Despite much investigation, there has been relatively little progress in the development of novel analgesic compounds to treat debilitating chronic pain conditions that affect millions of people. Inherent problems in behavioral screens to identify analgesic compounds include the use of expensive and time consuming low throughput rodent based assay as well as the use of behaviors that only measure the presence or absence of acute behavioral responses to noxious stimuli, whereas assays that measure an animal’s ability to choose between an innocuous and noxious environment overcome these setbacks. In this study, we aim to develop a high throughput two-choice thermal discrimination assay utilizing zebrafish (Danio rerio) larvae that can be used to screen for novel analgesic compounds. In order to do this, we will profile the ability of zebrafish larvae to discriminate between two temperatures. Individually arrayed 5 days post fertilization larvae will be placed in wells containing two distinct thermal zones and assayed to determine if they display a thermal preference between a testing temperature and a normal rearing temperature (28.5^oC). The testing temperature will range in one degree increments from noxious cold (10^oC) to noxious heat (38^oC). We will also test the limits of thermal preference by testing the ability of larvae to discriminate between two noxious temperatures, for example 36 vs 38^oC. To conclude if thermal preference is malleable based on the thermal environment to which larvae are acclimated, we will determine if prior incubation at a selected temperature other than 28.5^oC will alter the thermal preference of zebrafish larvae. Finally to determine if our assay may be appropriate for the use in a screen for novel analgesic compounds, we will investigate whether or not known noxious or analgesic compounds alter the thermal preference of zebrafish larvae.

Liver transplantation is a widely performed treatment for patients with end stage liver disease. Each day, the liver transplant waiting list grows longer due to a shortage of acceptable organs. Hepatic steatosis in donor livers is an increasing problem that contributes to discarding otherwise-transplantable livers. Numerous studies and surgeries have shown that high fat content in livers correlates with poor graft function and lower patient survival. Longer waiting lists are causing transplant centers to consider moderately steatotic livers as a part of extended donor criteria. However, a method is needed to objectively quantify fat content in livers in order to select suitable organs. Many studies over the past 30 years have characterized a correlation between fat content of livers and acoustic properties such as sound speed and attenuation. This research seeks to employ an acoustic caliper device to acquire acoustic measurements that can be used to quickly, accurately, and noninvasively evaluate the fat in transplant donor livers. Sound speed and attenuation were measured by transmitting short pulses of ultrasound at frequencies from 1-10 MHz through excised pig and cow livers. Liver thicknesses were measured with a dial caliper to permit sound speed estimates, and reference measurements were taken in water to calculate the additional attenuation created by the presence of the liver. In addition, a thermocouple was used to take precise temperature measurements in liver and water, since sound speed varies with temperature. Although fat content was not quantified, sound speed and attenuation did correlate with the presence of visually observable fat. However, significant variabilities between measurements were also found with regard to the preparation of the liver samples and tissue inhomogeneities. This work has led to ideas for an improved acoustic caliper device and for testing lipid emulsions as tissue phantoms for evaluating measurement capabilities.

 A new non-visual method of numeric entry into a smartphone is designed, implemented, and tested. Users tap the smartphone screen with one to three fingers or swipe the screen in order to enter numbers. No buttons are used—only simple, easy-to-remember gestures. A preliminary evaluation compares the method to a standard accessible numeric keyboard with a VoiceOver-like screen reader interface. Preliminary results indicate that users can enter numbers faster and with higher accuracy when using this method. The Tapulator, a complete calculator based on this non-visual numeric entry that uses simple gestures for arithmetic operations and other calculator actions is described.

The hair cells of the inner ear are the functional sensory units of hearing and balance in the auditory and vestibular system, respectively, and are therefore a major cause of deafness and balance deficiencies when lost after damage. Unlike in reptiles, birds and fish, existing studies suggest that the mammalian sensory epithelia of the inner ear lack the ability to regenerate hair cells after such damage. Here we focus on the cristae, which are a component of the vestibular system, and are responsible for sensing angular acceleration and rotation of the head.  Previous research has suggested that Notch activity, a crucial signaling pathway necessary for patterning the sensory epithelium, continues in adult mouse cristae, suggesting the potential for regeneration after damage.  We characterized a lesion protocol for this system through the administration of the ototoxin iminodipropionitrile (IDPN) to test whether spontaneous regeneration of hair cells is present in the mammalian cristae. We used confocal microscopy to image the layers of the cristae's sensory epithelium  after immunohistochemistry staining with markers specific to hair cells.  Each cristae's hair cells were counted, and an average was obtained for cristae for each of several time points after IDPN injection. Preliminary findings show a severe reduction in total hair cell number in the mouse cristae one week after IDPN injection, and continued loss of hair cells in subsequent weeks.  While we saw no evidence of spontaneous regeneration in the mouse cristae, this lesion protocol may be used in further hair cell regeneration studies using various manipulations or drugs, such as inhibition of Notch signaling.

Our study aims to determine how physiological measures of affect such as heart rate variability and galvanic skin response may be linked to other representations of emotion that arise naturally in text-based communication during online collaboration. Research in this area is important because people increasingly collaborate in a distributed manner across greater distances, and text-based communication is one of the primary means of facilitating these collaborations. The inspiration for this work comes from Aragon and Williams' (2011) paper, which introduces a complex systems model of collaborative creativity and describes ways that affect contributes to this model. In this study, we test and validate the model by using physiological measures to detect and explore the relationship between affect and creativity. Possible outcomes include correlations of creative events with physiological measures of affect and/or the occurrence of affect in chat messages.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human pathogenic γ-herpes virus and is the infectious cause of Kaposi’s sarcoma (KS), primary effusion lymphoma, and plasmablastic multicentric Castleman’s disease. The KS tumor is highly vascularized and is characterized by latently infected spindle cells of endothelial origin. While in the KS tumor 1-5% of the cells are lytic and producing more virus, greater than 90% are latently infected and not producing virus. The goal of this project is to investigate whether the latent genes expressed by KSHV are sufficient to alter endothelial cells to induce angiogenesis, metabolism, and oncogenic cell signaling. We will determine if latent genes alone induce these effects or if the small percentage of lytic genes play a paracrine role. In order to do this we are cloning the entire KSHV latent locus into a gutted adenovirus expression vector that eliminates the contamination from expression of adenovirus genes. The latent locus includes the 4 genes expressed during latency and the 12 virally encoded microRNAs. The adenovirus vector expressing the latent locus will be used to infect endothelial cells and we will examine the cells expressing the KSHV latent locus for changes in factors involved in angiogenesis, metabolic pathways and oncogenic cell signaling. If the latent genes recapitulate the pathways of a KS infection, this will indicate that latent gene expression alone, without the low percentage of lytic genes in the culture, are sufficient to induce many cellular pathways induced by KSHV and are relevant for the pathogenesis of KSHV in KS tumors.

Thermostability is the capacity of an organism to survive and thrive at high temperatures and is a characteristic with many biotechnological applications, such as food processing. We are seeking to understand the mechanisms of thermostability in viruses by using an RNA virus, phi-6 Cystovirus. We are evolving the phi-6 virus to become thermostable by exposing it to a target temperature of 61°C over many generations (wild type viruses grow at 25°C). In order to see if exposure to different intermediate temperatures affects the genetic pathway to thermostability, we will expose the phi-6 virus to different temperature regimes prior to reaching the target temperature. The temperature treatments are sudden, where the virus is exposed to the target temperature throughout the entire course of the experiment, moderate, where the virus is exposed to the target temperature halfway through the experiment, and gradual, where the virus is exposed to the target temperature on the final day of the experiment. The genome of phi-6 has already been characterized, which enables us to compare the genome of the evolved, thermostable phi-6 viruses to the ancestor and allows us to study the genetic basis of thermostability. In future studies, we hope to use these thermostable viruses to learn about costs and benefits of maintaining thermostability.

 The performance of machine learning (ML) classification algorithms in an open-ended problem with manual labels is difficult to assess, because errors can exist both in classification and manually labeled datasets. An additional dataset of manually re-annotated data can help to differentiate classifier and manually labeled data errors; however, there is currently no effective way to visualize all three datasets simultaneously. To address this problem, we introduce a new type of visualization called a confusion diamond. The confusion diamond consists of 8 triangles, with each comparing different dimensions of all three datasets. The arrangement of the triangles complement traditional confusion matrices and utilize many of their visual properties. The confusion diamond layout allows users to pre-attentively perceive data errors as distinct from classification errors, which makes the visualization easy to use. Multiple confusion diamonds can be analyzed at once, making it useful for datasets containing multiple labels, and giving researchers the ability to quickly identify which labels have errors and how to solve them. This work presents the key design elements of the confusion diamond visualization, relevant usage scenarios, and findings from semi-structured interviews with members of a research team interested in using the confusion diamond to help them visualize ML classification performance on large social media datasets.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has been used extensively to study the biology of aging, and several determinants of C. elegans longevity are conserved in higher order organisms. The specific mechanisms of longevity in C. elegans have not been completely identified and many pathways are still poorly understood. Dietary restriction (DR) is one promising pro-longevity intervention, though its mechanism is poorly defined. DR, a reduction in caloric intake in the absence of malnutrition, extends lifespan in C. elegans and in a wide range of other species, including yeast, flies, mice, and primates. However this extension is highly dependent on genetic background. To investigate this, we are performing a genome-wide screen of the Vidal RNA interference (RNAi) Library, which has a catalog of 11,511 clones. By knocking down specific genes within the C. elegans genome through RNAi, we are able to observe an extension or reduction in longevity in response to DR. We have screened over 1500 genes; 61 of which are short-lived and 67 of which are long-lived. We are currently following up on our collected data by measuring the full lifespans using the RNAi clones that significantly shortened or extended lifespans in our screen. By screening such a large number of genes, the widespread effect, and hopefully mechanism, of DR will be elucidated. We hope to see genes significantly affected by DR cluster into groups with common functionality, or along the same pathway, and then follow up on those specific pathways to clarify the mechanisms underlying DR. If these pathways are conserved, research findings relating to C. elegans may be applicable to other organisms. We hope to confirm these results and further investigate the interaction between DR and other potential longevity pathways.

Public transit plays an important role in the daily lives of blind individuals. Unable to drive, blind commuters must rely on buses, subways, and other methods of public transportation to travel from one destination to another. Although transit schedules and even real-time arrival information are now readily available and accessible, there are still barriers to overcome. For example, a blind bus rider may find it difficult to locate a designated bus stop. Prior research has shown that, given a set of information about a bus stop, blind commuters will find their bus stop with significantly greater ease and lesser time spent. Our goal is to create a database of useful and reliable information regarding every bus stop in Seattle and make that information accessible to blind individuals. In order to collect such a vast amount of data, we have turned to crowdsourcing, a process that involves dividing work amongst a large group of workers over the Internet. By providing workers with Google StreetView images (ground-level visuals on the appearance of a location) and simple, multiple-choice questions, we hope to collect accurate information to answer questions such as “How many benches are at the bus stop?” or “In what direction is the bus stop relative to the intersection?” After acquiring a sufficient set of bus stop data, we will test the usefulness of this information to blind commuters by providing them with access to our database and analyzing their feedback. We believe that our work in this field will improve the public transit experience for blind individuals by providing them with a feeling of confidence and independence during their commute. Our work in this field may lead to further research in utilizing crowdsourcing to solve accessibility issues.

Rhododendrons are plants native to the Northern hemisphere, comprising over 1000 species, most of which have eye-catching flowers. The native Rhododendron of the Pacific NW, R. macrophyllum, harbors a degree of DNA sequence variation exceptional for a single species. Our study focuses on RPB2d, the gene encoding the second largest subunit of RNA polymerase II. Within intron 4 of R. macrophyllum RPB2d, there exist four conserved haplotypes, which have a structured geographical distribution. (Puget Sound differs from Oregon Cascades differs from Oregon Coast.) This surprising pattern of intraspecies variation inspired my project of looking into the degree and pattern of genetic homogenization in these populations by recombination events within and near the RPB2d gene. Thus far, I have sequenced most introns throughout RPB2d and some of the non-coding region upstream. The results have shown that the intron 4 haplotypes are linked to specific variants in intron 1. However, the later introns (6-13, 13-15, and 23-24) are uncoupled from the intron 4 haplotypes and show increasing recombination. About 1 kb upstream, there is a 341 bp DNA sequence inversion in certain plants. An inversion is a segment of a chromosome that has been reattached on the same chromosome in the same location, but in the opposite direction. The inversion found in most RPB2d genes of R. macrophyllum is not found in other species or in haplotypes 1 and 4, the ones most similar to other species. The data continues to show that fewer recombination events occur in introns closer to the inversion. My research is now focused on how the inversion limits the rate of recombination. The next step will be to sample heavily from homozygous populations to see if there is a normal rate of recombination. This regulation could help explain the persistence of varied haplotypes in intron 4.

