Consolidated bioprocessing is the creation of one engineered organism that can handle a chain of chemical reactions that take a low cost feedstock, such as plant biomass, and synthesize high value products such as biofuel or medicine. This project aims to address the first stage of consolidated bioprocessing which is the digestion of the feedstock. Our design is an E. coli strain that can be in one of two states: the consumer state or the altruist state. In the consumer state, the cell carries out normal function by growing and dividing. In the altruist state, the cell produces a large payload of digestive enzymes that degrades plant biomass then lyses to release the payload to the environment where it can act. The consumer cells can then utilize the products of the digestion process to grow and divide. This system is made up of three major genetic components. The first component is a plasmid that designates whether the cell is in the consumer or the altruist state. This plasmid carries a genetic toggle switch that has two operons that produce transcription factors that co-repress each other. The second component is a plasmid that handles the production of digestive enzymes coupled with a lysis gene. Expression of this plasmid is only active if the cells are in the altruist state. The third component a genomic set of enzymes that handles the digestion of cellobiose, the product of the breakdown of cellulose. Initial results demonstrate that the engineered strain is able to grow solely on cellulose and current work focuses on the optimization of the system.

Chagas Disease is a tropical disease caused by the parasite Trypanosoma cruzi. An estimated 100 million people are at risk in 21 endemic countries and ~8 million persons are currently infected. Left untreated, Chagas disease can cause fatal heart rhythm abnormalities, a dilated heart, a dilated esophagus, and/or a dilated colon. The efficacy of current treatments decreases the longer the person is infected, and 40% of treated patients experience adverse reactions, demonstrating the need for new and improved drugs. A drug target has been identified in T.cruzi: the Methionyl-tRNA synthetase (MetRS). Like tRNA synthetase enzymes in other species, the T.cruzi MetRS is crucial for protein production and survival. To exploit this target in T.cruzi we developed a robust assay (Z' factor ≥ 0.5) to identify hits against the MetRS enzyme using a luminescence ATP detection method (Promega's Kinase-Glo™). Compounds that inhibit the T.cruzi MetRS enzyme will be tested for specificity by assaying the compound with the human MetRS homolog. Specific T.cruzi MetRS inhibitors will be tested for potency against intracellular T.cruzi amastigotes and cytotoxicity against mammalian cells. Compounds that are toxic to T. cruzi, not cytotoxic to mammalian cells, and have good pharmacological properties will be selected for hit--to--lead drug development. 

Trypanosoma cruzi is a protozoan parasite prevalent in South and Central America that causes life-long infection in humans. Approximately 30% of infected individuals develop a condition called Chagas disease which usually manifests as life-threatening cardiomyopathy or pathologies in the gastrointestinal system. Over 8 million people are believed to be infected. Unfortunately, current drugs for treating Chagas disease have low antiparasitic activity, are expensive, and are known to cause harmful side effects. As a result, research needs to focus on discovering new drug targets in Trypanosoma cruzi to lead to improved drugs. Most eukaryotic organisms synthesize sterols for essential biological functions that when manufactured incorrectly can lead to cell death. This research is investigating the effects of blocking the synthesis of ergosterol by deactivating an enzyme called sterol 14-demethylase. This enzyme catalyzes one of the intermediates of 20 steps of ergosterol synthesis. Inhibitors of sterol 14-demethylase, known as azoles, have been shown to be extremely active on T. cruzi in vitro and are now the center of research regarding anti T. cruzi drug discovery. We are developing novel azole compounds that target the sterol 14-demethylase homolog of T. cruzi to deprive the cell of its necessary sterols. However, further evidence shows that blocking sterol 14-demethylase can lead to an accumulation of sterol intermediates that are converted into toxic agents in the presence of another enzyme, ERG3 enzyme, which is normally active in a later step in the biosynthesis of ergosterol. The objective of this research project is to analyze the ERG3 homologs of T. cruzi. The putative homologs have been cloned and sequenced. No significant mutations were found. The research now is focused on engineering genetic knockouts of the ERG3 genes to test the hypothesis that the knockout parasites may be resistant to treatment with azole drugs.

