Our research seeks to more accurately define how the Pacific Northwest regional climate will look in fifty to one hundred years. Our results help quantify the uncertainty in our climate predictions, a major shortcoming in climate research. Our research is interesting because it utilizes a high resolution numerical weather prediction model to examine regional climate change here in the Pacific Northwest. We are creating a large set (an ensemble) of predictions of climate change in the Pacific Northwest. The tool used in our research is the Weather Research and Forecast (WRF) model. WRF requires General Circulation Model (GCM, also known as Global Climate Model) data to feed it information on the larger-scale climate. A big part of our project is collecting GCM data from a variety of research centers worldwide, and preparing it to be used as input data for WRF. We expect to complete our model simulations by the end of Spring Quarter 2018. These results could be of direct use to a number of local agencies and policy makers so that they can shape their decisions around the future changes that our local climate may experience. We also expect our data to be of great use for further research in quantifying climate prediction uncertainty.

Antibiotic resistance is a major public health problem. For example, in people with the genetic disease cystic fibrosis, Pseudomonas aeruginosa causes chronic lung infections that resist treatment to all existing antibiotics. Gallium is a promising new antimicrobial agent that works through a novel mechanism of action. Bacterial uptake systems are unable to distinguish gallium from iron due to their nearly identical ionic radii. Once inside, bacterial gallium is thought to disrupt iron-dependent processes because Ga³⁺ cannot be reduced in physiological conditions, and iron's biological functions depend on its capacity for redox cycling. Here I sought to better understand gallium’s mechanism of action by identifying gene mutations that decrease P. aeruginosa gallium sensitivity. To accomplish this, I used transposon (Tn) mutagenesis, which transfers a genetic element to random locations on the bacterial chromosome thereby inactivating genes. Of the 295,000 mutants generated, I selected for mutants capable of growing in the presence of increased gallium compared to the parental strain. Using a procedure known as rescue cloning, a restriction digest was performed on the chromosomal DNA of these mutants to produce individual fragments. One of these DNA fragments will contain the Tn insert and chromosomal DNA flanking that insert. The Tn insert encodes resistance to gentamycin and holds the capacity to replicate as a plasmid in E. coli. I ligated the isolated fragments and transformed E. coli, selecting for gentamycin resistance. I isolated this plasmid DNA from two mutants and their Tn insertion sites (and the genes inactivated by the Tn) were identified by sequencing the DNA surrounding the Tn inserts. These genes were found to be involved in iron transport. By completing these experiments, I hope to suggest hypotheses for the roles these genes play in gallium sensitivity and to provide data useful toward guiding its development as an antibacterial agent.

This paper analyzes a collection of letters and postcards hand written during World War II, authored by German crystallographer and volcanologists Ferdinand Bernauer to his son. The analysis draws on aspects of critical theory of the epistolary genre, while keeping in mind that the collection is a set of artifacts, not a fiction, as they were never published or intended as a work of literature. Their analysis provides a unique insight into the decisions and repercussions of a strong refutation of Nazism by a distinguished man of science residing in Hitler’s Germany. His greatest contributions to the academic world – proof of Continental Drift through his studies in Iceland and a crystal growing method now lost to mankind – attest to his considerable intellect and dedication. Yet it is his letters that uncover a life worthy of investigation for what they illuminate about the human psychological faculty of weighing the risk of death with the ethical reward of fighting for one’s values. The epistolary lens allows the reader to grasp aspects of the text not explicitly written such as intentional self-censorship, introspection upon one’s choices and moral stance, and stylistic choices made because the intended recipient was his son. The letters, unlike actual epistolary novels, need no omniscient narrator to provide a historical backbone upon which the narrative may play out, because it has been provided largely by World War II records. Therefore, this work facilitates a strong identification between Ferdinand Bernauer and the reader in an absorptive reading experience typical of an epistolary novel, without vast holes in historical perspective. This analysis explores a method of reading letters written under political oppression that enables one to understand the real difficulty in making the choice between standing against an immense malevolent power, and portraying one’s resistance quietly among the chaos.