Jane is blind and in need of a new wall oven for her apartment. As she shops for the new oven she discovers that almost all ovens have digital displays for showing the state of the oven, its temperature, and the time left on its timer. They also have buttons flush with the display panel for providing input. She knows from past experience that she can put Braille labels on the buttons and will eventually memorize which button does what, so input will not be a problem. However, how will she read the digital display? Through a friend she learns of a smartphone application called Appliance Reader, which can read digital displays for various appliances including some wall ovens. In order to implement the appliance reader, we have to utilize computer vision algorithms particularly those that match one image against another. The two images can be taken from different perspectives so geometric transformation needs to be inferred from the two images. Once the two images are matched properly, the digital data has to be interpreted to form what would be told to the user. The interpretation requires a machine learning algorithm because standard optical character recognition is not sufficient. A user interface suitable for blind users is needed to make the Appliance Reader practical. As a part of CSE Mobile Accessibility group, which develops smartphone application to aid handicapped people, the two of us have been developing Appliance Reader from the ground up. Under the guidance of Professor Ladner and our mentor, an Intel researcher, Bryan Russell, we have been researching and building Appliance Reader, which is a computer vision-based smartphone application.

Isochrysis galbana is a marine alga that produces large quantities of lipid per cell. Lipids contain extensive amounts of potential energy. Therefore, this haptophyte may be considered for use in biofuel production. The lipid body organelle—the site of neutral lipid storage—is involved in many cellular processes. To date, little is known about this organelle in I. galbana. Chloroplasts contain lipids stored within smaller structures, termed plastoglobuli. For the purpose of the study, lipid body and chloroplast isolation techniques have been developed for I. galbana.  While light intensity has shown no direct effect on I. galbana’s growth rate, we hypothesize different qualities of light will affect growth by altering cellular protein, carbohydrate and lipid content. We will culture I. galbana under red and blue wavelengths of light. Using chloroplast isolation techniques, as well as developed lipid body isolation procedures, we will analyze the lipids contained within I. galbana lipid bodies and chloroplasts when grown under different light qualities. We will also determine if these wavelengths of light induce a stress response concurrent with lipid production. Preliminary data shows that heat shock proteins 70 and 90 –proteins that maintain homeostasis within the cell—are present in I. galbana whole cell protein extracts as well as isolated lipid body proteins. Using Western blot techniques, the abundance of these stress proteins will be measured under different light conditions. These studies will provide information on growth and lipid production under different light qualities, and whether these wavelengths of light elicit a stress response in I. galbana, an alga with potential for applied biomass production.

Drilled oil is becoming increasingly expensive because it is a non-renewable resource that the planet depends on for energy and transportation. Algae may become the next source of global fuel. Chrysochromulina sp. is an ideal model for algal biofuel production because it has a high growth rate in culture, and the two lipid bodies (the organelles that store potential biofuel precursors) are easily detectable using cell biological techniques. The ability of Chrysochromulina sp. to change its lipid metabolism quickly in response to replicated environmental stress conditions is a crucial factor to better understanding its growth optimization and survival. Based on our preliminary data, this microalgae has the potential for production and commercialization of its lipid-based compounds. Guschina and Hardwood’s 2006 studies have shown that lipid production increases in algae under various environmental stress conditions, such as nitrogen limitation, salinity, or increased temperature. Thus, we have investigated the growth dynamics of Chrysochromulina sp. under the following stress conditions: increased salinity, depleted nitrate levels, depleted ammonium levels, and depleted phosphorous levels. For each of these experiments, flow cytometry was used to obtain cell counts and determine growth rate (K’). Neutral lipid content and lipid body size are essential measurements because they represent how efficiently Chrysochromulina sp. produces lipids (i.e. biofuel) and the characteristics of the different lipids being produced. Using Gas Chromatography/Mass Spectrometry (GC/MS), we have elucidated that the lipid profiles change under different growth conditions. The results attained from these studies provide relevant information about the physiology and ideal growth of lipid producing conditions of Chrysochromulina sp. Using this information, we will be able to determine the proteins involved in lipid biogenesis and apply genetic modification techniques to optimize lipid production for commercial use.

Adenoviruses (Ads) are the most exhaustively researched and well-understood viral platforms for gene therapy and oncolytic virotherapy, with potential applications including the treatment of numerous inborn and acquired genetic diseases, including cancer. However, when injected into the bloodstream, Ad vectors can bind several blood coagulation factors that facilitate the transduction of hepatocytes, sequestering the virus in the liver. Although both coagulation factors VII and X bind the Ad5 hexon with very high affinity, only FX appears to play a role in mediating Ad hepatocyte transduction in vivo. To understand the discrepancy between efficacy of FVII binding to the virus and its poor capacity at supporting cell entry, we conducted structural analyses on the Ad5-FVII complex. Using high-resolution cryo-electron microscopy followed by molecular dynamics flexible fitting (MDFF) simulations, we have determined that the FVII GLA-domain sits within a surface-exposed hexon trimer depression in a different orientation than found for FX, althouth several of the same hexon residues that mediate FX binding also mediate FVII binding to hexon. Furthermore, we found that two proximal FVII molecules on the viral surface may interact via their serine protease (SP) domains, burying potential heparin sulfate proteoglycan (HSPG) receptor binding residues within the dimer interface. Several viruses with point mutations in critical residues of the hexon protein were engineered and grown. Infection assays revealed cell transduction efficacy that varied independently of the strength of the interaction between the hexon and FVII. MDFF simulations revealed an altered interface between hexon and FVII that could affect the orientation of FVII in the pocket and, thus, the efficacy of dimer formation. Since the occluded HSPG domains are crucial for cell entry, our results suggest that differing binding orientations for FX and FVII GLA domains lead to disparate quaternary structures, which ultimately determine the cell-transduction efficacy of the complex.

Emerging from the political decline of the Islamic “Gunpowder Empires,” the Central Asian Nadir Shah assumed leadership of the former Safavid Empire before winning notable victories over his Ottoman and Mughal counterparts. In this paper, I attempt to grapple with the complex issue of Nadir Shah’s ethnic identity, which has alternately been presented as either Turkic or Persian. As such, I attempt to navigate the trend of nationalist historians to appropriate Nadir Shah into their own nation’s retroactive “national history,” relying not only on the works of such historians, but also those more removed from nationalist perspectives. Using readings spanning from the 18th century until the present day, I note the shifts in Nadir Shah’s ethnic identification across nations and time periods as well as the differing degree to which historians attempted to restrict the king’s identity to a single ethnic group. Highlighting political changes affecting Central Asia during the 18th century and their enablement of Nadir Shah’s navigation of class and race-based hierarchies, I then critique narrow national identifications of Nadir Shah and attempt to highlight nuances within the issue of pre-nation state ethnic identity. In conclusion, I assert that pre-nation-state identity may have been mutable and that Nadir Shah’s identity, rather than sitting on one side of a Turkic-Persian dichotomy, reflects a fluidity of nationality extant in 18th century Central Asia.

The pancreatic islet β-cell maintains blood glucose homeostasis by releasing insulin, a process that becomes dysfunctional in type 2 diabetes mellitus. Thus, maintaining normal β-cell function is critically important and occurs through many mechanisms including interactions with the surrounding extracellular matrix (ECM). We have recently shown that the polysaccharide hyaluronan is a normal component of the islet ECM. Under normal conditions, hyaluronan exists in a high molecular weight (HMW) form (>500 kDa) which exerts anti-inflammatory effects on surrounding cells. One study has shown that HMW hyaluronan can potentiate β-cell glucose-stimulated insulin release (GSIS). Our preliminary data suggest, in diabetic islets, hyaluronan can become fragmented. Low molecular weight (LMW) hyaluronan (<10 kDa) is pro-inflammatory. Furthermore, agents that can activate the same intracellular signaling pathways as LMW hyaluronan (e.g. free fatty acids and lipopolysaccharide) have been shown to impair GSIS, suggesting LMW hyaluronan may have a similar effect. I hypothesize that HMW hyaluronan potentiates GSIS, whereas LMW hyaluronan inhibits GSIS in vitro. Rat pancreatic islet cells were cultured on plates coated with HMW hyaluronan, LMW hyaluronan, or L-ornithine (control). After 48 hours GSIS was determined by comparing insulin release under basal (low glucose) and stimulated (high glucose) conditions. Our preliminary data show that as expected HMW hyaluronan potentiates GSIS 2 fold while LMW hyaluronan decreases GSIS by 30% compared to control. Thus it appears HMW hyaluronan is beneficial for β-cell function whereas LMW hyaluronan lacks this effect. Hence, hyaluronan fragmentation in the islet under diabetic conditions may contribute to impaired β-cell function.

Pathologic enlargement of the esophagus, termed megaesophagus, results in a failure to complete peristalsis leading to vomiting, severe weight loss and potential death. Megaesophagus is characterized in a number of disease states including parasitic (Chaga’s disease), autoimmune (myasthenia gravis), and neuromuscular (muscular dystrophies), however, a detailed cellular and mechanistic understanding is lacking. To address this deficit, we performed a comprehensive examination of the onset, pathology and cellular composition of megaesophagus in the mouse model of Duchenne muscular dystrophy (DMD). DMD affects ~1/3500 male births and is a catastrophic and ultimately fatal muscle wasting disease resulting from a mutation in the gene encoding the integral skeletal muscle protein dystrophin. Although poorly defined in human DMD patients, megaesophagus is documented in canine and mouse models of DMD. Advancements in DMD therapies has increased patient lifespan and underscored a need to assess the impact of the disease in a range of organ systems, including the esophagus. We find that older dystrophic mice can develop severe megaesophagus, but that the timing of onset depends on the nature of dystrophin expression in muscle and non-muscle tissues and the genetic background of the mice. Further studies are in progress to delineate the precise dystrophin expression pattern in different cell types of the esophagus that contribute to pathology. Work presented here will be essential for future work aimed at identifying new therapeutic targets for DMD.

 Flying organisms achieve flight stability by employing a multitude of control surfaces, most notably the wings. However, airframe deformations, such as abdominal motions in the hawk moth Manduca sexta, have recently been shown to play a significant role in stabilizing flight. In my research I have studied the control potential of abdominal deflections using a closed-loop flight arena. Tethered moths controlled the velocity of a projected black bar with their abdominal angle. Image velocity varied according to the difference between the abdominal angle and the set point (relative to the average abdominal angle), scaled by a gain factor. Experimental trials were performed for a ten-fold range of gains at three different set points and consisted of 60s periods during which the moths attempted to stabilize the drifting bar. The moths were capable of stabilizing the image for all experimental conditions, with the highest average performance (50%) occurring at the medium gain and the set point corresponding to the average abdominal angle. Poor performance (<50%) during low gain trials was characterized by steady state error, likely the result of the relatively low image velocities. For high gain trials, the decreased performance (<35%) was characterized by large abdominal oscillations. These results support an active and plastic role for the abdomen in flight control, but also tested the limits of the abdominal control circuit. To continue exploring the role of the abdominal motion I have been working on performing EMG recordings from the abdominal muscles during free flight. For this research the virtual arena serves as a controlled environment to collect preliminary data and to refine the experimental procedure and animal preparation. Finally, I will present my work developing a biometric controller for the Parrot AR.Drone quadrotor. This project utilized a Kinect and custom software to control the quadrotor with body postures.

Visual-Motor processing provides a mechanism for animals to translate visual cues into useful information for locomotion. My research aims to investigate this mechanism by analyzing the responses made by fruit flies to certain visual stimuli. I accomplish this by first tracking and quantifying fruit fly locomotion when two flies are present. By quantifying this behavior I can create a behavioral map of fly responses in relation to other fruit flies. I can then replace one of the flies with a cylindrical, fly-sized magnet that is readily controlled by me. This step is important because by controlling the magnet it is possible to reproduce large amounts of a specific behavior, instead of watching two flies interacting randomly and waiting for relevant interactions. It also will allow me to investigate the affects of object speed and angle of approach and create a behavioral map of the fly's locomotor response. Once this is accomplished it will then be possible to test fruit fly responses to visual stimuli presented on an LED screen surrounding the fly and to measure brain activity in the flies while these stimuli are displayed. Ultimately we hope to identify the neurons that are responsible for visual-motor processing and how they accomplish the transition from visual input to locomotor response.

Death has been a phenomena that has been dealt with in varying ways throughout human history. How we deal with physical death online, specifically through Facebook, has proved to be an insightful challenge to the current digital age. The use of Facebook as the link between the living and the dead highlights the level at which we appropriate our Facebook profiles as extensions of our physical body due to the desire for reinvention associated with death. While only a few studies exist directly related to death on Facebook, they expose communities of mourners interacting with each other and the deceased on a surprisingly horizontal level, and contacting the dead without necessitating time restraints or religious/spiritual intermediaries. Traditional death rituals usually have temporal limits on liminal periods of mourning, divided between private and public time, but Facebook offers us unlimited access to memories of the deceased. The choice to mourn communally and virtually problematizes not only our personal relationships with the deceased, but also the legal ownership of our feelings and sentiments. The final element of death on Facebook examined in this paper is the spiritual space that deceased profiles occupy. For the secular and religious, these profiles give a virtually physical body to what might traditionally be called a spirit, which challenges the finality and permanence of death. In studying the posts and stories that decorate the profile walls of the deceased, it is clear that this virtual aspect of ourselves has very real elements, and it is exactly this questioning of reality that death demands of us in the first place. 

Mobile devices have become ubiquitous in today’s society, yet their role in artistic practice is often relegated to documentation, communication, and play. I have spent the last quarter as the TA for Media, Time, and Technology, a course designed to explore the use of mobile devices in contemporary arts research and practice. Student assignments ranged from concrete conceptual exercises to thought experiments designed to explore and develop idealized future works. My research draws on my experiences in this class as well as the development of my own artistic practice, focusing not only on emerging technologies but also the exploration and reconceptualization of historical works of art. Through the marriage of technological exploration and historical interrogation I hope to develop a personal practice that is grounded in the rich lineage of art yet always looking forward.