In conjunction with Sustainable Seattle, my project seeks to identify barriers to recycling and composting in Seattle in order to ultimately increase the amount of recycling and composting that takes place. My research questions attempt to address this in two parts. First, if Seattle residents participate in recycling and composting, what is preventing them from recycling and composting more? Second, what changes can be made by the government or policy makers in order to encourage more recycling and composting? To identify barriers, I have read past reports studying difficulties in implementing recycling, and also behavioral studies identifying which methods can lead to behavioral change. From this, I discerned that the ideal methodology would consist of conducting interviews of consumers and testing multiple waste infrastructure systems at Seattle Tower using different plans on each floor to determine obstructions to commercial recycling in Seattle. Next, to identify possible behavioral modifications for more renewable action, I will conduct consumer interviews as well as the Seattle Tower project, both of which are still in progress. Initial research suggests that common recycling problems for consumers are a lack of standardized labeling on packaging and products. The results of this study and recommendations about labeling will be posted on the Sustainable Seattle website, and the Seattle Tower will be given infrastructure recommendations for improved efficiency based on the results of what was most effective. Ideally the implications of this project will lead to increased recycling and composting rates in Seattle. This can lower the costs of waste disposal for the public, businesses, as well as the city. Recycling and composting is much more cost effective and environmentally responsible than hauling trash each day to a landfill, and therefore should be utilized as much as possible. 

During the colonial period in Korea the Japanese tried to squelch Korean identity, enfolding the Korean people within their expanding empire. As Japan’s policies and actions on the peninsula became increasingly oppressive, the Western world naively accepted Japan’s benign explanations. However, not everyone disregarded the warning signs in East Asia. Thomas F. Millard, an American journalist, was struck by the Korean plight and took up the Korean cause for independence in his writings. Millard was not only immersed in the affairs of East Asia, he was passionate about what he wrote. As Japanese historians and politicians worked to distort the Korean voice so that it reflected a supposed need for and acceptance of Japanese assistance, Millard attempted to expose their deception. In response to Japanese domination, Korean nationalism rose. Koreans formed a national identity centered on their shared heritage. Identity needs both internal and external recognition. Though the Korean people never identified as Japanese, during the colonial period the rest of the world saw them as Japanese citizens, not Koreans. In 1948 the United Nations agreed that nationality was a human right. Though the Universal Declaration of Human Rights was far from being conceived in the early 1900s some people already believed in the rights that it would solidify; Millard was one of those people. He saw Japan’s actions as criminal, and he wrote about just that. As the Japanese tried to muffle Korea’s voice, Millard became a conduit for that voice. In a present-day world that still wrestles with the vestiges of imperialism, Millard’s work is a testament to the importance of listening to the multitude of voices that continue to sound around the globe.

The Holocaust is an example of an Encounter with the ‘Other’ in world history. It is plain to see that the Nazis viewed the Jews as “other”, but the converse is also true, and to see the Nazis as the “other” is still common today. However, is Nazism an aberration, an “other”, an outsider to the historical process? And even more than that, are individual Nazis, including Adolf Eichmann “other”? There are primary sources from the time regarding Eichmann’s personality and actions. These primary sources include trial and interrogation transcripts as well as literature about Eichmann from the time. There are also secondary sources to see what scholars think about Eichmann. My paper is organized by the questions I ask about the Eichmann texts and the understandings I form through those documents. Some of these questions might be formed as: Is Nazism an aberration, an “other”, an outsider to the historical process? And even more than that, are individual Nazis “other”? Specifically, who is Adolf Eichmann and what problem does he pose for humanity? What does Eichmann as an individual perpetrator mean for history? Is Eichmann the Other? Or, what do we have in common with “evil” and its banality? My primary hope for researching this topic was to understand, in some small way, human evil. To know about Adolf Eichmann and his evil is not to understand him or excuse him. His personality and character (or lack thereof) remains befuddling to me. In terms of what this paper accomplishes, I I address “otherness” as a concept which alienates perpetrator from victim as well as victim from perpetrator, and (for this essay) the perpetrator as Other for the rest of society.

