Escherichia coli can contaminate food, such as beef and spinach, causing illnesses ranging from recurring diarrhea to death in susceptible individuals. They may produce a toxin typically associated with another harmful bacterium known as Shigella spp. This toxin is referred to as “Shiga-toxin”, and E. coli that produce this toxin are known as STECs. While E. coli O157 remains the most common STEC in the United States there are over 100 STECs that can be just as harmful. My project, conducted in collaboration with individuals at CSUEB and the USFDA, involves identifying chemical indicators that would help detect different strains of non-O157 E. coli in contaminated foods, and determining if these STECS are vulnerable to a coliphage and a Bdellovibrio sp. invasion. Specifically, nine different STECs were chosen to be screened for their ability to produce catecholate siderophores (phenolates), such as enterobactin, chelating compounds important for some pathogenic bacteria in the acquisition of iron. The same STECs were screened for the presence of acyl homoserine lactones, chemicals involved in bacterial communication, also known as quorum sensing, and expression of virulence factors. Assays were conducted to determine if a coliphage and a Bdellovibrio sp. can infect these STECs. Detection and identification of non-O157 STECS is not easy, which hinders determination of their origin and, subsequently, how to eliminate them. If new tools, such as the use of predators or chemical indicators were developed to facilitate detection of these bacteria, then determination of the source of contamination (e.g., packing plants and fields) may occur, and quicken measures that prevent or, at least, decrease further infection, disease and/or death.

In the failed state of Somalia, piracy is increasing exponentially, impacting all nations that engage in trade or travel in the region. The economic and human cost of piracy is sure to rise as the situation devolves into maritime terrorism. Although current anti-piracy efforts are moderately successful, there is room for improvement. This report has thoroughly researched the situation to create one comprehensive source for understanding piracy in the Horn of Africa. Research methods included reviewing secondary source material, in addition to interviews with professionals working in counter-piracy efforts and with Somalis in the United States. The coordinator of the U.S. Office of Counter-Piracy and Maritime Security, Donna Hopkins, and her team provided assistance through interviews, contacts with people working in Somalia and around the world, and other valuable sources. The research was compiled into a task force report that provides recommendations for the U.S. State Department, and for other organizations involved in counter-piracy efforts. The following recommendations provide alternative courses of action to address current imperfections in the international architecture’s counter-piracy campaign. Social media should be utilized to launch an anti-piracy information campaign that engages the vast Somali Diaspora. Because piracy is a land-based problem with a maritime manifestation, a grassroots approach to address the land-based causes of poverty and political instability must be instated. National prosecutions and detentions should be supported and facilitated through new international legal frameworks. Passive defensive measures must be expanded on commercial vessels and armed guards should be utilized. The three parallel naval forces patrolling the area are insufficient protection against pirates and must be consolidated into one international force. Finally, the U.S. must expand its diplomatic engagement with regional governments in Somalia through imams and clan elders if the fight against piracy is to succeed.

In a population of organisms, traits that increase fitness, or the ability to survive and reproduce, are passed on to progeny and become more prominent in the gene pool. It has been similarly shown that E. coli with a phenotype that increases or decreases fitness given an environmental pressure can be tuned to correspondingly decrease or increase fitness through random mutagenesis and selection of desired mutants. However, this process does not focus mutations and does not confer the maximum fitness benefit or cost per mutation. In order to isolate mutation location and effect, and increase the rate at which evolution occurs, I am applying a tool called Simple Sequence Repeats (SSRs). SSRs are repeats of short nucleotide sequences (AAA…, ATATAT…, etc.) that have been shown to mutate primarily in the form of insertions or deletions of an SSR and have a rate of mutation four to five orders of magnitude higher than non-repeat sequences. Most importantly, it has been shown that when SSRs are inserted between the ribosome binding site (RBS) and start of a gene, every insertion or deletion has a discrete, predictable effect on the level of expression for that gene. We have inserted SSR spacers in front of lacZ, lacY, and lacI in the lac system, and will show that we can rapidly optimize the expression of the genes. This could serve as a significant tool for tuning other, novel behaviors in bacteria.