Non Hodgkin Lymphoma (NHL) affects over 70,000 people in the United States each year. Inappropriate activation of B-cell receptor signaling is associated with NHL, and therapies that target this pathway are showing promising results in the clinic. One example is ibrutinib, a small molecule inhibitor of Bruton’s Tyrosine Kinase (BTK). While ibrutinib has improved the prognosis for the majority of NHL patients, many patients eventually develop resistance to ibrutinib due to mutations in BTK and its downstream target Phospholipase C gamma 2 (PLCG2). Thus, it is important to understand how these mutations affect cell signaling, with the ultimate goal of designing new strategies to mitigate ibrutinib resistance. Previous studies in the James Lab suggest that long-term treatment of cultured cell lines with ibrutinib leads to increased cellular reactive oxygen species (ROS). However, it is unclear if ROS contribute to ibrutinib resistance or if they are a byproduct of other mechanisms that drive resistance. I hypothesize that cells with mutations commonly seen in ibrutinib-resistant patients may also exhibit increased ROS, and these cells may be sensitive to small molecules that regulate ROS. In order to test this hypothesis, I used CRISPR/cas9 gene editing with homology-directed repair to generate lymphoma cell lines that express a leucine-to-phenylalanine mutation in PLCG2. This is a well-described mutation in ibrutinib-resistant patients. Preliminary analysis shows that these mutant cells are resistant to ibrutinib. I measured ROS in these cells by ROS-Glo luminescence. Then, I cultured the cells with ROS-regulating compounds including SOD-1 and NADPH oxidase inhibitors to assess how ROS affect their viability. My goal is to clarify the role of ROS in the acquisition of ibrutinib resistance. These studies may provide justification for further pre-clinical evaluation of small molecule regulators of ROS in lymphoma, especially among subjects with acquired ibrutinib resistance.

Imidacloprid is the most widely used agricultural pesticide in the world. As a neonicotinoid, it is highly potent against insects while having low toxicity to mammals. One area of concern is its effect on honeybees, possibly playing a role in colony collapse disorder (CCD), a sudden disappearance of worker bees from an otherwise healthy population. Bees are known to be sensitive to imidacloprid at high concentrations, but what is unclear is whether chronic exposure to the low doses used to protect crops can also be harmful. The E.U. and Canada have taken steps to ban the use of imidacloprid as a precaution, and the U.S. EPA has warned users of its potential for leaching into groundwater. It may also persist for years in soil, pollen, and nectar. Due to its low volatility, high boiling point, and high solubility in polar solvents, imidacloprid can be difficult to reliably detect in environmental samples using current gas chromatography (GC) methods. To address this, we evaluated GC-MS detection of imidacloprid in methanol standards, spiked water, and spiked honey. Samples were evaluated after clean-up with both C18 solid-phase extraction (SPE) and dispersive solid-phase extraction (DSPE) methods, using two different GC columns (Restek Rxi-5MS and Rtx-1701). Samples were also subjected to a range of pH conditions, and ionized by both electron ionization (EI) and chemical ionization (CI). As GC is typically faster, cheaper, and easier than liquid chromatography, currently the method of choice for detection of neonicotinoids, our research could benefit the study of imidacloprid toxicity by reducing the attendant time, cost, and training requirements. Preliminary results have shown reliable detection of imidacloprid-urea, the main hydrolysis compound of imidacloprid, at pH 5-7.

Probiotics are an area of significant clinical research, as they have been shown to aid digestion, fight infections, and even mitigate irritable bowel syndrome; however, probiotic colonies must grow and thrive in the extreme pH environment present in the human digestive tract. Previous experiments focused on the production of the surface protein Elongation Factor Thermally Unstable (EF-Tu), which attaches to glycoproteins lining the intestines and has been used as a biomarker for probiotic health. The growth of several strains of probiotics (Lactobacillus bulgaricus, Lactobacillus acidophilus, and Bifidobacterium longum) in both lysogeny broth (LB) and De Man, Rogosa, and Sharpe broth (MRS) was evaluated. The fractions of secreted, cytosolic, and membrane proteins were quantified using a Bradford assay. Finally, the relative amount of EF-Tu was determined by tryptic digestion and liquid chromatography-mass spectrometry (LC-MS). LB was found to be a more effective growth medium for the range of bacteria tested, with 24 hours at 37°C the ideal incubation period. To isolate the secreted proteins, the supernatant was drawn off the broth culture after centrifugation. Freeze-thaw lysis was used to extract cytosolic proteins, with three cycles determined as optimal for protein recover. Sodium deoxycholate was used to separate proteins from the membrane. All three protein samples were separated and run through a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the EF-Tu bands (roughly 43 kDa) were excised and sent for analysis by LC-MS. Using the information obtained from this research study, pharmaceutical companies can create more suitably tailored probiotic products, and make them more accessible and understandable to the general population. 