Force-feedback (haptic) displays are becoming increasingly common in consumer technology. Perceptual thresholds for haptic interaction are important for determining how much force output is required from a haptic display to provide detectable feedback to the user. In this presentation, I will talk about an experiment measuring force perceptual thresholds for single and multi-finger interaction with haptic targets. Using the UW Multi-Finger Haptic Display, subjects feel two targets on a virtual surface. One target produces center-attractive force when in contact with the subject’s finger while the other produces no force. A thresholding algorithm presents the subject a new set of haptic targets with a smaller maximum force after correctly selecting the force-producing target twice. Eventually the subject can no longer detect a target and the max force is adjusted upward. This type of forced-choice algorithm causes the threshold force to converge to where the user successfully detects the stimulus 71% of the time. In this experiment, three new methods of presenting time-correlated haptic feedback to the subject were tested. Statistical analysis shows significantly lower minimum force thresholds for multi-finger methods providing time-correlated feedback (mean thresholds of 22.94, 21.75, 22.13 mN) when compared to the single finger and multi-finger spatially correlated methods (29.24 and 33.37 mN respectively). These results suggest a neural summation effect when haptic targets are arranged to provide time-correlated stimulation to multiple fingers at once.

While timely and specific diagnosis can enable optimal treatment of patients, modern-day diagnostics are not optimized for the needs of low-resource settings due to: 1) low sensitivity, 2) slowness in providing results, and 3) dependence on electricity, chemical reagents, and trained clinicians, at a high cost. The result is frequent poor diagnosis, which can increase the rate of negative health outcomes such as patient mortality and evolution of drug resistant pathogens. There is therefore a pressing need for diagnostics that are sensitive, accurate, rapid, and inexpensive. The field of synthetic biology holds much promise in this area due to the ease and affordability of genomic re-engineering and replicating of single-cell organism like Escherichia coli and Saccharomyces cerevisiae. To address this need and opportunity, we have engineered a prototype detector in E. coli that utilizes two DNA-based modules to sense a low concentration of target analyte and produce a large output response. Module 1 detects small concentrations of target analyte by switching cells from an ON to OFF state and Module 2 significantly amplifies an output signal by enabling ON cells to grow rapidly. By seeding these cells in an environment with a non-detector strain that grows faster than OFF cells but slower than ON cells, we hope to demonstrate that this genetic logic enables a detector strain that conditionally and significantly takes over the population after detection event. This approach holds much promise for a multiplexed diagnostic, as different strains of detector cells sensing a specific pathogenic marker could be co-cultured, with the result that only the cells that detect a target analyte grow. As a cell that is a diagnostic could be easily replicated and simple to use, this approach could be extremely feasible for a low-resource setting.

Lignin serves as the natural glue that holds plant fibers together. It constitutes roughly 15 to 30 percent of cellulosic biomass weight. Lignin is the second most abundant natural polymer on earth. Despite lignin’s ample availability across the globe, it has largely been discarded as waste or burned as low value fuel; especially in the pulp and paper industry. Lignocellulosic biomass based biorefineries typically involve substantial capital cost with many negative externalities such as biomass and crude oil price fluctuation as well as changes in government policies (tax deduction, government-backed loans, subsidies, etc.). Therefore, producing added value fuels and chemicals from lignin, the major biomass pretreatment by-product, via commercially viable routes is essential to increase the profitability and the sustainability of these biorefineries. One of the leading biorefinery developers based in Colorado, ZeaChem, is currently investigating the possibility of producing hydrogen gas for their ethanol production by gasifying lignin residues. This research project focuses on modeling the lignin gasification process (using Aspen Plus, a commercial process simulation software) and determining the techno economic feasibility of producing hydrogen from lignin residues. The research approach is explained and some of the research findings - including the hydrogen gas yield, overall mechanical energy and heat consumption, optimum operating conditions, and capital cost - is presented.

Those who have studied genetics have a keen understanding of the fact that incredibly small changes can have tremendous, and often tragic, consequences. In patients with Fragile X Syndrome (FXS), a repeating sequence of genetic information (CGG) in an X chromosomal region is repeated more often than in the general population. This results in a spectrum of conditions including intellectual disability, anxiety, and physical features that deviate from the norm. About one fifth of people who have FXS also have seizures; the underlying cause of seizures in this fraction of FXS individuals is unknown. We hypothesize that FXS individuals who have seizures will have specific genetic variants –duplications and deletions of genetic information –that are not seen in FXS individuals without seizures. The experimental group is composed of DNA samples of FXS patients with seizures, while the control group will be composed of DNA samples of FXS patients without seizures. In order to find genetic variants, I utilize a genomic technique called array-comparative genomic hybridization (array-CGH). In array-CGH, DNA samples are tagged with fluorescent dyes to contrast patient DNA (tagged with pink dye) from control DNA (tagged with blue dye). The ultimate product of this technique is a quantitative representation of any differences between patient and control DNA, which can be processed and analyzed by computer software. By comparing the genomes of these patients, we will determine if there are common genetic variants across the experimental group that are distinct from the control group. If such variants are found, the next step is to associate specific genes with the subset. Establishing an association between a gene and the FXS and seizure phenotype is essential because it deepens our understanding of the condition and provides a foundation for future therapeutic treatments.