Ischemic preconditioning (IPC) is a robust neuroprotective phenomenon in which a brief ischemic exposure increases resistance to the injurious effects of subsequent prolonged ischemia. Characterizing the cellular and molecular mechanisms of IPC is an active area of investigation in stroke research. Microglia are the resident immune cells of the central nervous system and are the primary cell type responsible for post-stroke neuroinflammation in the brain. Several lines of evidence support a role for microglia in IPC. Previous research from the Weinstein laboratory has demonstrated using ex vivo flow cytometry that IPC induces a robust increase in the number of microglia/macrophages that can be identified and sorted from the preconditioned (ipsilateral) cortex. However, it is uncertain if this increase reflects a true change in cell number (due to proliferation or migration) or if it reflects a novel characteristic of the preconditioned microglia to better survive the enzymatic and mechanical tissue digestion that precedes the flow cytometry processing. In order to address this question, we are carrying out quantitative immunofluorescent microscopy on tissue sections from preconditioned or naïve mouse brains that have been stained with a fluorochrome-conjugated antibody (anti-Iba1) specific for identifying microglia/macrophages. My contribution to this work will include tissue preparation and sectioning, antibody staining, microscopy image capture and cell quantification using custom calibrated image analysis software. Answering this initial yet fundamental question will be an important starting point for additional investigations into the function and phenotype of microglia/macrophages in the preconditioned brain and may shed light new light on the mechanism of IPC.

During embryonic neural development mouse hindbrains exhibit spontaneous depolarizations that propagate from cell to cell across the tissue in waves. There is a discrete, anatomically identifiable population of cells responsible for generating these waves: the cells of the initiation zone (InZ). Spontaneous waves of activity have been shown to affect neuronal migration, pathfinding, and differentiation, among other developmental sequences. Spontaneous depolarizing events, in general, allow passage of ions into and out of the cell; of developmental importance are calcium ions, which are involved with regulating gene expression. During critical windows of development these calcium fluxes help to determine the adult fate of a neuron. The spontaneous and variable nature of the propagating neuronal events makes them difficult to manipulate experimentally. Were these propagating events able to be elicited experimentally and discretely that would provide both a vehicle for further study and an indication of what properties of the initiator population allows the cells to generate propagating events. I have used a combination of several techniques, including calcium-dye imaging, whole-cell patch clamp, and extracellular electrical stimulation, to probe the details of the cells’ activity. These experiments have identified separate populations of cells that are capable of initiating propagating waves, both spontaneously and elicited by external electrical stimulation. One population includes the previously identified cells of the InZ, and another population exists at the isthmus between the midbrain and hindbrain. Cells in these populations respond differentially to a variety of electrical stimuli. Within each of these initiator populations may exist single, highly-networked neurons capable of initiating propagating electrical activity evoked by intracellular stimulation, which is the subject of further investigation.

Rio de Janeiro is infamous for violence. In the city’s large, informal shantytowns known as favelas, violent drug gangs rule with impunity while a corrupt and murderous police force contributes to distrust of formal government. With Rio scheduled to host the World Cup in 2014 and the Olympics in 2016, pressure to tame the favelas and reduce violent crime is high. Facing the need to try something new, in 2008 Rio’s leadership introduced an innovative, community-oriented policing program known as UPP in an attempt to “pacify” the favelas. The program represents a paradigm shift in policing methods, focused on breaking down the adversarial relationships between favela residents and police and encouraging community involvement and integration. It has shown impressive progress: in favelas that have received UPPs, murder rates have fallen to nearly zero. However, much remains to be done--the UPPs are an expensive fraction of the police force, and skeptics worry that if the UPP’s leave, all progress will be lost. The challenge, then, and the focus of this Task Force, is to ensure that the favelas are integrated and supported in a way that preserves the progress that has been made and fosters permanent change. Each of the 11 chapters within this Task Force presents policy recommendations prepared for the Public Security Secretary of Rio de Janeiro José Beltrame, and as such, are tailored to his position and responsibilities. Subjects include reforms within the police force, such as improvements in police training, organization, and working conditions, as well as policies intended to promote integration of the favelas into the formal city, including methods to improve education and access to healthcare, implementing programs to target at-risk youth, and providing economic opportunities for favela residents.

Since the international ban on the ivory trade in 1989, environmental science has been constructed as a ‘value-neutral’ and ‘objective’ realm in which elephant conservation is expressed as a universal reality. However, preservationist formulations of environmental science have been privileged over their utilitarian counterparts, precluding many Southern African countries from employing science-based arguments in favor of sustainable ivory harvesting. Concurrently, the relatively recent global transition from consuming elephant bodies to consuming elephant imageries has facilitated the production of a wildlife-based international tourism market, as well as reinforced the belief in delineated conservation areas. In this context, transfrontier conservation areas (TFCAs) have gained traction as a means of simultaneously restoring historic elephant ranges and promoting regional tourism to benefit African elephants and economies alike. As a result, environmental science as a political tool has been utilized to produce elephants as either regional or global resources, silencing local and national claims to elephant populations and related management. In the process, placed-based frustrations for local communities associated with problematic elephant populations and related human-wildlife conflict have been subsumed under the importance of the regional environment and national economic development. To explore science’s role in framing and understanding environmental problems, I analyze how scientific concepts such as biodiversity and the precautionary principle are utilized in international conservation policy and the elephant management debate, with particular emphasis on Botswana. I aim to show that science is neither ‘objective’ nor ‘neutral’ but instead is a field with international politics that recommend vastly different philosophical and ethical ways of understanding humanity’s role in the environment.

The successful growth of a plant is dependent on many variables, such as location, extent of available resources, competition by other plants, and reproductive success; what if there was a way a plant could almost guarantee survival and success? Many invasive species have found ways to be very successful; Alliaria petiolata (garlic mustard) has developed allelopathy, which can decrease surrounding plants germination rates and ability to grow. In my experiment I will attempt to determine if different populations of garlic mustard have variations in allelopathic strength. At least ten samples of garlic mustard from three different locations throughout Seattle will be grown, along with ten control lettuce plants. The three sites for this experiment are Seola Park, Woodland Park, and the University of Washington. Throughout the experiment I will try to figure out if the lettuce seeds germination rate and growth abilities are affected as a result of the garlic mustard. I will plant lettuce seeds in with garlic mustard and analyze the germination rates compared to the control samples, and at the end of my experiment I will harvest the lettuce and garlic mustard samples, and they will be biomassed. The data collected will be analyzed for significant results. My hypothesis for this experiment is that the sites in which the garlic mustard has been present the longest will have the greatest negative effect on the lettuce plants; but, because I don't know how long each population has been established I believe the Seola Park location, because it had the largest garlic mustard population, will have the largest effect on the lettuce.

This project strives to address the surprisingly high rates of eating disorders (ED) in southern Italy. Determinants for ED in southern Italy are characterized by perfectionist tendencies and parental criticism, rather than the low self-esteem and body image problems most often associated with ED. The high prevalence of ED and unique composition of symptoms and psychological determinants among southern Italian women point to a compelling paradox: Given the widely supported theory that the development of eating disorders in a population may be ascribed not only to media-stimulated body image concerns but also to processes of modernization and industrialization, why do women in southern Italy, which has not conformed to or successfully undergone these processes, exhibit relatively high rates of eating disorders? This thesis argues that ED in southern Italy is a symptom of transgenerational transmission of trauma related to collective regional fear of the end of WWII and loss of the precarious economic certainty of the Allied occupation. This fear and eventual loss could not be openly mourned because of its conflict with the Resistance and liberation myths that sustain WWII memory in the popular national narrative. To substantiate this claim, I will further propose that both culture-specific ED pathologies and acclaimed films represent ciphered articulations of the psychically “indigestible” nature of this conflict. I will analyze selected films of acclaimed southern Italian directors: Ettore Scola’s La famiglia, Giuseppe Tornatore’s Cinema Paradiso, Francesco Rosi’s Tre fratelli, and Mario Martone’s L’amore molesto. Each film is analyzed anasemically—that is, completely wthin the bounds of its auditory, visual, and cinematographic language—before connecting it to historical and social contexts.

Tribal communities often lack access to high-quality healthcare, prevention programs, cancer education, cancer screening tests, and cancer clinical trials. As a result, American Indians and Alaska Natives experience the worst cancer-related disparities and the poorest survival rates of all racial and ethnic groups in the U.S. Comic books have long been used as an educational tool to improve public health. In 2008, the Native Comic Book Project was launched as a youth-focused community education project of Native People for Cancer Control, a Community Network Program Center funded by the National Cancer Institute. The purpose of this ongoing project is to use comic book creation as a way to educate Native youth about cancer, especially methods of cancer prevention. Along with basic art skills, participants learn about traditional foods and wellness, non-ceremonial tobacco use, human papillomavirus, and obesity prevention. Modeled after Dr. Michael Bitz’s Comic Book Project, the Native Comic Book Project has been adapted for both urban and reservation-based youth by incorporating Native storytelling and traditional values. Its ultimate goal is to promote healthy decision-making for Native youth and their communities. We are currently conducting a formal evaluation of this project by using pre- and post-intervention assessments to measure knowledge, habits, and decision-making among youth participants regarding tobacco use, healthy eating, exercise, and human papillomavirus. The Native Comic Book Project has been implemented at 10 sites and has enrolled 55 participants. Data will be fully analyzed in Summer 2013.  Future planned activities for research and education include developing more youth-oriented health interventions.

College students (usually between the ages of 18 and 24) are in a transitional period in their lives in which they are expected to adapt to many overwhelming and sudden changes. Mental health problems that college students experience most commonly arise from extreme academic, relationship, financial, and psychosocial strains. Despite these high rates of stressors and mental health problems, these same college students demonstrate a persistently low level of perceived need for help and actual service utilization which leads to non-treatment seeking behaviors. This study examined common barriers that prevent college students from seeking mental health treatment by using standardized measures. Two hundred and fifty college students in the Psychology Subject Pool (PSP) at the University of Washington volunteered to participate in the study which was conducted by using online survey software. Information collected included past/current mental disorder diagnoses and mental health treatment, duration of the treatment, and knowledge of on-campus resources (e.g., Counseling Center, Hall Health). Further, participants answered seven questionnaires about attitudes toward, barriers to, and fear of treatment, in addition to participants’ ambivalence concerning emotional expressiveness. The results are primarily descriptive in nature and focused on exploring common barriers, attitudes, and issues that interfere with reaching out to and receiving mental health treatment. By identifying the barriers we hope to bring the target consumer audience closer to effective treatments via distributing our findings to local counseling centers. We also examined how demographic variables relate to college students’ attitudes toward mental health treatment which will help local universities and colleges to tailor advertising efforts with greater specificity to ensure that college students in need of mental health services are aware of and engaged in available services.

President Obama’s recent inaugural speech included the call for equal rights for members of the GLBT community. Among the issues transgender people especially face are access restrictions to transition related health care. This project examines the implications for transgendered people in terms of access to gender-confirming surgical procedures and how this affects their quality of life. The sites of this study were the United States and Germany, two countries using the same parameters to determine if a person suffers from Gender Identity Dysphoria, the clinical diagnosis required to undergo gender-confirming procedures. In the U.S. these procedures are widely not covered because they are labeled elective or cosmetic. German law on the other hand mandates full health insurance coverage as surgery is considered a necessary and effective treatment of a clinically established condition. This project uses quantitative data collected with questionnaires and qualitative data in the form of life history interviews. It compares the results of the National Transgender Discrimination Study (n=6,540) with responses to the same questionnaire by transgender Germans (n=88). Findings indicate that access to transition related care via health insurance improves the mental and physical well-being of individuals whereas access limited due to lack of coverage can be detrimental. Life histories provided by five individuals, three Americans and two Germans, evaluated in open and closed coding according to the Grounded Theory approach, also indicate lower rates of depression and drug use in people after gender-confirming procedures. Yet due to the small size of the German sample these findings may not be representative. Furthermore, even though gender-confirming procedures are effective, they should not be understood as mandatory for everyone diagnosed with Gender Identity Dysphoria.

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.

In this project, we explore conditions that are critical for de novo evolution of cooperation and altruism in a bacterial system. Previous theoretical work has shown that there is a competitive advantage to defection and selfishness, but the ubiquity of cooperation in nature suggests that cooperation may be adaptive. Here, we aim to uncover environments and genetic conditions that actually promote greater cooperation. We use an engineered cooperative strain of Escherichia coli. Through a bistable genetic switch, single cells have the capability to be either a producer cell that makes the cellulase enzyme to break down cellulose, or a consumer cell that eats the byproducts of cellulose breakdown. Our first aim is to characterize the social dynamics of this synthetic system and determine if a social dilemma is in fact occurring—i.e., is there a cost to producing cellulase, and can cheaters that do not produce cellulase displace the cooperators that do? Our second aim is to evolve the system over many generations and analyze how it changes. Uncovering the conditions favoring higher cooperation (e.g., greater production of cellulase) provides insight into how cooperation can evolve and how populations circumvent social dilemmas. We hope to use evolution as a tool to tune the synthetic genetic circuit for maximal cellulose breakdown. On a broader scale, these conditions that favor cooperation may have practical applications in waste degradation and biofuel production.