Huntington’s disease (HD) is a genetic neurodegenerative disease caused by a polyglutamine expansion in the protein huntingtin. The age of symptom onset has been correlated with the length of this repeat. Adults with a repeat length of greater than 35 polyglutamines typically present symptoms between the ages of 40 and 50. Greater than 60 polyglutamine repeats causes a rapidly progressing early onset of the disease in adolescents. While both types of HD result in cognitive deficits and neuron loss, 25-35% of early-onset HD patients exhibit epileptic seizures that become less responsive to anti-epileptics over time. One of the earliest molecular changes in HD is the loss of the cannabinoid receptor 1 (CB1) before symptom onset. Activation of CB1 inhibits the release of neurotransmitters and protects neurons from cell death, whereas genetic deletion in HD models exacerbates motor symptoms. Additionally, activation of CB1 prevents the onset of seizures. The goal of this study was to determine if loss of CB1 activation increased epileptic activity, decreased survival, and whether elevating endocannabinoid (CB1 agonists) levels reversed these effects in early-onset HD. R6/2 mice (a rapidly progressing mouse model of early-onset HD) were treated with SR141716A (a CB1 antagonist), WWL123 (elevates the level of endocannabinoids), or vehicle (a control) and the frequency and severity of spontaneous and handling-induced seizures were analyzed. Preliminary results demonstrated that R6/2 mice treated with SR141716A had a decreased lifespan and more frequent seizures whereas WWL123 decreased seizure severity. This suggests that the endocannabinoid system has a regulatory effect on seizures associated with early-onset HD. Future experiments in the lab will further characterize the role of endocannabinoids and CB1 in early-onset HD seizures as a novel therapeutic pathway.

Tuning the gene expression of biomolecular pathways is an area of intense interest and value to synthetic biologists. Through the use of a mixture controlled turbidostat (MCT), we have been able to streamline, and in many cases enable the processes involved in this area of study. A turbidostat is a continuous culture bioreactor that maintains constant culture volume and cell density while diluting with fresh media to keep cells in an exponential growth phase. With the addition of mixture control, the turbidostat can vary the chemical environment within the culture vessel without altering cell density levels. Tuning gene expression in Escherichia coli is a simple matter when using a MCT. Currently, an area of global interest is the industrial production of butanol with E. coli. The first step towards this is enabling E. coli to live in rich butanol environments. Because butanol is naturally toxic to E. coli, an increase in gene expressions of the biomolecular pathways responsible for detoxification of butanol is required. By placing laboratory grade E. coli into our MCT, the concentration of butanol can be increased over time while the cell density remains constant. This will allow fitness increasing mutations within the biomolecular pathways responsible for detoxification of butanol to prevail over many generations, with the hope of eventually converging to a final population of E. coli that subsists in highly concentrated butanol environments. Through increasing E. coli’s resistance to butanol, we hope to illustrate the usefulness of MCTs in tuning gene expression of biomolecular pathways.

Indole-3-acetic acid (IAA), or auxin, is a plant growth hormone that is vital to the regulation of growth and development in plants. Although auxin’s function in plants is to control growth, it potentially regulates any gene in other organisms, making it a powerful regulatory tool. By investigating the synthesis of auxin, it can be generated in organisms besides plants. The goal of this project is to take the first step towards that reality through the synthesis of auxin in Saccharomyces cerevisiae (baker’s yeast). Due to the thorough characterization of S. cerevisiae, the organism is an optimal choice because the mechanisms involved with the manipulation of its gene networks are well understood. This project integrated the tryptophan-2-monooxyngenase (iaaM) and indole-3-acetamide hydrolase (iaaH) genes from Agrobacterium tumefaciens into the yeast genome. The enzymes produced from these genes convert tryptophan into auxin. In an attempt to reproduce this synthesis pathway, the two genes were amplified and integrated into the yeast genome through the use of the Gateway® cloning system and integrating vectors. Checking for successful auxin synthesis utilizes the auxin signaling pathway that was built in S. cerevisiae by the Klavins Lab. This system involves interactions between two proteins, an AFB (auxin signaling F-box) and an Aux/IAA (a transcriptional regulator of auxin responsive genes). The presence of auxin triggers the degradation of an Aux/IAA. By fluorescently tagging an Aux/IAA, the expression of the protein was observed. A decrease in fluorescence indicates the production of auxin. Successful auxin synthesis in yeast creates further possibilities for establishing regulatory systems based on the use of auxin in non-plant cells.

Exoplanets are planets outside our solar system. As they do not emit light of their own, these objects have historically been much harder to find and study than stars. One detection method exploits the slight dimming of a star caused by a planet passing across its face. In astronomy, these events are termed transits, and they cause distinct u-shaped dips in the lightcurve—a plot of observed brightness versus time—of their host star. In recent years, the Kepler Space Telescope has provided lightcurve data on a fixed group of stars that is unprecedented in precision. Our research is primarily concerned with searching released Kepler data for previously undiscovered exoplanets. Due to the vast quantity of information, we have developed a pipeline of computer code to expedite the search process. The code, written in python, detrends variations in quiescent star brightness to normalize each lightcurve around a fixed, flat value. This readies the data for input to a Quasi-Periodic Automated Transit Search algorithm (QATS). Thus far, we have identified a handful of planet candidates as well as a number of undocumented binary star systems. Current and future tasks involve automating QATS output classification to further improve the efficiency of the planet finding process.