 Understanding the heterogeneity in neural activity will help develop theories in neural dynamics and coding - how sensory information is encoded via spike times and patterns. For example, the correlation structure of spiking activity in neural populations, which affects the collective coding capacity, is heavily dependent on the heterogeneous distribution of firing rates across neural networks. A natural cause of heterogeneity in a network arises from the non-regular connectivity between neurons. To capture this natural heterogeneity, we used an adapted Erdos-Renyi node-probability model to construct random architectures for both purely excitatory and excitatory-inhibitory networks of different sizes. The Erdos-Renyi model flips a weighted coin defined by a probability parameter to determine whether a path between nodes exists. However, we need to construct directed graphs, so we adapted the Erdos-Renyi model to construct directed paths based on the probability parameter. For each randomly generated network we examined the coherent structure of the steady-states that persist as the connection strengths between neurons are uniformly increased. In purely excitatory networks, we saw that the steady-state growth is very linear with respect to the connection strength of the network. As a result, stronger connection strengths lead to stronger heterogeneity between the firing rates within the network. We are using the coefficients in the linear approximations to steady-state growth to define the coherent structure of each network. We are continuing towards finding a relationship between the frequencies of three-cell connectivity motifs and this coherent structure since realistic neural network connectivity patterns are not purely random, but have significantly prevalent motifs that define neural architecture. We are specifically looking at how different frequencies of chain, divergent and convergent three-cell motifs affect the first and second order moments of the coherent structure that represents the heterogeneity.

Synapses, the connections between neurons, are not constant in strength but instead are plastic, adjusting due to various biophysical rules. While various types of plasticity have been identified, the interaction of different plasticity rules in a network of neurons in not well understood. We consider the behavior of two experimentally observed forms of synaptic plasticity, homeostatic and spike time dependent plasticity (STDP), and seek to exploit these rules to tune a model neural network towards a state in which activity throughout the network maintains dependence on its inputs. Our network consists of firing neurons arranged in layers connected in a sparse, feed-forward manner. Reliable propagation of layer-wide firing rates without all neurons firing together (synchrony) is required to achieve graded transmission of input rates, that is, signal transmission. Plasticity is introduced to our network through mathematical learning rules. One of our learning rules, STDP strengthens causal linkages by increasing or decreasing synaptic weights depending on the timing of pre- and postsynaptic activity. STDP by itself is often unstable, causing synaptic weights to grow without bound. We therefore attempt to stabilize STDP with a homeostatic rule, adjusting the weights of all of a neuron’s incoming synapses in order to achieve some intrinsic firing rate. While current literature has focused primarily on the behavior of these rules in single neurons or in rate-based systems, we examine the interactions between these rules in a spiking, stochastic system with dynamic inputs, and their effectiveness in tuning such a network towards reliable rate propagation. Our hope is to shed light on the development of cortical networks able to perform successful computation.

The National Cancer Institute estimated that there will be approximately 72,570 new cases of bladder cancer in the United States during the year 2013, and 15,210 deaths due to the disease. The high recurrence rate of 50-70% of treated bladder cancer creates a necessity for life long surveillance, which in turn makes bladder cancer the most expensive cancer for a per-patient cost—89,000 to 202,000 USD per patient—from diagnosis to death. This research aims to combine the cancer specific biomarker 5-Aminolevulinic acid (5-ALA) and the Scanning Fiber Endoscope (SFE) to fluorescently detect and treat cancerous legions in a controlled setting within the bladder, and to find any correlations that might exist between our dependent and independent variables, allowing us to optimize cell death. The Scanning Fiber Endoscope (SFE) created in the Human Photonics Lab (HPL) at the University of Washington was used to apply therapy at 405 nm to A549 cancerous cells previously administered with 5-ALA. Cells were stained with LIVE/DEAD® stain and analyzed under a confocal microscope. The results show that PDT of A549 cancer cells with 405nm light and 5-ALA induces Protoporphyrin IX (PpIX) was successful. Varying sizes in cell death were produced when varying combinations of duration of therapy and light intensity were applied. A correlation between light intensity and duration of therapy was found. An increased time of exposure and a decreased light intensity yields a larger area of cell death than a decreased time of exposure and an increased light intensity. Recurrence of cancers is mediated by cancerous cells that are overlooked in the treatment process, therefor optimizing cell death will allow for a decrease in costs associated with bladder cancer due to a decrease in recurrence rate.