The nuclear transport pathway performs the fundamental function of moving cargo between the cytoplasm and nucleus in all eukaryotes. Nuclear transport is carried out through a highly conserved mechanism across all eukaryotes. Yet, in Drosophila, several components of the nuclear transport apparatus, including importins, evolve rapidly under positive selection. Genetic conflict with selfish elements has been suggested as a possible cause for this pattern of rapid evolution. Here, we present a comprehensive phylogenomic analysis of importin gene evolution in Drosophila. Importins are adapter molecules that directly mediate the transport of cargo into the nucleus. Our analysis reveals a recurrent pattern of gain and loss of importin paralogs across independent lineages of Drosophila. Interestingly, we discovered that almost all new copies of importins have acquired a testes-specific expression pattern since their birth through gene duplication. This pattern of repeated gains of testes-specific copies of importins suggests a function in suppressing genetic conflicts such as segregation distortion in the male germline. Segregation distorters such as SD in Drosophila melanogaster act by impairing nuclear transport in the testes. We are currently performing functional tests for the hypothesis that an increased dosage of these testes-specific importins may serve a role in suppressing segregation distortion in the male germline by restoring nuclear transport during spermatogenesis.

Recent reports have shown that metabolic signatures are highly characteristic for a cell and may act as a leading cause for cell fate changes. In particular, we have shown that during development from naïve to primed embryonic stem cells, the cells undergo a dramatic transition from metabolically bivalent to highly glycolytic. However, this state of inert mitochondria rapidly changes to highly potent mitochondria during further differentiation. It is not yet understood how and why the pluripotent cells enter the highly glycolytic, metabolically cancer-like (Warburg effect) stage and how a differentiating cell leaves this stage. We have now performed metabolomics profiling of naïve and primed pluripotent cells using mass spectrometry. Interestingly, primed cells accumulate the tryptophan degradation product kynurenine. Kynurenine (KY) can act as a ligand for the transcription factor AHR. In cancer cells AHR activation by KY incudes growth while in surrounding T-cells KY based AHR activation will inhibit T-regulatory cells. My hypothesis is that kynurenine is a key metabolite in determining stem cell stage and in helping protect the primed stage embryo against the mother's immune cells. I will now test this hypothesis by analyzing the levels of the key KY metabolism enzymes by qPCR. My prediction is that the tryptophan degrading enzyme IDO1 will be unregulated in the primed stem cells. To test whether kynurenine can change the state of naïve stem cells, I will add different concentrations of kynurenine to the stem cell media and test potential cell fate changes. Since primed ESC-like metabolism is also observed in cancer cells, it is of great interest to understand how the cellular transitions in and out of this metabolic state are controlled during normal development. These studies will shed light on the function and regulation of metabolism during normal and disease developments.

It is well known that mammals contain several types of phospholipase A2. The cytosolic phospholipases A2 (cPLA2s) are one type, and are composed of six enzymes: cPLA2alpha, cPLA2beta, cPLA2gamma, cPLA2delta, cPLA2epsilon, and cPLA2zeta. There has been significant interest in the cPLA2alpha isoform because of the enzyme's 10 fold preference for the hydrolysis at the sn-2 position of the glycerol backbone in phospholipids, resulting in the liberation of arachidonic acid. Arachidonic acid serves as a precursor for several highly regulated inflammatory mediators that play an important role in asthma, atherosclerosis, and arthritis. To address these problems, inhibitors that target cPLA2alpha have been developed to serve as anti-inflammatory therapeutics. Wyeth pharmaceuticals has released a class of indole inhibitors that inhibit cPLA2zeta at <10 nM IC50. However, a recent study has shown that in cPLA2alpha-/-stimulated lung fibroblasts arachidonic acid production is lessened but still present. cPLA2zeta has been identified as the other enzyme involved in the release of arachidonic acid. To further our limited understanding of cPLA2zeta's role in the inflammatory process, we are developing selective and potent inhibitors of the cPLA2zeta isoform. Our synthetic strategy is to modify the scaffold of Wyeth's cPLA2alpha inhibitor. Analysis of the structure-activity relationships and 3D molecular modeling has revealed 4 different places on the scaffold to introduce substitutions. To date, we have generated and assayed a panel of inhibitors. These inhibitors display low nanomolar potency, but have only minimal selectivity for cPLA2zeta. Thus, our main focus is to increase the selectivity of our inhibitors towards cPLA2zeta in order to further our understanding of cPLA2zeta's role in arachidonic acid production.

 Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has been used extensively to study the biology of aging, and several determinants of C. elegans longevity are conserved in higher order organisms. The specific mechanisms of longevity in C. elegans have not been completely identified and many pathways are still poorly understood. Dietary restriction is one promising pro-longevity intervention, though its mechanism is poorly defined. DR, a reduction in caloric intake in the absence of malnutrition, extends lifespan in C. elegans and in a wide range of other species, including yeast, flies, mice, and primates. However, this extension is highly dependent on genetic background. To investigate this, we are performing a genome-wide screen of the Vidal RNA interference (RNAi) Library, which has a catalog of 11,511 clones. By knocking down specific genes within the C. elegans genome through RNAi, we are able to observe an extension or reduction in longevity in response to DR. We have screened over 1500 genes, 61 of which are short-lived and 67 of which are long-lived. We are currently following up on our collected data by measuring the full lifespans using the RNAi clones that significantly shortened or extended lifespans in our screen. By screening such a large number of genes, the widespread effect, and hopefully mechanism, of DR will be elucidated. We hope to see genes significantly affected by DR cluster into groups with common functionality, or along the same pathway, and then follow up on those specific pathways to clarify the mechanisms underlying DR. If these pathways are conserved, research findings relating to C. elegans may be applicable to other organisms. We hope to confirm these results and further investigate the interaction between DR and other potential longevity pathways.

Ethosuximide is a succinimide anticonvulsant used in the clinical treatment of petit mal epileptic seizures in humans. Although its exact mechanism is not fully elucidated, it is believed to inhibit T-type calcium channels. In addition to its medical use, ethosuximide has been found to increase lifespan in the model organism, the nematode C. elegans. Nematodes exhibit lifespan extension when treated with ethosuximide at concentrations below a toxic threshold. While the published experimental results on ethosuximide-treated nematodes are promising, we have discovered that ethosuximide breaks down in the presence of 254 nm ultra-violet (UV) light, forming a toxic by-product. This change in ethosuximide that results in toxicity is specifically caused by irradiation, not heat. Acute exposure of C. elegans to ethosuximide treated with UV light results in death at lower dosages compared to nematodes treated with the control, native compound. UV-treated ethosuximide toxicity is independent of chemosensation (i.e. the sensing of chemical stimuli) as evidenced by the fact that chemosensory mutants do not show enhanced resistance to the UV-treated drug. Some human patients taking ethosuximide experience a butterfly rash under the eyes following exposure to sunlight. We speculate that this rash may be indicative of a similar mechanism of UV-induced cytotoxicity occurring in these patients. Taken together, these studies will help explicate the mechanism of toxicity resulting from exposure of ethosuximide to UV light, as well as the mechanism of action of native ethosuximide.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by broad social and cognitive impairments and the presence of stereotyped, repetitive behaviors. Our research examined the hypothesis that impairments in neural signal routing in ASD underpin behavioral symptoms. Specifically, we investigated structural and functional differences in the basal ganglia (BG), a group of subcortical nuclei that regulate signals to frontal cortex. The BG is known to regulate movements, balance, and posture and their abnormal development has been related to motor impairments in ASD. It is necessary to extend these investigations, as previous research did not account for the relation between BG structure and function, or the role of the BG in complex cognitive tasks. In the current experiment, anatomical tracings of two of the key input stations of the BG (the caudate and putamen) were generated for 31 individuals, including 14 with ASD and 17 controls. To determine whether the size of the BG nuclei correlated with their functional influence over cortical regions, we correlated our volumetric measures with indices of effective connectivity, measured using dynamic causal modeling analyses of an fMRI data set. Our results indicated that across all individuals, there were reliable negative correlations between the size of the BG and its modulation of synchronization between several cortical regions. This finding was particularly evident in the individuals with ASD. However, inconsistent with previous investigations, between groups t-tests showed that there were no significant differences in absolute BG volume between groups. Future analyses will account for total brain volume to see whether absolute value or relative value of the BG account for differences in network-level signal routing properties in individuals with ASD. We will also correlate these findings with behavioral measures to establish its relevance for the symptoms of ASD.

Chemostats are a continuous culture system in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients. Unlike batch cultures, the selective pressure imposed in a chemostat is constant. This has made them a desirable tool for evolution and competition experiments. Traditionally, chemostat experiments with S. cerevisiae have been constrained due to the limited number of cultures that can be run at once. In order to solve this problem our team is working with a new culturing system that allows 64 chemostats to be run simultaneously. In our experiment we evolved 96 biological replicates of haploid MATa yeast cells under either constant sulfur, phosphate, or glucose limiting environments for 300 generations. We are now using a variety of techniques including array-comparative genomic hybridization and whole genome sequencing of evolved populations to characterize mutations that have been selected for during the course of these evolution experiments. These techniques have revealed that amplification of genes that code for nutrient transporters are a common mutation that occur in the evolved populations over the course of the experiments. Therefore, we will also be experimenting with strains that have the most common amplified genes knocked-out in the hopes of identifying alternative pathways for nutrient-limited fitness increases. Changes in the genome can be correlated to changes in relative fitness, which we assayed through competition with a green fluorescent protein (GFP) expressing ancestral strain every 50 generations. Ultimately it is our hope that higher replicate number will better reveal the spectrum of genes important for adaptive growth in specific nutrient-limited environments.

Autism is a disorder characterized by deficits in social and communication skills and repetitive behaviors. Fifty-two percent of the children with autism are treated with medication for some of the common co-morbid behaviors – e.g., anxiety, aggression, and hyperactivity. However, only 49-69% of these children respond positively to the medication, and about 18-26% of these children experience side effects such as insomnia, sedation, and increased appetite. An extensive series of trial-and-error might be needed for a patient, which could be time-consuming and stressful for both the patient and parents. A reliable method is needed to predict the effectiveness of the pharmacological treatment. This study used electroencephalography (EEG) to detect early changes in the brain that we cannot yet observe in behavior. Children who are diagnosed with autism and starting a medication (e.g. stimulant, antidepressant, or atypical antipsychotics) participated in this study. EEG was recorded while the subjects participated in a social processing task, a resting task, and the Go-NoGo inhibition task. The participant’s caregiver completed three questionnaires: Child and Adolescent Symptom Inventory, Aberrant Behavior Checklist, and Social Communication Questionnaire. This project analyzed the subject’s change in EEG and behavioral data over three different time points (T1= before, T2= one week after, T3= one month after the start of medication). Multiple aspects of each individual’s EEG signal were examined by comparing the difference from T1-T2 and T1-T3 against the null hypotheses of no change (or comparing change to 0). The goal of this project is to search changes in the EEG signal that is related to positive (or negative) behavioral changes in this population. This study will provide an important first step for understanding changes in the brain in response to medications, which may provide useful information to professionals during the process of choosing the most effective medication for children with autism.

Superior spatial abilities in Autism Spectrum Disorder (ASD) are popularly associated with savant behaviors, and have been demonstrated in previous studies including tests of spatial intelligence. Our study examines participant's performance on a classical mental rotation task, which provides an objective measure of spatial ability. We seek to demonstrate how differences in reaction time (RT) and brain responses of adults with ASD differ from controls in this task. Previous research in typical individuals demonstrates that as the degree of rotation increases, participants are slower to make orientation decisions. These RT measures suggest individuals mentally rotate a second image to a reference image in order to make a decision. Our study tested age and IQ matched ASD and typical controls in a task that required a decision about the orientation of images of block alphabet letters. Participants were asked to decide whether the second of two images was identical to or a mirror of the first image. Images were presented at one of four angles. Dense array electroencephalography (EEG) and RT were recorded. Thirty-five male adults (17 in ASD, 18 in control) participated. RT, IQ, angle of images, and accuracy were variables for behavioral analysis. EEG data was analyzed in the time/frequency domain using open source and in-house software. EEG and RT measures from each trial of the task were compared based on subject group, types of images (normal or mirrored), and angle of images. Our analysis extends to include investigating brain activity prior to the pressing of the response key (the time that participants are making a decision). No general advantage in mean RT was seen in ASD group, however, high performance IQ (PIQ) participants in ASD group performed faster over the course of the experiment. This suggests high PIQ autistics may learn faster in this particular task.

Current research in Himalayan geodynamics focuses on the complex relationship between surface erosion and active rock uplift. This study focuses on the Eastern Himalayas, where the Yarlung-Tsangpo River abruptly turns southward, incising over a 2 km deep gorge through the easternmost Himalayan mountains to join the Brahmaputra River in Assam. Because geologists have primarily concentrated on just the surface geology, they have struggled to explain this massive gorge despite over a decade of research. Our research takes a new approach to this aging problem by examining the sedimentary record immediately downstream of this gorge. In fact, sedimentary rocks of the eastern Siwalik foothills preserve the full record of Himalayan uplift as well as the evolution of the river systems draining the Tibetan Plateau. Here we focus our research, interpreting the sedimentary record through both detrital zircon U-Pb dating, which is a radiometric dating technique, and modal analysis, which is an evaluation of the minerals present within the samples from these Siwalik units. Past research results suggested a stable sedimentary provenance sourcing from both Himalayan and Tibetan source rocks, but new higher resolution modal analysis will add valuable insight into the competitive influences of progressive roofing of the high Himalayas and sediment recycling in the Himalayan foreland. Furthermore, this research provides valuable new data for a region of the Himalayas where there is currently a deficiency of information from the sedimentary record. This information is necessary for a full comparison of landscape evolution westward across the great Himalayan arc, and also shows a regional relationship between climate and tectonics.