In the search for habitable planets, density is crucial in allowing us to determine whether a planet is terrestrial or gaseous. We developed a method of measuring the flux from a star during a planetary transit to constrain the density and the mass of the planet. This method depends on our ability to detect a moon in the transit and chart minor fluctuations in moon velocity and moon position with respect to the planet. To determine the precision with which we can measure the density of a planet, we have created a model which produces a synthetic light curve from which we can attempt to extrapolate planet parameters. However, we have found that the accuracy of planet density calculations is highly dependent on the signal to noise ratio of the sample due to the fact that moons have very small signals. We explore methods with which to detect moon signals to improve upon our ability to determine the density. It may still be necessary to combine our efforts with other methods, such as spectra analysis of Doppler shifts, to confidently differentiate potential life-bearing planets from those which are not habitable.

Parkinson’s disease is a motor disorder characterized by tremors, rigidity, and inability to initiate movement. One therapy for non-drug responsive cases is high amplitude, high frequency stimulation to the basal ganglia’s subthalamic nucleus (STN), via surgically implanted electrodes. While the mechanisms of how this therapy restores proper motor function remains under debate, one hypothesis is that the currents disrupt rhythmic, synchronous firing patterns present in basal ganglia structures of people with Parkinson’s disease. Our goal is to explore the effects of low amplitude, low frequency stimulation to the basal ganglia nucleus globus pallidus externus (Gpe), specifically whether this type of stimulation can disrupt pathological firing synchrony in GPe and STN. We use a conductance-based model of individual neurons in multicellular networks with varying patterns of short, depolarizing currents to mimic deep brain stimulation. Through this work we hope to discover novel patterns of deep-brain stimulation with the potential to eliminate pathological firing patterns of cells in the basal ganglia.

Paleontological evidence has shown that during large scale extinction events, hydrogen sulfide (H2S) rises to abnormal levels in Earth’s atmosphere. To survive these extinction events, an organism would be required to counter-act the negative effects of H2S by evolving unique adaptations. It can be hypothesized that many of these unique adaptations still exist in present-day lineages. Modern plants, for example, may have developed a method of utilizing H2S in order to survive, and possibly thrive, in what would be considered a highly toxic atmosphere. Through experiments involving species Phaseolus vulgaris and Pisum sativum, it was discovered that applications of H2S to seedlings increased growth at rates of up to 200% within 1 week. In addition to the increases in growth, increases in plant mass and photosynthetic activity were also observed. In order to better understand the effects of H2S on P. vulgaris and P. sativum, seeds of the species were also exposed to H2S, under the same controlled conditions. The effects of the seed experiment were similar to the initial seedling tests. Time to germination decreased with exposure, and the overall rates of germination increased. Large scale implications of these findings present benefits such as greater crop yields and perhaps a more efficient method of bio-fuel production. Continuing research of H2S and its effects on plant life could someday create a powerful means to deal with our ever-expanding human population…and the ever-expanding human footprint that goes along with it.

While necessary for many biological functions, essential heavy metals, such as copper and zinc, are highly toxic when elevated, requiring their concentrations to be tightly regulated.  This need to maintain homeostasis has led to a variety of adaptive mechanisms that chemically sequester excess heavy metal ions.  One such mechanism is the biosynthesis of phytochelatins by the enzyme phytochelatin synthase (PCS), found in plants, algae and several fungal species.  Induced in cells of certain organisms exposed to elevated levels of heavy metals, phytochelatins also bind non-essential heavy metals such as cadmium and arsenic, offering protection against anthropogenic toxins.  Despite their demonstrated ability to synthesize phytochelatins, the PCS gene has not been investigated in conifers.  The simplest method of probing genomc DNA (gDNA) for specific genes is the polymerase chain reaction (PCR) method.  However, lack of sequencing data presents a challenge in designing universal primers for conifers.  The objective of this project was to develop degenerate primers capable of amplifying the PCS gene in a selection of conifers native to Washington State.  The degenerate primers were designed from known and putative PCS sequences of non-conifer plant species obtained from a BLAST query.  The sequences were aligned and primers selected from regions of high consensus, while controlling for degeneracy.  The primers were used in PCR trials to probe the gDNA of thirteen species of conifers.  To date, I have amplified and sequenced PCR gene segements of 267 and 133 base-pairs in Pinus ponderosa and Pinus contorta, respectively; adding two species of conifers to the list of PCS genes sequenced, and allowing me to design primers with increased specificity for conifers for use in subsequent PCR trials.  Future studies could develop molecular assays for expression of the PCS gene for use in assessing metals stress at the cellular level for conifers in contaminated sites.  