Exoplanets are planets outside our solar system, and the current explosion in exoplanet discoveries is revolutionizing our understanding of the potential for extraterrestrial life. This prolific era of detections has stemmed largely from the unprecedented observing capabilities of NASA's Kepler Space Telescope. The Kepler Spacecraft collects high precision time-series photometric data on a fixed group of approximately 160,000 stars. The data are represented by temporal lightcurves (i.e. brightness vs. time) that can be used to detect transiting exoplanets, the topic of our research. Transits are events where an orbiting planet partially eclipses its host star, casting a small shadow on the telescope. To detect transit signals, we rely on the Quasi-Periodic Automated Transit Search Algorithm (QATS). As an automated tool, QATS provides a crucial means to reduce the Kepler dataset to a manageable size. However, since the algorithm is sensitive to stellar variability, eclipsing binary stars, and systematic artifacts of the spacecraft, additional analysis is required to separate true detections from false positives. Determining the best way to do this is the present focus of our work. Concurrently, we are exploring the potential for QATS not only to determine orbital period, but also to constrain transit depth and duration (properties related to the size of the exoplanet and to the density of the stellar host). While this increases the complexity of the QATS algorithm and the amount of output to manage, it provides greater potential for a fully automated transit search process with results that are more descriptive of the exoplanet systems detected.

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 metals, such as biosynthesis of phytochelatin by the enzyme phytochelatin synthase (PCS), found in plants, algae and several fungal species.  Induced in cells of organisms exposed to elevated heavy metals, phytochelatins also bind non-essential heavy metals such as cadmium and arsenic, offering protection against anthropogenic toxins.  In previous research, Douglas-fir (Pseudotsuga menziesii) seedlings exposed to municipal solids containing heavy metals also had elevated phytochelatins.  The objective of this project was the discovery and characterization of the PCS gene in conifers.  Degenerate primers were designed based on published sequences for PCS from other non-conifer plants including Arabidopsis, then used with the polymerase chain reaction method to search for the PCS gene in genomic DNA extracted from several conifer species.  PCS gene segments of 267 and 133 base-pairs in length were successfully amplified and sequenced from ponderosa pine (Pinus ponderosa) and lodgepole pine (Pinus contorta), respectively.  More recently, using an alternative approach employing complimentary DNA (cDNA) and DNA primers that were designed based on this newly obtained sequence data, an additional 354 base-pair PCS gene segment was amplified and sequenced from Douglas-fir.  Protein alignments between the newly obtained conifer sequences and published PCS sequences from other plants revealed a strong degree of consensus, suggesting that our newly obtained sequences were indeed from the PCS gene.  This constitutes the first report of PCS gene sequences from conifers.  Ultimately, a better understanding of the PCS gene in conifers could enhance the use of phytochelatins as bioindicators of metals stress in conifer forests exposed to anthropogenic metals pollution. 

Nicotinamide adenine dinucleotide (NAD) is used in many biological processes such as metabolism and cellular signaling. Recently NAD is found to have an important role in many signaling pathways. As NADs role in cellular pathways is being discovered, it is important to be able to detect and quantify the amount of NAD in vivo and in vitro by nuclear magnetic resonance (NMR) analysis. NAD is found in cells at low concentrations and its peak intensity in a phosphorus spectrum is much smaller than that of other phosphorus containing molecules like ATP. By decoupling and Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) experiments we hope to isolate and increase the signal of the characteristic phosphorus nuclei found in NAD molecules. Isolating and increasing the signal to noise of the NAD peak will prove that we have the right peak and allow us to quantify the amount of NAD in vivo. This will provide information about health risks of a subject.