The neurotransmitter dopamine is a key component of the limbic sysem, which plays a critical role in emotional processing, learning and memory, and reward processing.  Disruptions in dopamine synthesis, release, and reuptake underlie many neurological disorders, learning deficits, and diseases.  Previous work from our lab established the importance of the glutamatergic NMDA (N-methyl-D-aspartate) receptor in maintaining normal phasic activity in dopamine neurons of the ventral tegmental area (VTA) and showed that mutations of this receptor cause deficits in fear-related learning, highlighting the importance of dopamine in fear-related learning.  In this project we use in-vivo electrophysiology to record from dopamine neurons of the VTA during Pavlovian fear conditioning.  The paradigm consists of two types of trials where one cue (CS+) is paired with a mild footshock (0.3mA, 0.5 sec), while the other trials utilize a cue (CS-) which predicts the absence of a footshock.  Our preliminary data show plastic differences in neuronal activity patterns across training days associated with both conditioned and unconditioned stimuli, where initially there is increased activity in response to the fearful stimulus, but across days of conditioning, this response shifts to being associated with the conditioned stimulus.

Although every cell in the human body has nearly the same DNA sequence, there are many different cell types performing different functions; for example, lymphoblast cells which differentiate into white blood cells or endothelial cells which provide a lining throughout the human vascular system. But cells do not always function as they should, for example, overproduction of white blood cells can be caused by lymphoblastic leukemia, a type of cancer. I intend to use data on the sensitivity of parts of the genome to deoxyribonuclease (DNase) digestion to explain the differences in cell behavior. Regions of the genome structure that are hypersensitive to DNase have already been shown to affect gene expression. I want to determine if portions of the genome which are mildly sensitive, or mesosensitive, to DNase play a role in cell differentiation. My hypothesis is that DNase can identify large scale regions, on the order of thousands of base pairs, that harbor cell type specific genes. I used Segway, software which uses a dynamic Bayesian network, to segment the genome based on DNase sensitivity data into regions of low sensitivity, mesosensitivity, and hypersensitivity on six different cell types. From my segmentations I removed the hypersensitive regions from considerations, and compared the low and mesosensitive regions to gene expression data. I found that regions Segway labels mesosensitive had higher levels of expression than low sensitivity regions. I expect to find that many of the regions responsible for cell type specific behavior are mesosensitive. If this is the case, I hope the results will be robust enough such that anyone with access to DNase data can determine what genes are active in a population of cells, enabling them to better predict the behavior of the cells. In furthering our understanding of gene regulation, we may better understand diseases such as leukemia.

M dwarf stars are classified by their small radii and relatively cool surface temperatures. Their internal structures are known to be almost entirely convective, and this extreme convection causes a tangling of the stars' magnetic field lines. Upon realignment of the magnetic field lines, an incredible amount of energy is released from the star in what is called a flare. This research project is an ongoing observation of the flare activity on the M dwarf stars GJ1243 and GJ1245. Two months of flare activity was observed using the flux measurements made by NASA's Kepler telescope. The frequency, duration and types of flares were recorded and analyzed with the aid of the computer program "Flares by Eye." It has been observed that most flares have short rise times and long decay times, the intensities of which seem to follow a power law. Continued research on this topic will help us learn more about the physics behind the ignition of these stellar explosions, how the flares decay, and how the stars become less active as they age.

Ubiquitin is a protein modifier that is essential for many eukaryotic cellular processes. Attachment of ubiquitin to substrate proteins (ubiquitination) occurs in a three-step cascade that involves a ubiquitin activase, ubiquitin conjugase, and ubiquitin ligase; it is the ligase that targets substrate proteins. One challenge for the ubiquitin field is identification of substrate cohorts for ubiquitin ligases. Although ubiquitination only occurs naturally in eukaryotic organisms, we previously demonstrated that it is possible to reconstitute the ubiqutination cascade in E. coli, allowing for analysis of ubiquitination outside of its natural environment. Our project consists of the construction of a protein complementation assay, using two fragments of dihydrofolate reductase (DHFR) to detect the ubiquitination of proteins in E. coli, outside of the natural eukaryotic environment of ubiquitin. To perform the assay, I transformed all of the essential components of the ubiquitination pathway, including individual fragments of DHFR fused to ubiquitin and a substrate protein of interest, into E. coli cells. The assay tests for the covalent attachment of ubiquitin to the substrate protein through growth on selective medium, which is possible only through the combination of the separated fragments of DHFR. After construction and transformation of vectors using a known yeast ubiquitin ligase and substrate partner, I have found that ubiquitin fused to a fragment of DHFR functionally attaches to other proteins via a known ubiquitin ligase. Transformation of entire cDNA libraries into the bacterial cell in place of a ubiquitin ligase will allow for high throughput screening of ligases with homologous function as well as discovery of new ubiquitin ligase substrates. As no functional screening mechanisms presently exist, our system to efficiently screen for ubiquitin ligases and substrates provides a novel tool to address a significant challenge faced by the ubiquitin field.

The mission of the Cell Biomechanics Lab led by Professor Nathan Sniadecki is to “understand how mechanics affects human biology and disease at the fundamental level of biological cells.” To do this, microstructures are created in this lab to interact with cells on their native micrometer length scales. These microstructures, made out of polydimethylsiloxane (PDMS), take the shape of micropost arrays on which cells are placed. When the cells attach to the posts they exert forces which bend the microposts. This bending is measured and used to calculate the forces generated by individual cells. It is important to measure these forces to fundamentally understand cell interactions within living tissue. This research increases the sensitivity and function of micropost arrays by decreasing the diameter (thus reducing the stiffness) of the posts to the nanometer scale using PDMS etchant, tetrabutylammonium fluoride (TBAF). With nanometer diameters, researchers gain the ability to measure forces produced by individual filopodia. Difficulties with this approach include ensuring homogenous etching of the arrays, and controlling the etch rate. This research overcomes both of these difficulties. To ensure a homogenous etch, the etchant is circulated evenly among the posts using a flow chamber. Etching rate is controlled by adjusting the etchant flow rate and time. Experiments are currently in progress. So far, etch rates have been determined for different flow rates using single block and post structures. This knowledge has been used to design the micropost array experiments. These experiments will be conducted in the coming weeks.

Iron-oxide magnetic nanoparticles (MNPs) with tuned magnetic properties can enable novel diagnostic and therapeutic technologies such as magnetic particle imaging and hyperthermia. In order to optimize the magnetic properties of MNPs, organic synthesis methods are required. However, these organic synthesis methods render the nanoparticles hydrophobic and unusable for biomedical applications. To make MNPs functional in biomedical applications whilst retaining their magnetic functionality, they must be transferred to the aqueous phase using a biocompatible polymer. In this work, MNPs will be coated using a biocompatible PEG-ylated amphiphilic polymer: poly (maleic anhydride-alt-1-octadecene) or PMAO-PEG. To ensure the PMAO-PEG is a well suited polymer coating for MNPs, various molecular weights and molar ratios of PMAO-PEG are synthesized and characterized to study the effects of PEG surface density and molecular weight on MNPs. By altering the density of the polymer used to coat and phase transfer MNPs, effects such as the solubility, circulating life, colloidal stability, dosage frequency, and proteolytic degradation rates of MNPs in the biomedical applications are studied using data obtained from Gel Permeation Chromatography (GPC) and Fourier Transform Infrared Spectroscopy (FTIR). Molecular weight distributions of the various polymers are obtained from the GPC to evaluate the conjugation success after PEG-ylation. In addition, FTIR was used to identify known and unknown materials in the samples, determine the quality and consistency of samples, and to determine the amount of components in samples.

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.

CO₂ separation from flue gases constitutes to nearly 40% of the CO₂ emission in the United States. Developments resulting from this work address factors key to increasing the transport properties in polymer membrane systems. In particular, this work explores method to engineer porous structure to enhance the selectivity of polymeric membrane. The polymer system of interest in this study is poly(l-trimethylsilyl-1-propyne) (PTMSP), a glassy polymer of extraordinary high free volume compared to conventional polymers. It is “reverse-selective” for CO₂ separation from flue gases such as nitrogen or hydrogen, i.e., transports CO₂ gas faster in respect to lighter flue gases. Based on prior studies that showed that enhanced transport properties for PTMSP if constrained to thin films, here we present our data for PTMSP if constrained in nanoporous functionalized anopore alumina oxides (AAO) “sieve” membranes. Impregnation was achieved via vacuum solution impregnation. The overall CO₂ permeability of impregnated membranes was found to be ~80,000 Barrer, which is almost two times higher than that of bulk PTMSP membranes. Selectivities of CO₂/Helium and CO₂/Nitrogen in impregnated membranes were found to be ~7.0 and 9.0, respectively; these are also noticeably higher than the corresponding selectivities of 6.0 and 6.6 found in bulk membranes. Furthermore, we investigated the temporal stability of these membranes that are notorious for their fast aging in the form of ultrathin films. The origin for physical aging is the collapse of the free volume. A two week aging study of PTMSP revealed a significantly slowed down physical aging process if confined in nanoporous AAO by about a factor of 20 over thin films. The exposure to the radial constraint within the pores helped stabilize the free volume and reduce the occurrence of free volume collapse.

Contemporary US society has in many ways written off self-mutilation, the deliberate damage or modification of one’s body tissue without intention of suicide, as a pathological act whose roots lie in mental illness and destruction beyond any sort of rationality. Often, self-mutilation is linked to suicidal behavior, but even when it isn’t it bears a transgressive nature and is frequently met with repulsion. In order to understand and examine the effects of the transgression of self-mutilation on individuals and larger communities, I took into account some ways in which individuals in specifically religious contexts have been marginalized by these practices. Parts of the Lakota Sun dance involving self-mutilation were banned in the US between the years 1895-1978 and the Modern Primitives, a group that emerged in the late 1960s and 70s seeking to reclaim self-mutilating religious rituals were met with extreme ostracism. In my research I explored spiritual self-mutilation as a vehicle for connection to something beyond an individual’s bodily limits, examined the physical and psychological capacities humans have to endure intense physical sensation, and looked at the implications of the process of physical healing as a powerful and direct metaphor for spiritual healing. Taking all of these into consideration, with the help of the narrative of the Modern Primitives, I sought to understand the way in which, for many individuals, the brutal destruction and modification of skin and flesh becomes an act of creation, of reconstruction and life-affirmation. If we allow ourselves to reconsider our cultural taboos surrounding self-mutilation in a religious context, we begin to see that there may be a stronger, deeper connection between spiritual self-mutilation and pathological self-harm than we can realize, without taking a closer look at the roots of both.

Poly(3-hexylthiophene) (P3HT) and 6,6-phenyl-C60 butyric acid methyl ester (PCBM) are model materials used to study and understand the performance of polymer solar cells. A critical design parameter in improving device performance is the structural morphology of the active layer. Traditional processing of organic solar cells involves the deposition of a P3HT/PCBM composite film from a common solvent and then post-processing treatments (i.e. annealing) to influence the extent of phase segregation and improve the percolation of electron and hole transport pathways throughout the film volume. Therefore, the electronic properties of the resulting solar cells are inherently tied to how the film is processed (e.g. choice of solvent, annealing temperature, film thickness). While process optimization has led to improvements in laboratory performance, it is challenging to extend the same principles to improve the performance of large scale roll-to-roll processes because deposition conditions vary significantly. Increasingly, researchers recognize the need for methods that decouple film processing from active layer structure and device performance. My research involves the synthesis of P3HT/PCBM composite nanoparticles (CNPs) in aqueous dispersion as a means to circumvent the dependence of active layer structure on the specific mode of deposition of the P3HT/PCBM film. I have synthesized CNPs with variable content of P3HT/PCBM and different preparation procedures while also controlling particle size. The characterization of these particles focuses on spectroscopy, small angle X-ray scattering (SAXS), dynamic light scattering and the performance evaluation of devices produced with CNP active layers. This project will tie device performance to CNP structure regardless of active layer processing, and assist in identifying CNPs with optimized morphology without the need for annealing or post-processing.

When cells migrate, they undergo a complex process of signaling pathways coordination and interaction with their surrounding environment. Actin filaments polymerize at the leading edge of the cell membrane, pushing it forward and allowing for the cell to bind to new sites in the extracellular matrix. In order to sophisticatedly study the process of cell migration, one must pay close attention to the initial binding points, or nascent adhesions, that can either fall apart early and cause the front of the cell to collapse, or grow into the larger focal adhesions that provide the mechanical support for the cell in the extracellular matrix. Though tools have been created to study the mechanics of cell forces, there is a distinct lack of ability to measure the extremely small forces created by cells, such as the contraction of a single filopodia. This work serves to develop such a tool, in the form of polydimethylsiloxane (PDMS) nanopost arrays, via soft lithography manufacturing techniques. These nanoposts can be treated as tiny cantilever beams by which deflection can be immediately translated to force, using known dimensional and material properties of the posts. Fabrication of the posts consists of a single-cast of PDMS onto silicon negative molds and curing the posts at 110° C at low vacuum pressure. It has been determined that vacuum pressure is necessary to achieve the maximum post aspect ratio, due to surface tension issues between the liquid PDMS and the silicon chip. The gas permeability of PDMS allows for the air contained in the holes of the master chip to escape and be replaced by liquid PDMS while curing. The success rate of nanopost fabrication is characterized by relating post aspect ratios to vacuum pressure of the curing environment, which are found to correlate.

