to screen species for QSI compounds. In this study, fungi representing diverse ecological niches were collected from local forests around the Puget Sound and isolated on potato dextrose agar plates. To screen fungal isolates we used C. violaceum, a bacterium that forms purple colonies when able to quorum-sense and changes to white when a QSI is present. Our results suggest fungi are an underexploited and unexplored source of novel bioactive molecules that could provide a method to inhibit virulent effects of bacteria without damaging an organism’s microbiome.

In the US more than 100 million people are living with diabetes or pre-diabetes. The economic burden caused by these conditions, including medical costs, is approximately $327 billion annually. Conventionally, transgenic Escherichia coli has been the primary source of commercial insulin production, a process that requires extensive purification to ensure shelf stability and complete removal of contaminants. This study seeks to establish an alternative mode of insulin production using polyethelyne glycol (PEG) and agrobacterium to transform the oyster mushroom, Pleurotus ostreatus, with the human insulin gene. P. ostreatus is a valuable target for genetic transformation due to its lack of endotoxins, rapid growth, and fully sequenced genome. P. ostreatus was transformed using PEG and agrobacterium with a plasmid containing the human insulin gene and a carboxin resistance gene. Transformed cells were selected using carboxin, extracted, and regenerated on plates composed of yeast extract, malt extract, and glucose (YMG). Integration of the human insulin gene in to the mushroom genome was confirmed through PCR analysis of the transformants. Successful transformation of P.ostreatus offers a new avenue for insulin production, potentially diversifying the market and treatment options for diabetics.

Mercury (Hg) is widely known to be a neurotoxin. Mercury in our environment is found in many different forms, and the key difference between them is evident in the way they are absorbed by organisms. Hydrophobic methylmercury (Me-Hg) readily bioaccumulates in the tissues of all organisms, leading to Hg exposures involving higher doses. Recent research shows that sulfate-reducing bacteria (SRBs) play a role in methylating mercury only when they possess the hgcAB gene cluster. Gaining a more comprehensive understanding of the aqueous conditions required for SRBs to thrive and consequently methylate inorganic mercury is essential for addressing the ongoing problems associated with Hg toxicity. In the initial phase of this study, ion chromatography was used to quantify sulfate (SO42-) concentrations along an urban river in an industrial region of Seattle (the Duwamish/Green River Watershed). The measurements revealed that SO42- concentrations as high as 5300 ppm were present at several sites along the lower portion of the Duwamish River. A number of previous studies also showed significant amounts of mercury in this region’s sediments and fish tissues. The secondary phase of this study involved an investigation determining whether the measured high sulfate concentrations were related to the production of Me-Hg and additionally examined if SRBs likewise played a role. River sediment was analyzed for the presence of the hgcAB gene cluster. Ongoing studies are focused on quantifying Me-Hg at sites where SRBs were found and on the identification of key indicators for mercury methylation conditions along a watershed. This study provides further insight into the relationship between mercury, sulfates, and SRBs when found in combination in an aqueous environment.