Spruce-fir forests in the Appalachian Mountains are experiencing decline; in particular, red spruce (Picea rubens) has largely been eliminated from the higher part of its elevational range. Appalachian forests are downwind from coal-fired power plants and other industrial sources of pollutants in the Midwest, forming a gradient of heavy metals in forest soils reflecting many decades of deposition. Soils tend to reflect historical deposition, while accumulation of metals on lichens reflect more recent deposition. Phytochelatins (metal-binding peptides) provide a more sensitive and direct indication of metals stress at the cellular level. We studied tree stress responses in six sites along this depositional gradient by measuring metals concentrations in soil and on the growing edges of two lichen species (Hypogymnia physodes and Imshaugia aleurites), and identified correlations with metals uptake into red spruce foliage and phytochelatin production in roots and foliage. Metals in soils and foliage were measured using inductively coupled plasma mass spectrometry (ICP-MS), while phytochelatins were detected using high-performance liquid chromatography (HPLC). There was a strong correlation between soil metals and foliar uptake for cadmium (Cd), copper (Cu), mercury (Hg), lead (Pb), and zinc (Zn), while the correlation between lichen metals and foliar metals was only significant for Pb, suggesting that historical metal deposition is a stronger factor than more recent deposition in determining foliar uptake. Recent metals deposition (measured on lichens) for Cd, Cu, and Zn were above what a US Forest Service survey determined as typical background levels in North American forests, implying that despite tighter industrial regulations, forests are continuing to be exposed to toxic metals. In preliminary analyses, phytochelatins were detectable in foliar samples, suggesting that these trees are experiencing metals stress at the cellular level; however, HPLC analysis is still in progress. Our results suggest that red spruce decline in high elevation forests may be related to metals stress.

Treating tumors with radiation requires a trade-off between the necessary dose to eliminate the tumor and the quantity which the healthy tissue can withstand. Radiation therapy with protons allows for a large amount of dose to be delivered to the target area while only delivering a relatively small amount of dose to the surrounding healthy tissue. Utilizing this new method guarantees improved patient safety and treatment success. At the UW Department of Radiation Oncology, a proton cyclotron is being refined to provide a unique system for laboratory experiments in radiation. The purpose of these measurements was to gain further insight into dose distribution of the beam, its lateral spread through air, and its dose delivery efficiency into biological medium. Gafchromic EBT3 film, utilized in conjunction with a rectangular water phantom to simulate biological tissue, measured various characteristics of the beam. When exposed to the beam, the film darkend due to energy-catalyzed cross linking of the polymer. The opacity change in the film was calibrated against a known dose from a clinical beam. Using the film, the dose of the beam was measured at incremental depths leading up to, and behind, the Bragg peak. From the data acquired, it was discovered that the EBT3 film has an under-response of up to 30% dose within the Bragg peak, likely due to effects of linear energy transfer. Under-response of the EBT3 film is prohibitive for measuring absolute Bragg peak dose values, but the relative dose distributions obtained from these experiments provides important insight into the accuracy of Monte Carlo simulations for proton pencil beams.

Biosolids applications can increase heavy metals in soils, which may impact seedling survival in managed forests. Western white pine (Pinus monticola) seedlings, planted in a biosolids-treated site at Pack Forest in the lower foothills of the Washington Cascades, have experienced heavy mortality which has prevented reforestation of the site even after 30 years of replanting. The purpose of this study was to determine whether these seedlings are exhibiting metals stress. Soil and foliar metal content was measured using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Phytochelatins and glutathione, compounds that are induced by metals stress and serve as bioindicators of metals stress, were measured in freshly emerged needles using high performance liquid chromatography (HPLC). Soil metals, at 16 ppm for cadmium, 293.5 ppm for copper, 585 ppm for zinc, and 509.5 ppm for lead, were substantially elevated at this site compared to comparable untreated sites, and furthermore, these were significantly higher than at other biosolids-treated sites. Foliar cadmium was 2.6 ppm, copper was 10 ppm, zinc was 177 ppm, and lead was undetectable; of these, all but lead was significantly elevated in seedlings growing in the biosolids-treated site compared to control seedlings. While phytochelatins and glutathione measurements are not yet complete, based on preliminary results it appears that seedlings are producing elevated levels of these compounds. The elevated foliar metals, in particular the highly toxic cadmium, suggest that these seedlings are experiencing metals stress. Further analysis of phytochelatins levels in seedlings in this biosolids-treated site may help to explain why these seedlings are experiencing such high mortality rates.