Quantitative mass spectrometry (MS)-based proteomics is a powerful approach to measure relative expression changes across thousands of proteins in a complex mixture. Commonly, stable isotopes like ¹³C and ¹⁵N are incorporated into proteins and peptides by metabolic labeling (ML) or chemical labeling (CL) to produce “heavy” labeled forms. Mixtures of light and heavy peptides can be identified and quantified by MS. In ML, proteins are labeled in vivo by growing cells with isotope labeled metabolic precursors, while peptides are chemically labeled in vitro in CL. These two labeling approaches have distinct workflows and their own strengths and limitations. In a typical proteomics experiment, complex mixtures of peptides are separated on reversed phase liquid chromatography (LC) and sampled in real-time by the mass spectrometer for peptide identification and quantification. We propose to analyze mixtures of chemically and metabolically labeled peptides in single LC-MS runs, comparing each method’s quantitative accuracy and precision, and other factors, such as analytical losses from sample processing without errors introduced by stochastic sampling. We compare stable isotope labeling by amino acids in cell culture (SILAC) and reductive dimethylation CL by mixing equal amounts of peptides from each approach.Preliminary results indicate minimal differences between ML and CL in quantifying proteins (median of >2 peptide ratios). With individual peptides, however, CL showed 13% larger variance than the ML. We observed signal losses in CL samples compared to ML samples, and fewer quantified peptides and proteins in CL (6005 peptides, 897 proteins) than in ML (7448 peptides, 1059 proteins). We attribute the differences to the extra labeling step in CL, and will perform further analyses with ratio mixtures of heavy and light labeled peptides to clarify the source of variance. Our comparison of CL and ML will guide future optimization and applications of both quantitative proteomics labeling methods.

Cardiovascular disease is the leading cause of death worldwide, and current treatments for heart failure are limited to slowing the disease progression or transplanting a donor heart. One potential approach to restore heart function is to transplant human embryonic stem cell-derived cardiomyocytes (hESC-CMs). An independent and similarly promising approach is to overexpress the enzyme ribonucleotide reductase (RR), the enzyme responsible for the production of the small molecule deoxy-ATP, which has been previously shown to improve contractility and diffuse between cardiomyocytes via gap junctions. We hypothesize that by overexpressing RR in hESC-CMs and then transplanting the latter into infarcted hearts, we will improve global cardiac function by increasing dATP content throughout the myocardium. To begin testing this hypothesis, I will test the efficacy of an RR adenovirus in hESC-CMs in vitro using quantitative PCR and immunostaining, and I will then correlate the subsequent improvement in fractional shortening following in vivo transplantation to the amount of successfully engrafted cells. To date, I have successfully developed an immunofluorescence staining protocol for RR. I am currently working on designing an immunohistochemistry protocol to identify graft tissue and stain for RR and GFP and localize RR in relation to the infarct. After confirming the protocol, I plan to apply it to the fixed rat heart slices to assist in the proof of concept of the transplantation of hESC-CMs overexpressing RR as a novel therapeutic method.

The Moon does not have a dipolar magnetic field, like that of the Earth. Instead, the Lunar surface has areas of magnetized crust called magnetic anomalies. Many of these magnetic anomalies are coincident with features called lunar swirls - regions with high-albedo or high-reflectance material. As many of these lunar swirls often appear in the same location as areas of relatively intense magnetic fields, one of the leading hypotheses explaining the formation of the lunar swirls is that the surface is shielded from the surface darkening solar wind by the magnetic anomalies. We seek to further understand the connection between the visual high albedo and the magnetic anomalies by creating three-dimensional models of the magnetic fields and particle deflection around well-known lunar swirls. We do this through the use of a three-dimensional particle tracking program. The particle tracking program simulates solar wind interacting with the magnetic field and determines the final location of the solar wind particles on the surface of the Moon. This work focuses on the results for simulations of the Mare Ingenii swirl region. Preliminary results show that the particles are in fact deflected, and as expected the smaller electrons are more highly deflected. Better understanding of the origin of the Lunar swirls not only allows us to gain insight into the process of space weathering and magnetic shielding but can also offer clues for methods to mitigate the harmful effects of solar wind and particle radiation during human space travel and at lunar or Martian human colonies.

In aquariums and zoos across the world marine mammals are among the most popular exhibits. Caretakers strive to provide the least stressful and natural environments for the animals. Analysis of (steroid) hormones in excrement has proven to be an effective, non-invasive method. It allows animal keepers to monitor physiological health associated with reproduction and the stress response in captive animals. Prolactin (PRL) is a non-steroidal, non-tropic peptide hormone most commonly associated with milk production in nursing females. However, it has a multitude of normal physiological functions such as: regulation of the immune system, osmotic balance, angiogenesis, an indicator of parental care (in both males and females), sexual maturity in males, and near-term pregnancy in females. Chronically high or low levels of PRL have been associated with a number of ailments. For instance, impotence reduces the secretion of estrogens and testosterone. To date, no studies have been documented on prolactin PRL levels in captive northern fur seals, (Callorhinus ursinus), harbor seals (Phoca vitulina), or sea otters (Enhydra lutris). Using an enzyme-immunoassay (EIA) kit, previously tested only in human and elephant serum analysis, we investigated PRL levels of several samples collected from captive marine mammals (Northern fur seal blood serum, harbor seal saliva, and sea otter urine/fecal samples). Due to the helical structure of PRL, fecal samples were not expected to survive intact after transit through the digestive tract; therefore, were not expected to produce positive results. Here we report preliminary results of the EIA PRL assay on several different types of samples, as well as several longitudinal samples from the same individual, to show significant changes in this hormone over time. If detectable in urine, fecal matter, and/or saliva, this assay could prove to be a useful non-invasive method for determining health in captive marine mammals.

We estimate a Gaussian graphical model corresponding to a covariance matrix using machine learning techniques. A motivating application is to identify genes associated with cancer in the human genome. Suppose we have genetic data from two people, one of whom is healthy, while the other has cancer. Our goal is to identify gene expressions that differ between datasets and contribute to cancer. We model each dataset as a Gaussian graphical model, which is a network of edges and nodes. Each node represents a random variable, or gene. Each edge represents the dependence between the two variables it connects. An edge with zero value indicates the two variables are independent when conditioned on the rest of the variables. Such networks are analogously represented as inverse covariance matrices. Our goal is to use empirical genetic information from cancer patients to recover the true inverse covariance matrices. We suppose that the inverse covariance matrices have a certain structure and we set up a problem to encourage that structure. Structure refers to the relative magnitudes of matrix elements. A structure of interest is a hub node, which is highly connected to other nodes. These nodes play a regulatory role in gene expression and may be related to cancer. Our goal is to identify hub nodes in the difference between two inverse covariance matrices. We formulate this question as a convex optimization problem. We encourage the solution to have a certain structure by adding regularizer terms to the objective function. We recover the overall structure of the inverse matrices, while simultaneously encouraging the solution to have specific structures of interest. An alternating directions method of multipliers (ADMM) algorithm is used to solve the optimization problem. We run the algorithm on synthetic genetic data, and on real gene expression data to identify cancer related genes.

During the Pinochet dictatorship in Chile (1973-1990), attempts to exclude leftist-oriented thinkers resulted in the exile of over one million Chileans from their homeland. In what was perceived to be a temporary absence, Chilean exiles faced unusual dilemmas while waiting for democracy and normalcy to return to Chile. This period of uncertainty was especially pronounced for those exiles arriving in the U.S., a nation that contributed to destabilizing the socialist government and securing the military coup in Chile. This research seeks to examine the ways national identities or concepts of the homeland emerge in testimonies and cultural productions of these exiles. Primary sources include: testimonies of exiles, essays, short film, as well as the 9/11 Collective- a group of exiles responding to the shared date of national tragedy in the U.S. and Chile. Theoretical concepts surrounding diaspora, exile, identity, and memory provide an analytical lens to better understand the fluidity of nation, home, and identity in the self-representations of Chilean exiles in the U.S. This research highlights the permanent impacts that power dynamics impose on identity processes, as well as the role of culture to overcome these obstacles. I conclude with suggestions for possible avenues to both complicate and contribute to conversations surrounding the ongoing identity negotiations for Chilean exiles in the U.S.

Stem cell based therapeutics is a promising venture to regenerate a damaged heart after a myocardial infarction and to prevent the progression to heart failure. However, we currently do not have a clear understanding of the maturation processes of cardiomyocytes, and this has been one of the main obstacles limiting progress in the field. Here, we develop a device that simulates the mechanical conditions that human cardiomyocytes experience during their growth phase. The aim of the device is to apply a constant mechanical stress on cardiac tissue to test the effects of creep on cardiomyocyte size and alignment. Cardiac tissue used in this study is obtained by mixing a collagen solution with cardiomyocytes derived from the directed differentiation of human embryonic stem cells and pipetted into PDMS molds held in 6-well plates (6wp). Testing of initial designs incorporating a moving magnet below the 6wp coupled with a metallic base within the plate have resulted in too much friction, leading to uneven stretching of the cardiac tissue and the breakage of all constructs tested within 10 days. In response, a revised design is utilized, where a direct screw-driven platform suspended on top of the 6wp controls the mechanical stress applied to the cardiac tissue. This device effectively stretches the constructs to 110% of their initial length over a period of 14 days, without the breakage of any of the four constructs tested. Here we show that mechanical creep over an extended period of time affects cardiac morphology, where increased cardiomyocyte size and a greater degree of cell alignment within the cardiac tissue are observed with stretch versus non-stretched controls. This project provides insight on the mechanical cues involved in the maturation of cardiomyocytes, with the device offering a platform for in vitro modeling of human cardiac tissue.

In this presentation I will argue that regardless of whether owned by the state, private capital or publicly traded on Chinese stock exchanges, Chinese corporations doing business outside of China inevitably become instruments of China's foreign policy. In a feedback loop in which both the state and the enterprise benefit (sometimes in counter-intuitive ways), the actions and fates of corporation and state become inextricably linked in a way that could not be replicated outside the unique world of "capitalism with Chinese characteristics". From the alleged links between telecommunications equipment manufacturer Huawei and the People’s Liberation Army to the official designation of Chinese IT industry hero Lenovo as a foreign-owned enterprise within China, divining the precise ownership of Chinese firms presents a Gordian knot to international customers and governments alike, encouraging suspicion and nationalist rhetoric in response to Chinese contract bids in developed economies, while simultaneously paving the way to lucrative deals in developing nations. But what does Chinese state support for industry really mean for non-Chinese trans-national firms? What is the significance of the state-encouraged acquisition spree that has seen Chinese firms purchase IBM’s PC business, Volvo and the firm that manufacturers London’s iconic black cabs, among others? How should other trans-national firms compete with Chinese bids for major international infrastructure projects and what is really at stake beyond the initial projects in question? This presentation, based upon an examination of the ownership and activities of Chinese firms competing in international business, will offer an alternative lens through which to view the international activities of Chinese enterprises, suggesting that to truly evaluate the potential profit and loss of any specific commercial activity one must first look beyond the letterhead.

Like their Victorian forebears, Modernist authors have always placed keen emphasis on women’s hair, often presenting their luscious locks in vivid and scrupulous detail. Aldous Huxley's Crome Yellow (1920) uses hair as a tool of social commentary and trope of the fairytale epic to challenge and even reverse traditional Victorian gender roles and sexuality norms. The tension between the private and public self is also explored; what Modernist women could not say aloud, they expressed through the style, color and length of their hair. Crome Yellow constantly alludes to the hair of the female protagonists Jenny Mullion and Mary Bracegirdle, yet the intentions behind the loaded descriptions of their physical appearance are left up in the air. The characterization of an infamous Fallen Woman and modern flapper girl allows distinctions to be made between the Victorian and Modernist eras that determine the emergence of new gender ideals. This paper will use a close reading of Crome Yellow and Freudian theories on sexuality to explore the multifaceted significance of hair in the Victorian era so we may fully appreciate the past’s influence on modernist eras. A woman’s hair became a phallic image used to engender fear in men, which posits a move towards gender equality and the emergence of the suffragette. Hair was no longer just a physical attribute but a body politic that reflected changes taking place in modernist British society of the entre deux guerres period.