Edible fungi can accumulate certain toxic metals, including cadmium, lead, and copper, in a species dependent manner. Forests that have been treated with biosolids have elevated soil metals, which could lead to additional accumulation in mushrooms growing in treated sites. This can be a possible safety hazard for people who gather wild mushrooms for consumption. My objective was to determine metal concentrations in several species of edible mushrooms and whether these species accumulate metals above background levels in their soil substrate. Collections of edible mushrooms from biosolid treated forest and control sites were analyzed for toxic metal concentrations. Mushroom concentrations of cadmium and silver were significantly higher in biosolids treated forests, while the other metals were not significantly different. The overall results showed that metals uptake was more strongly influenced by species than by biosolids application. Gomphidius subroseus exceeded the European Union regulatory upper limit with Cd and reached it with Cu. Metals in all other mushrooms in this study were below recommended safe annual limits for moderate levels of consumption.

Synthetic biology holds great promise regarding the production of important compounds, and the degradation of harmful ones. Last summer, we harnessed the power of synthetic biology to meet the world’s needs for fuel and medicine. We constructed a strain of Escherichia coli that produces a variety of alkanes, the main constituents of diesel fuel, by introducing a pair of genes recently shown convert fatty acid synthesis intermediates into alkanes. Using this method, we were able to produce on average 160 mg of alkanes per liter. Also, we identified a protease with gluten-degradation potential, and then reengineered it to have 100 times increased gluten-degrading activity, allowing for the breakdown of gluten in the digestive track when taken in pill form. Finally, to enable next-generation cloning of standard biological parts, BioBrick vectors optimized for Gibson assembly, an emerging DNA cloning technique, were constructed and used to construct the Magnetosome Toolkit: genes for biofabrication of magnetic particles.

The National Cancer Institute estimates that there will be 73,510 new cases of bladder cancer in the United States during the year 2012, and an expected 14,880 deaths due to the disease. With a 50-70% chance of recurrence, bladder cancer is one of the most expensive cancers to treat. If detected and treated early, the cost of this cancer could be dramatically reduced. Some current methods of detection are conducted by using a cystoscope inserted through the urethra to image the interior wall of the bladder. These cystoscopes can be fairly large—ranging from 3.3-5.3mm in diameter—and sometimes require general anesthetics, which increase costs due to additional bodies within the operating room. In contrast, the scanning fiber endoscope (SFE) developed in our lab is 1.2-1.6mm in diameter; at this small size the cost of anesthetics can be greatly reduced. In addition the SFE produces high resolution images by using red, green, and blue diode laser light from a single scanning optical fiber. Our research aims to combine the cancer specific biomarker 5-Aminolevulinic acid (5-ALA) and the SFE to fluorescently detect and treat cancerous legions. Once inside a cell 5-ALA converts to Protoporphyrin IX (PPIX), which has a fluorescent excitation peak at 405nm; the corresponding emission peak at 635nm causes cancerous regions to appear red. This is possible because 5-ALA has a high binding affinity to cancerous cells, allowing for a contrast to be seen between healthy and abnormal cells during fluorescent imaging. With prolonged 405nm light exposure and an increase in laser power, we can also cause death of cancerous cells with photodynamic therapy (PDT). PDT causes the photosensitive and cancer bound PpIX to release a singlet oxygen molecule, which in turn destroys vital organelles within the cancer cells, leading to the eventual death of the cancerous mass.

The reduction of CO₂ has become a global priority as the concerns of climate change and the adverse effects of atmospheric carbon dioxide on the environment continue to rise. One method of reducing CO₂ emissions is through conversion and utilization as a chemical feedstock. CO₂ is cheap, nontoxic, and can be recycled to produce valuable new products, making it an attractive feedstock. The increasing importance of CO₂ utilization processes requires a better understanding of the underlying mechanisms of its conversion. The industrial conversion of CO₂ is typically achieved using a heterogeneous catalyst, usually consisting of small transition metal particles supported on a high surface area metal oxide. Our project focuses on elucidating the mechanism of the reverse water-gas shift (RWGS) reaction on platinum, an extensively studied catalyst with a highly active surface. Our goals are (1) to quantitatively measure the RWGS rate under conditions of low H coverage and (2) to compare the measured rate to the rate of CO₂ dissociation (the rate-limiting step according to the surface redox mechanism) and to the theoretical rate calculated using a micro-kinetic model based on the associative mechanism. The information we gather from the investigation will clarify whether the RWGS reaction proceeds on platinum via the surface redox mechanism or the associative mechanism.