Binary star systems offer a deeper insight in regards to the properties of the stars in those systems, as opposed to the observation of a single star. Information from binary stars can help to better understand the lifecycle of stars, the interation among stars, and more accurate predictions as to the mass and radius of certain stars. This study analyzed the radial velocity and lightcurve data from eight binary star systems. Data were collected from the Kepler Space Telescope MAST database. A variety of IDL routines were used in analyzing the lightcurve data. When looking at the lightcurve data we were able to make a breakdown of the curves, determining what portions of light were contributed by Doppler beaming, ellipsoidal deformation and reflection off of the stars. These IDL routines are essentially bits of code used to analyze the data in a systematic manner. We were also able to make rough estimates of the masses and radii of some of the stars using simplified versions of Doppler beaming equations. From the results we were able to identify heartbeat star systems, eclipsing and non-eclipsing binary star systems as well as the periods of the orbits of these systems. Starspots proved to make the interpretation of lightcurves more difficult as they introduced inconsistent changes into the lightcurve data by showing a decrease in magnitude as if from an ecclipsing object but instead from the starspot itself. Future work includes resolving more accurate radii and masses by taking into account the inclination of the orbits by looking more in depth at the radial velocities.

Current estimates place pancreatic adenocarcinoma within the top ten deadliest diagnosed cancers in the US and #1 for mortality (~98%, five years after diagnosis). Consequently, early detection and the study of its pathology are paramount. Typically, this is accomplished through microscopy of flat specimens that are taken from tissue core (TC) biopsies (d=400-1200 µm) of a patient's pancreas. In the Human Photonics Laboratory, we are attempting to enhance diagnostic sensitivity by using a 3D microscopy system, originally designed for single cells, to image entire 400 μm diameter TCs. Imaging thick, opaque TCs requires specimens to be optically cleared and stained. Therefore a microfluidic device (MD) was developed to prepare pancreas (pTCs) and agarose hydrogel (aTCs) cores in an "all-in-one" approach that simplifies, automates and preserves the 3D structure of TCs. Flow and diffusion properties for bulk tissue in MDs deviate from the well-characterized properties of single cells. Subsequently, designing the initial MD required investigating histological dye diffusion and fluid flow with TCs. We found transparent 1% agarose were a suitable pancreas substitute for observation/measurement of volumetric diffusion in an open chamber. In comparison, pTCs needed to be cut open for subsequent analysis. Initial results demonstrate that by 30 min, most dyes completely diffused through larger (L=10 mm, d=2 mm) aTCs, but concentration profiles between dyes varied. Although the density and stiffness of aTCs matched pTCs, diffusion differed, as dye in pancreas samples diffused only 12% as far as in agarose. Properties of dye diffusion into aTCs with flow were then studied on a smaller scale in a test microfluidic channel (L=5.2 cm, d=720 µm). A slow flow rate of 80 µL/s was created by a variable-height gravity line system. Initial results demonstrate that slow flow rates significantly attenuated dye diffusion into aTCs, but allowed the aTCs to remain stationary.

Barrett’s Esophagus (BE) is an inflammation of the lower esophagus. This inflammation can progress to cancer through a stage called High Grade Dysplasia (HGD). One approach to early detection of HGD is the use of light emitting “beacons “consisting of fluorescently labeled peptides targeted to HGD tissue biomarkers. Our laser based Scanning Fiber Endoscope (SFE) possesses the ability to capture live images of the esophagus during examinations and also excite and locate fluorescently tagged targets. Refining SFE fluorescence imaging for clinical use requires mathematical algorithms such as image stitching and distance compensation. Image stitching combines a series of partial views of a 3-D surface into a composite view of the entire surface. Distance compensation addresses the issue of fluorescence signal reduction as the distance between the light emitting beacon and endoscope increases. Phantoms are synthetic mimics of disease conditions used to refine and calibrate new medical devices. This research was directed toward the development of a low-cost, flexible, and durable esophagus phantom with color matching to provide a realistic model of BE. The color of inflamed BE tissue and healthy tissue were matched using quantitative combinations of commercial acrylic paints to create recipes for both tissue types. The resulting paint mixtures were applied to the interior of a latex cast cylinder mimicking the dimensions of an adult esophagus. The painted latex models were shown to a panel of gastroenterologists for approval. The end result was the development of a color realistic phantom onto which fluorescent targets could be affixed. The phantom was imaged using the SFE and demonstrates 2D stitching with distance compensation. (C. Yang, V. Hou, L. Nelson, E. Seibel. J. Biomed. Optics, Feb 2013) Future work extends similar techniques to develop brain tissue phantoms for the calibration of the SFE after integration onto the RAVEN surgical robot.