The fear of being unimportant, being erased, and of being finite, grips humans and motivates us to prove that we matter, that we exist. In an attempt to create truth and authentic understandings, our society seeks evidence, proof, and facts, even while acknowledging (and often ignoring) that these “facts” may be hard to establish. In my paper I explore how post-modern novels, Everything is Illuminated and Extremely Loud and Incredibly Close by Jonathan Safran Foer and Austerlitz by W.G. Sebald, utilize multi-modal textual souvenirs to function as lodestones and guide readers through fictionalized versions of real historical traumas. These novels can be read as scrapbooks of memory which investigate the social importance and definition of “truth” in the face of traumatic events. My project questions whether or not truth can be found in fiction, non-fiction or even photographs as it looks at the way modern technologies have impacted how authors choose to create historical fiction. The multi-modal nature of these novels forces readers to reflect upon authorial intent and authority in the construction of memory and post memory. Furthermore, by looking at the narratives the authors create about traumatic events such as the Holocaust, the bombing of Dresden, Hiroshima, and the attacks of 9-11, my paper reveals the ways in which collective memories and national narratives can arise from trauma and how these events in turn create social identity for individuals within and across groups. An analysis of the postmodern methods the authors use in an attempt to create this authenticity, along with the expectations society holds concerning their construction, and the impact of individual and cultural desires on the creation of these narratives, uncovers the many fallacies and complexities that come with testimony and the social construction of authentic truth.

Dravet syndrome (DS), also known as severe myoclonic epilepsy of infancy (SMEI), is a rare genetic form of epilepsy, caused by a mutation in the neuronal voltage-gated sodium channel, NaV1.1, encoded by the SCN1A gene. This form of epilepsy is characterized by febrile seizures at infancy that often change with age into afebrile seizures, which include generalized tonic-clonic and myoclonic seizures. In most cases, DS is a debilitating disorder that renders patients with decreased cognitive function and impaired development. To study this disorder, a mouse model of DS, with heterozygous deletion of the SCN1A gene (Scn1a +/-), was developed, demonstrating a phenocopy of the DS condition, with similar seizure patterns and atypical behaviors. In vitro work in the model demonstrates selective loss of sodium current and excitability in hippocampal GABAergic interneurons. However, it remains unknown if similar changes in neocortical GABAergic interneuron excitability occur. We hypothesize that fast spiking neocortical interneurons are responsible for control of cortical excitability via GABAergic inhibition and that reduced excitability of fast spiking interneurons associated with heterozygous loss of NaV1.1 contributes to hyperexcitability and seizures. To explore this hypothesis, we will compare neocortical interneuron excitability measured from single action potential parameters and repetitive action potential firing patterns in unaffected wild type mice and the DS mouse model, using pre-existing single-cell recordings. Excitability will be determined from the peak amplitude, width at half height, threshold for single action potentials, and from total number of action potentials in induced neuronal spike trains. If this hypothesis is correct, we expect that there will be atypical action potentials and firing patterns showing reduced excitability. This experiment will provide more insight into DS epilepsy and better therapeutic targets for the disorder.

Serotonin (5-HT) is implicated in numerous emotional behaviors, including depression and anxiety. One form of serotonin receptors is the 5-HT_1B autoreceptor, which is involved in regulating 5-HT transmission and uptake. 5-HT_1B autoreceptors are located on serotonergic neurons and are pharmacologically indistinguishable from 5-HT_1B heteroreceptors located on non-serotonergic neurons. Distinguishing between activation of the two receptor types is important as they have different functions. A novel strategy to accomplish this is to use an engineered receptor to mimic the 5-HT_1B autoreceptor using a modified DREADD receptor (Designer Receptor Exclusively Activated by a Designer Drug). The DREADD-1B receptor will mimic the 5-HT_1B autoreceptor function with pharmacological specificity. The DREADD-1B receptor is G_i/o-coupled G-protein coupled receptor that is activated by the selective ligand clozapine-N-oxide. When G_i/o-coupled GPCRs are activated, decreases in intracellular adenylyl cyclase and, subsequently, cAMP levels are measured. It is expected that when the DREADD-1B receptor is activated, a decrease in cAMP levels will be observed. To verify this, I created a stable DREADD-1B cell line and will use transient transfections of a luciferase plasmid to measure cAMP levels via proportional luminescence using luciferin. Optimizing the conditions for receptor activation and observing sufficient decreases in cAMP levels will confirm the general functionality of the receptor. If there are large variations in the downstream biochemistry between the DREADD-1B receptor and the 5-HT_1B autoreceptor, using the DREADD-1B receptor as a surrogate for the 5-HT_1B autoreceptor could have profound effects on behavioral data obtained in other projects. Creating and characterizing the DREADD-1B receptor in an in vitro setting will provide valuable information and tools for future projects involving the 5-HT_1B autoreceptor and this novel tool.

Francisella noatunensis is an emerging fish pathogen that causes disease and mortality for several economically important fish including tilapia and Atlantic cod. The Francisella Pathogenicity Island (FPI) is a region of the genome known to govern virulence for the genus. PdpA is one of the genes found in the FPI that has been implicated in the ability of Francisella tularensis, the human pathogen, to replicate in infected phagocytes. A recent study by our laboratory using zebrafish as a model host indicated that pdpA is important for the ability of F. noatunensis to cause mortality in infected zebrafish—thus the functional role of the pdpA protein appears to be conserved for the genus. Our goal is to compare the innate immune response of zebrafish during infection with either wild type or pdpA knockout strains of F. noatunensis to determine if the host immune response is altered. To accomplish this, we are monitoring the expression of key proinflammatory genes (IL1B and TNFA) using quantitative RTPCR during acute Francisella infection in zebrafish. The results from this study will provide additional information about the potential use of the F. noatunensis pdpA knockouts as live vaccine candidates for fish.

Diatoms are unicellular photosynthetic algae, or phytoplankton, and account for approximately 20% of global primary production. The pennate diatom Pseudo-nitzschia can produce a neurotoxin called domoic acid (DA) that bio-accumulates in the tissues of shellfish when this diatom blooms. DA poisoning causes life-threatening conditions in mammals and humans when these shellfish are ingested. Parameters such as shellfish exposure length and bloom toxicity can be found by understanding what regulates these diatom communities. One mechanism of bloom regulation that we know little about is that of viral infection, despite viruses being the most abundant predator in the ocean. After a toxic Pseudo-nitzschia bloom at Sunset Beach, Oregon was sampled in 2009, the first Pseudo-nitzschia infecting virus (PmDNAV) was isolated by infecting the host P. multiseries Clnn-16. I hypothesize that there are many different viruses that can infect the genus Pseudo-nitzschia in addition to the PmDNAV. Over the past year, I worked to isolate additional Pseudo-nitzschia viruses from three blooms collected in the Pacific Northwest: Penn Cove, Friday Harbor, and Sunset Beach, Oregon. I have identified potential viruses in 12 of 48 infection experiments using these bloom samples. Three strains of Pseudo-nitzschia: GGA2, GGA3, and GGB1, which represent the species P. multiseries and P. cingulata, were infected by putative viruses from two blooms, while many others were only infected by one or none. The data suggests that independent of species, certain strains of Pseudo-nitzschia are more permissive to viral infection than others. As well, in June 2012 I isolated seven new strains of Pseudo-nitzschia from Golden Gardens, WA. I genetically identified these strains by extracting DNA, running PCR, and sequencing the Internal Transcribed Spacer region. I will test these strains to determine how permissive they are, and how effective they will be to use as hosts when working to isolate new viruses.

Pseudo-nitzschia is a genus of phytoplankton widely dispersed throughout the world known for its production of a neurotoxin called domoic acid. As there is not a lot known about which species of Pseudo-nitzschia are associated with the neurotoxin, research on domoic acid is very limited. Little is known about the evolutionary relationships within the genus, but in 2002, the genus was broken into two clades (Lundholm, et al. 2002). Recently, the validity of these clades was tested by the sequencing of the Ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) gene in 14 species of Pseudo-nitzschia (Guannel, unpublished data). As there are currently 37 known species, the goal of this project is to sequence DNA, using the RuBisCO gene, from more species in order to fill out more of the phylogenetic tree. Cultures of 6 strains of species lacking known RuBisCO sequences were grown to mid-exponential phase and then had their DNA extracted. After the DNA was isolated, polymerase chain reaction (PCR) was conducted in order to amplify the gene. Before the DNA was sent for sequencing, the PCR products were run on an agarose gel confirming that the RuBisCO gene had in fact been successfully isolated and amplified. After the DNA is sequenced and placed on the phylogenetic tree, the physical characteristics (cell size, photopigments, etc.) of species making up each clade are then compared. This provides additional support as closely related organisms are likely to have similar traits. For example, we predicted that species with a larger cell size ( > 3 µm), would fall into Clade I, while smaller cells ( ≤ 3 µm) would be placed in Clade II. Through the addition of these species to the phylogenetic tree, further research can be conducted to pinpoint where the production of domoic acid lies in the evolutionary history of Pseudo-nitzschia.

Viruses require host cell metabolism to provide the necessary energy and biosynthetic precursors for viral replication. Recently, our lab conducted a global metabolic study to determine how vaccinia virus (VACV), a large double-stranded DNA virus, alters host cell metabolism. VACV is a model system to study poxvirus infections and was used as the live vaccine against smallpox. In our study, dual mass spectrometry platforms were used to analyze a broad range of metabolites from mock and VACV-infected fibroblast cells harvested at multiple time points within 24 hours of infection. One of the most notable metabolic alterations observed during VACV infection was increased glutamine utilization, or glutaminolysis. We have since shown that under conditions of glutamine starvation there is an approximate 90% decrease in VACV production in fibroblasts, thus revealing an essential role for glutamine during infection. Furthermore, this decrease in virus production could be rescued by the addition of tri-carboxylic acid (TCA) cycle intermediates, suggesting glutamine’s use as an anaplerotic, or replacement, substrate for the TCA cycle during infection. To determine at what stage(s) in the VACV life cycle glutamine is required, quantitative real-time RT-PCR and western blot analyses were performed to examine early and late viral transcript and protein levels. We have found that glutamine deprivation does not inhibit viral gene expression, but does significantly impact viral protein synthesis. Importantly, the addition of TCA cycle intermediates recovered viral protein levels, highlighting the need for glutamine carbon to maintain the TCA cycle at this stage in the VACV life cycle. This study reveals the robust metabolic program implemented by vaccinia virus during the course of infection and provides insight into the role metabolic reprogramming plays during viral replication. Ultimately, studies such as these may help to identify novel therapeutic methods to specifically target infected cells and inhibit viral replication.

Mycobacterium tuberculosis infection is a leading cause of death worldwide, but the full details of how the human immune system responds to these bacteria are not known. Our lab studies CD1 proteins, which allow T-cells to recognize and respond to lipids which are the major constituents of the mycobacterial cell wall. We recently discovered that low expression of CD1A on dendritic cells is present in ~10% of the population and is associated with increased susceptibility to tuberculosis in Vietnam. In addition to low surface protein expression, CD1A transcription is also impaired in CD1A-deficient individuals. The molecular mechanism of CD1A-deficiency is unknown. I hypothesized that CD1A expression is regulated by a single nucleotide polymorphism (SNP) that alters transcription factor binding. I cloned and analyzed the CD1A 5’ untranslated region (UTR) from four CD1A-deficient and five control individuals. There was one SNP, rs366316, that showed an association with CD1A-deficiency. I genotyped 163 individuals and examined the association of the rs366316 genotypes with CD1A expression. I found that the C/C genotype (N=9) was associated with CD1A-deficiency in comparison to the C/T and T/T genotypes (N=154, p<0.01). Based on this association, I examined whether the C/C genotype regulated transcriptional activity. I isolated 5’UTR sequences in normal and deficient individuals that were identical except for rs366316. I used site-directed mutagenesis to generate C to T mutants and T to C mutants. I made separate luciferase expression vectors for each of the four 5’UTR constructs and found that the normal and mutated 5’UTRs with a C allele showed 44% less expression than the normal and mutated 5’UTRs with a T allele (p<0.001). These data show that a SNP in the 5’UTR of CD1a is causally associated with human CD1A-deficiency. Together, these data suggest that CD1A-deficiency is common, regulated by a SNP, and important in MTB pathogenesis.

Administration of Anti-Retroviral Therapy (ART) slows disease progression and reduces HIV-1 viremia to near undetectable levels. Poor adherence or withdrawal of therapy often results in rapid rebound of viremia with the potential for the establishment of drug resistant variants. HIV is known to establish latency in long-lived central memory T-cells (CM) and is often referred to as “the reservoir”. However, we now understand that there is both latent and actively replicating HIV-1 that comprises reservoir sites. Using sequence information and computational tools we can define reservoirs as cells or tissues harboring HIV-1 with genotypic features of reduced temporal structure, high diversity within the population, and low divergence from their most recent common ancestor (MRCA). This is because reservoirs are populated with virus seeded at both early and late periods of infection. If a reservoir is established in a site with reduced drug penetrance, “drug-sanctuaries”, continued viral replication and diversification could occur. This may result in the accumulation of drug resistant variants that lead to therapy failure. Here we hypothesize that tissue and cellular sites with phenotypes reflective of reservoirs may also be drug restricted providing a boundary for unrestricted viral replication. To test this hypothesis we sequenced single genome template derived amplicons (SGA) of env and pol regions of HIV-1. Presented is data from the autopsy of subject S104. S104 presented with multi-drug resistant genotypes in all tissues sampled. Bioinformatical analysis measuring viral genetic diversity within and divergence from the MRCA of the viral population of S104 demonstrated reservoir phenotypes in tissue virus isolated from lung. We continue to expand our characterization of reservoirs and compartments to clearly define sites that must be targeted to eradicate HIV-1 in the host.