The goal of our project is to create an electronic device capable of early detection of pancreatic cancer (PC) with high selectivity and sensitivity. PC projects a very low survival rate often due to late-stage cancer diagnosis. Recent research has established that there are PC biomarkers prevalent throughout the body for several years before symptoms emerge. The consequent wider time window presents an opportunity for these biomarkers to be detected at their initial low concentrations thus allowing for early diagnosis. Our device uses a modular sensing construct consisting of an immobilized probe molecularly bound to the surface of the sensor device. Detection occurs when a target biomarker specifically binds to the probe and changes the electrical properties of the sensing surface that is measured quantitatively. Validating the functionality of the sensing construct and its properties is accomplished through a variety of molecular adsorption and binding techniques that assess each step; from probe immobilization to target detection. Using this modular design, research is underway to develop an array of sensors, thus potentially revolutionizing rapid medical diagnostics to provide long-term health monitoring of PC and other cancers. 

Eukaryotic ribosome biogenesis is an essential and energetically demanding cellular process that must be tightly regulated. In S. cerevisiae, this regulation is partially achieved via ubiquitination of RPA190, the largest subunit of RNA polymerase I, and further mediated by the deubiquitinating enzyme (DUB) Ubp10. Human USP36, the ortholog of Ubp10, rescues RPA190 stability in ubp10 delta yeast cells, indicating DUB activity may be conserved across eukaryotes. As such, there is considerable interest in deciphering the mechanism by which RPA190 and human RPA194 are ubiquitinated. Previous work identified three lysines (K405, K408, and K410) as possible ubiquitination sites. However, whether one or a combination of these lysines are ubiquitinated has yet to be determined. We created single lysine to arginine mutant RPA190 plasmids for each site and transformed them into rpa190 delta cells. These constructs may be used to help determine which lysine(s) are targeted by Ubp10 and whether those site(s) are functionally analogous in RPA194. This will yield a more complete understanding of the importance of DUB activity in the context of eukaryotic ribosome biogenesis.

The microbial community residing in the gastrointestinal tract, called the gut microbiota, plays an important role in host energy homeostasis. Since energy homeostasis is essential for survival, it is plausible that neural circuits involved in maintaining host energy homeostasis would evolve to shape the composition of the gut microbiota in alignment with the energy needs of the host. In humans and mice, neurons in the arcuate nucleus of the hypothalamus that express agouti-related protein (AgRP neurons) play an important role in stimulating food intake and maintaining energy balance. AgRP neurons are maximally active during starvation conditions; thus, we are investigating whether they help conserve energy under these conditions by altering the composition of the gut microbiota to a state with a greater capacity for energy harvest. To test this hypothesis, we expressed an excitatory designer receptor, the hM3Dq receptor, in the AgRP neurons of mice and then analyzed the bacterial composition and energy harvest capacity of the gut microbiota of these mice following pharmacological activation of the hM3Dq receptor relative to unstimulated control mice. We assessed the bacterial composition of the gut microbiota using 16S rRNA amplification, sequencing, and characterization, and we assessed the energy harvest capacity of the gut microbiota by determining the energy content of the mouse fecal matter using bomb calorimetry. Our results show how AgRP neuronal activity influences the composition and energy harvest capacity of the gut microbiota. These results reveal that the central nervous system can shape the gut microbiota to promote energy balance. This finding contributes to a better understanding of why and how gut microbiota changes occur, which may be relevant to understanding metabolic diseases such as obesity.