The field of synthetic biology aims to modularize gene-networks within microorganisms for the purpose of using them as well characterized parts in building novel, useful biological systems. Realizing this goal is vital because biology’s inherent practicality holds the key to needed energy efficient technologies. As the human population increases, energy efficient technologies will become more and more a necessity. The first step in realizing this vision of adapting biology into an engineering discipline is through properly characterizing gene expression and enzyme function within engineered and natural biological systems. To do this, I have developed a turbidostat with mixture control and fluorescence sensing capabilities. A turbidostat is a continuous culture bioreactor that maintains constant culture volume and microbe density through a feedback loop between turbidity measurements and dilution with fresh media. The constant culture environment and microbe density allows the cultured microbes to reach a “steady state” where growth and all environmental conditions are kept constant. By maintaining microbes at steady state, variables that effect microbial processes are minimized, which facilitates accurate examining of gene-network dynamics. The mixture control ability of the turbidostat allows for the concentration of a given molecule within the culture environment to be modulated through time without altering any other environment variables. Through mixture control, a molecule that acts as an input to a gene-network (i.e. an inducer molecule that initializes a gene-network) can be varied in concentration through time within the culture environment. Since most gene-networks utilize fluorescent protein production as their output, the turbidostat’s fluorometer (a fluorescence detector) can monitor the output of a gene-network while the input to the network is modulated. We believe that the ability of this turbidostat to look at the input-output dynamics of gene-networks under defined environmental conditions will yield enhanced behavioral understandings, aiding to the modularity of these systems.

Observations suggest that the intracluster medium in galaxy clusters is strongly influenced by supermassive black holes residing in cluster centers. We investigate the consequences a black hole can have on the thermodynamics of the gas in galaxy clusters by comparing the properties of a simulated galaxy cluster without black holes to X-ray observations of real clusters, which have black holes. For our data we used Python to manipulate a cosmological simulation made with ChaNGa, a code capable of simulating gravity, thermodynamics, and other things. Our cluster simulation includes gravity, hydrodynamics, gas cooling, star formation, and stellar feedback in a fully cosmological setting. However, this simulation lacks black hole formation, growth, and feedback, allowing us to pinpoint the effects of these processes on the cluster by comparing to observations. In our investigation, we found only minor differences in the density, temperature, and pressures of the gas between the observed and simulated galaxy clusters in the cluster outskirts. However, we found significant deviations between the simulation and observations near the cluster centers, where a large spike in star formation is present in the simulated cluster, not seen in the observations. Our results suggest that black hole formation and feedback in galaxy clusters most strongly affect the cluster in the central regions, where it can significantly decrease star formation by heating the gas.

Proton radiation therapy is an emerging modality for the treatment of cancer. Clinical use of protons is growing around the world because of their favorable energy deposition patterns in tissues, but many unknowns remain in terms of the radiobiology of proton radiotherapy. Therefore, more preclinical research is needed. To this end the UWMC is developing a first-of-its kind experimental proton beam specifically designed for preclinical laboratory studies. The goal of this experiment is to compare biological endpoints of DNA damage and repair to radiation dose deposition patterns of the proton beam. As the proton beam passes through tissues it loses energy exponentially. At the end of its track it reaches very high levels of linear energy transfer, LET, i.e. – the energy deposited per unit length. We hypothesize that cells within high-LET regions of the beam are subject to more DNA damage. A549 lung cancer cells were grown to confluence in chamber slides, irradiated with protons or ¹³⁷Cs gamma rays (control), and fixed in methanol at 0, 15, 30, 60, 120, and 1440 minutes post-irradiation. DNA double-strand breaks were indirectly measured by immunohistochemical staining for γH2AX, a histone protein modified at sites of double strand breaks. The resulting biodosimetric assay will provide valuable information concerning DNA damage as a function of varying LET. Future work will extend the results to an in vivo model of mouse brain tissue with the goal of analyzing normal tissue toxicity in high-LET regions of the proton beam. This research has implications for current cancer treatment planning by, for example, avoiding proton beams directed at an organ at risk, or correcting normal tissue toxicity tolerances currently accepted by the academic community.