The modern United States military is an all-volunteer force and the men and women who enlist or commission are subjected to multiple combat deployments. Many veterans have spent more than five years in a hostile fire zone. This means that the current generation is spending more time on the battlefield than any American generation before it. Methods of Traumatic Event Management (TEM) are employed by the Armed Forces to increase protective factors for servicemembers against the traumas of war. Despite these efforts, the veteran population continues to exhibit a high prevalence of military trauma disorders to include post-traumatic stress, post-traumatic epilepsy, post-war depression, and military sexual trauma. Furthermore, TEM indoctrinates servicemembers with new social regularities, traditions, and norms; combat deepens the meaning of all of these and results in a profound cultural shift. Combat culture stands in stark contrast to civilian and academic culture and includes stigmas which impede returning veterans’ ability to adapt to a peacetime environment. Through phenomenological interviewing, participant observation, and archival research, this qualitative study focuses on the lived experiences of returning veterans in the academic setting and seeks to understand how the presence of combat culture, military trauma disorders, academic culture, classroom pedagogies, and university policy all interact to affect veterans’ ability to adapt to the social environment of the college classroom. Specifically, this research develops a case study on the dynamics of the reintegration process of combat veterans who are returning to college at the University of Washington Bothell. Furthermore, this project demonstrates the integration of community, state, and federal services to meet the needs of readjusting veterans and their families. The transferability of this work is evident through recent interest and attention from leaders of the Department of Veterans Affairs and the Department of Defense.

The mechanism to direct auditory attention consciously is not well understood. For hearing impaired people, it is very hard to direct attention efficiently between conversations with background noise. My project investigated the differences between maintaining and switching attention prompted by visual cues. In the behavioral task, two streams were presented simultaneously, one with high pitch and the other, low. Each stream consisted two triplets of spoken letters and the two triplets were separated by a silent gap that varied in duration. In the beginning of each trial, subjects were prompted by two spoken letters which stream (high or low) to pay attention to initially. Simultaneously, a visual cue directed subjects to either stay or switch between two streams during the silent gap period. Subjects were asked to respond when they heard the second ‘E’ from one of two streams that they attended to as quickly and accurately as possible. Preliminary results suggest that subjects needed more time to respond when they had to switch between streams. As expected, there were no differences between subjects attending to the high or low pitch stream. We also hypothesized that the shorter the gap period, the longer the response time will be—I hope to test this hypothesis through more in-depth data analysis. The result from this project will be analyzed together with neural data obtained using magnetoencephalography where subjects will be performing a similar task. The findings of this project will provide insight into the underlying mechanism of switching of auditory attention—a task that hearing impaired people find difficult to perform in a noisy environment, even with the help of hearing aids or other augmentative devices.

The treatment of a disease is often based on that disease's progression. In cancer, such information is derived from indicators such as the location and extent of tumor within tissue. In addition to determining the presence of a disease, biopsies are often taken with the goal of finding these indicators. Unlike cell biopsy, samples from tissue biopsy preserve biological architecture, allowing these indicators to be more easily determined. For this project, Thin Needle Core Biopsy (TNCB) is used to obtain small cylindrical tissue samples 250µm in diameter and up to 2cm in length. Tissue samples are imaged with Optical Projection Tomography Microscopy (OPTM), which is currently used to generate high-resolution three-dimensional (3D) images of individual cells. Imaging through the tissue is facilitated by axial scanning through the optically cleared tissue; however, the microscope has limited lateral field of view and the TNCB specimen must be imaged in a series of 3D images. This project has three focuses. First, stitch a series of OPTM images using morphological operations, histogram manipulation, and phase correlation. Second, perform image reconstruction to create a single continuous 3D image of all cells within the biopsy. Third, present the 3D images to pathologists in a user-friendly interface. In addition to displaying the 3D image, the computer interface allows clinicians to view cross-sections of the 3D image in the x-, y- , and z-planes to simulate pathological tissue slices. The results allow pathologists to analyze the entire tissue sample, not just samples of cells, and have potential to reduce sources of error in making diagnoses.