 A significant challenge in intravaginal vaccination against mucosal pathogens concerns the immune privileged environment in the genital tract, which prevents migration of immune cells in the absence of inflammation. Dendritic cells (DC), the major antigen-presenting cell to T cells in lymph nodes, play a particularly important role in generating an effective immune response to vaccination. Our goal is to explore a new intravaginal vaccination strategy utilizing chemokines - short biological peptides that activate cells - to recruit DCs. We intravaginally administered CpG, MIP-3 Alpha and Beta-Defensin 2 to female C57BI/6J mice (Jackson Laboratories). Mice were euthanized after 24 hours and cells were isolated from both the spleen and reproductive tract. DCs were quantified using fluorescent activated cell sorting (FACS). We have demonstrated that DCs are actively recruited to vaginal mucosal tissues following intravaginal administration of the chemotactic peptides MIP-3 Alpha and Beta-Defensin 2. Future work will involve time course studies of DC recruitment and the delivery of nanoparticle encapsulated chemokines.

The primate immune system has evolved to counter viral attacks. Yet, after several cross species transmission events of SIV, HIV has infected about 60 million people since the beginning of its epidemic. This is in part due to the ongoing evolutionary arms race between antiviral proteins and viral antagonists. SAMHD1 is an antiviral protein that is degraded by the Vpr protein expressed in HIV and SIV. In this evolutionary arms race, SAMHD1 evolves to escape degradation, while Vpr evolves to bind to these new variants of SAMHD1. African Green Monkeys (AGM) are divided into phenotypically distinct and geographically isolated subspecies, each infected with their own species-specific SIVagm, making them ideal to study. A cloned SAMHD1 gene, Tan4, from AGM has been found to be resistant to Vpr from a number of different viruses, except for one called the tan1 Vpr. Sequencing Tan4 revealed that one residue of its nuclear localization signal was mutated. Since nuclear localization of SAMHD1 is required for degradation by Vpr, we hypothesized Tan4 evolved to be cytoplasmic in order to resist degradation. Using immunoflouresence, our results confirmed Tan4 to be in the cytoplasm. Moreover, we found that tan1 Vpr and Vpr from another AGM subspecies, vervet, share similar gene sequences, but only tan1 can degrade Tan4. Currently, I am constructing chimeras between the two Vpr proteins to find the residues in tan1 responsible for degradation of Tan4. Likewise, two Vpr proteins of the sabeus monkey share similar gene sequences yet cannot degrade the same SAMHD1. My ongoing work explores the mechanisms used by the tan1 and sabeus Vpr proteins to evolve in order to successfully degrade SAMHD1 despite its attempts at escape. This will elucidate a better understanding of the ongoing evolutionary arms race for both primate host and virus survival.

Hemophilia A is characterized by a deficiency of factor VIII, a protein necessary for the formation of blood clots. Treatment involves infusions of replacement clotting factor but about one-third of patients develop inhibitors to the clotting factor, resulting in reduced efficacy of the infusion and increased cost of treatment. Immunosuppressant drugs have been found to reduce an inhibitor response, but their off-target effects can complicate treatment further. Alternatively, T regulatory cells have a naturally suppressive function and their proliferation in vivo in response to treatment with IL-2 has been studied previously in the Hemophilia A mouse model. This project aims to expand a population of factor-VIII specific Treg cells in vitro by co-stimulation with anti-CD3 and anti-Crry, in addition to IL-2 treatment. Treg cells are harvested from the spleen, and then isolated by magnetic separation and FACS Aria sorting for CD25high cells. Analysis by flow cytometry has indicated that the expanded cells maintain Foxp3 expression – an intracellular marker for Treg cells – and further experiments are being done to analyze the suppressive functionality of the expanded cells compared to fresh Treg cells. Future expansions will be focused on factor VIII-specific Tregs using the Hemophilia A mouse model and the techniques mentioned previously. The goal is to generate an expanded population of these suppressive cells that show specificity to only factor VIII antigen. This method of expansion has translational potential for suppressing the inhibitory immune response to treatment in human Hemophilia patients. Treating patients with their own expanded cells can reduce possible rejection of the treatment and provide promising long-term results.

Angiogenesis is the development of new blood vessels from pre-existing vasculature and is a hallmark of Kaposi’s sarcoma (KS), a highly vascularized neoplasm prevalent among immunosuppressed individuals. The etiological agent of KS, Kaposi’s sarcoma-associated herpesvirus (KSHV), induces angiogenic phenotypes in endothelial cells during latent infection. Interestingly, KSHV infection leads to down-regulation of transforming growth factor-beta 2 (TGF-β2), an anti-angiogenic cytokine. In addition to six viral genes, KSHV also encodes 17 recently discovered microRNAs (miRNAs) during latent infection. These endogenously encoded RNAs of 19-23 nucleotides post-transcriptionally regulate gene expression through basepairing interactions with the 3’ untranslated region (UTR) of target messenger RNAs (mRNAs) and either mark them for degradation or prevent ribosomal binding. These viral miRNAs appear to directly contribute to pathogenesis and angiogenesis by targeting host gene expression. We screened the miRNAs of KSHV to determine if any are involved in the down-regulation of TGF-β2 during KSHV infection. We found that two KSHV miRNAs, miR-K3 and miR-K8, are independently sufficient to downregulate TGF-β2. To determine if these miRNAs directly target the 3’UTR of TGF-β2 as opposed to altering genes that affect TGF-β2 expression, we used a TGF-β2 3’UTR luciferase reporter construct. We also used competitive inhibitors of miR-K3 and miR-K8 to determine if these miRNAs are necessary for the down-regulation of TGF-β2. By identifying specific targets of KSHV miRNAs such as TGF-β2, we can improve our understanding of how KSHV is able to maintain latent infection and target host gene expression to alter endothelial cells.

Corals are important marine animals that provide significant habitats for thousands of marine teleost and invertebrate species, natural barriers to protect coastal communities, and sources of income for people in more than 100 countries around the world. Despite their vital importance, however, little is known about their taxonomy. Currently, they are classified based on their calcium-based skeleton morphology and coloration, which often causes problems because of high phenotypic variability within species. Thus, I used DNA sequencing in my research to identify species of the genus Lobophyllia from the Indo-Pacific Ocean. Specifically, I tested whether color morphs from different areas in Indonesia actually represent different species or mere color variations. For that purpose, I conducted PCR amplification of the mitochondrial DNA cytochrome oxidase gene and two nuclear genes in 42 colonies of 14 color morphs of Lobophyllia sp. The PCR products were sequenced in both directions at the High Throughput Sequencing Center of the University of Washington, and phylogenetic trees including known species were used to determine the species identities. I expected that color morphs would be genetically differentiated and therefore might represent different species. Regardless of the outcome, this project has helped to understand the biodiversity in coral reefs better, and is also useful to identify appropriate units for coral management and conservation.

Phylogenetics is the study of evolutionary relationships and in order to infer these relationships in plants, various loci that are phylogenetically useful in nuclear as well as chloroplast DNAs should be utilized. Presently, the use of chloroplast loci is widespread in phylogenetic studies whereas the nuclear genome is still somewhat under-utilized. The pentatricopeptide repeat (PPR) gene family is a large group of protein coding nuclear genes that has been shown to be highly informative in the inference of evolutionary relationships among closely related species. There are multiple reasons that make these genes useful to these types of studies. The PPR gene family is very large which offers researchers multiple loci that are available for phylogenetic analyses. This is important because having multiple loci is imperative to answering important phylogenetic questions. A large portion of PPR genes are intronless making it possible to sequence and align data with little or no difficulty. They also have a high rate of evolution, and are single-copy in most plant genomes. 127 of these loci have been identified as phylogenetically useful but of these only 5 have been developed. This research looks to develop 10 more loci that will specifically target the Lamiales. To do this, 10 primer sets were tested using a sampling of species across the Lamiales. The primers were used to amplify these loci in order to ascertain their usefulness for a wide range of species and to obtain sequence data for each species. The sequences obtained were aligned and the alignments were used to design new primers. These new primer sets will allow for new data to be more easily collected that has been either difficult to obtain or previously unavailable and will increase the number of nuclear loci available for phylogenetic studies.

Lipid bodies are cellular organelles involved in lipid production and storage within cells. Until recently, lipid bodies were thought of as inert storage units rather than complex organelles that play a large role in lipid biogenesis. The freshwater haptophyte Chrysochromulina sp. has served as an excellent model organism for research on lipid bodies because of its high growth rate, fully sequenced genome and two large, distinct lipid bodies. In algae and other eukaryotes, heat shock proteins can be investigated to detect when the cell is stressed. In many species of algae, when the cell population is placed under stress, the cells produce large lipid bodies. A classic stress response has also been detected in Chrysochromulina sp. after discovering genes coding for heat shock proteins (HSP 70 and HSP 90). Previous studies show that HSPs appear to be induced under high salinity stress in Chrysochromulina sp. The current study examines two important aspects of the correlation between salinity stress and lipid body production: what concentration of salt and what time frame is needed to cause haptophyte cells to become stressed enough to produce large lipid bodies. Previous experiments show that adding salt to algal media during log phase of growth fails to produce large lipid bodies while adding salt initially to media does not produce significant biomass to allow protein assays. Since HSPs are induced under high salinity concentrations, the second part of this study aims to determine a correlation between lipid body size, lipid production and how/when HSP proteins are induced. This study uses flow cytometry, the neutral lipid stain BODIPY 505/515 and Western Blotting to examine the relationship between lipid bodies and HSPs. Future research projects may include manipulation of stress response pathways that are relevant for biofuel applications.

The presence of fungal symbionts residing in the root tissue of plants is a well-documented occurrence, yet questions regarding the identification and comparison of fungal partners in mycorrhizal relationships have been largely unanswered. Ericoid mycorrhizae, an example of a mycorrhizal relationship, are found in host plants within the order Ericales. Ericales, which contains such familiar species as persimmon, blueberry and Rhododendron, are able to persist in edaphic conditions due to their fungal symbionts. These symbionts form hyphal coils inside plant cell membranes, and thereby exchange crucial nutrients with the host plant. This project aims to evaluate the specificity between fungal communities and their host Rhododendron species, with the expectation that differing communities may exist, even in closely related hosts. Using known techniques to extract fungal DNA from the root systems of Rhododendron species in varying conditions and proximity, this DNA is then used to generate species based communities within specific Rhododendron hosts. By using type-cultures and genomic sequencing, comparisons of the presence or absence of fungal species within host roots can be made. This will shed light on infection intensity, and specificity between roots and fungal symbionts. Anticipated results of high levels of specificity between host plant and fungus could prompt questions regarding the importance of fungal symbionts in genus Rhododendron, especially with regards to the speciation between individual host plants.

Darwin and Wallace recognized natural selection as an essential force in evolution over 150 years ago. Today, we still investigate the processes that drive evolution and the origin of new species. Two sister species of wildflower in the genus Mimulus (monkeyflowers) present an ideal case for studying the genetic and ecological basis of speciation. M. lewisii and M. cardinalis coexist in overlapping ranges in the Sierra Nevada Mountains, and although they successfully interbreed in the laboratory, hybrids are extremely rare in the wild. Each flower species is visited by a unique pollinator – M. cardinalis by hummingbirds, M. lewisii by bumblebees. This indicates that traits influencing pollinator choice and pollination success account for reproductive isolation. We hypothesize that these interspecific differences can be traced to single genes with large effects on flower traits, such as color, that pollinators use in making the choice of which species to visit in the field. If our hypothesis is supported, this would demonstrate that single genes of large phenotypic effect are directly responsible for the origin of new species. Field work indicated that hummingbirds prefer red flowers (high amounts of purple and yellow pigment) while bumblebees prefer pale pink (small amounts of purple pigment). Using a genetic technique called positional cloning, my main contribution to the project was to locate a novel gene that regulates the amount of purple pigment (anthocyanin) in Mimulus flowers. We named this gene ROSE INTENSITY1 (ROI1), verified its identity and function by creating transgenic plants carrying the alternative M. lewisii and M. cardinalis alleles, and showed that ROI1 represses transcription of the genes that encode enzymes for anthocyanin biosynthesis. Our findings are being published in the May issue of Genetics. Future directions include quantifying the effect of ROI1 on pollinator visitation in the Sierra Nevada Mountains.

Asian Americans are not only seen as less American than White Americans, but are also subject to the pervasive ‘perpetual foreigner’ stereotype. Is weight a factor that affects how American Asian Americans are perceived to be? In study 1, participants were randomly assigned to evaluate one of three pictures of Asian American women on perceptions of American identity. Between conditions, the photos were edited to create a heavier and a lower weight version of each woman, thereby controlling for other cues to American identity, such as hair and clothing. Results showed that heavier Asian American women were perceived as more American than their lower weight counterparts. In study 2, using the same methods, we tested whether weight would affect perceptions of the legal status of Asian American men. Results revealed that the heavier versions of the Asian American men were perceived as more American and as less likely to be in the US illegally than the lower weight versions of the same men. Taken together, these studies provide evidence that being overweight can increase recognition of Asian Americans’ American identity and may help protect against discrimination based on racial stereotypes.

Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous species of pathogenic bacteria and a major cause of hospital-acquired pneumonia. Immune responses to P. aeruginosa infection in mice are mediated by a cytosolic multiprotein complex called the NLRC4 inflammasome. The NLRC4 inflammasome assembles upon ligand recognition leading to the release of the pro-inflammatory cytokine interleukin (IL)-1β and culminating in cell death. P. aeruginosa elicits a flagellin-dependent release of IL-1β and cell death in mouse macrophages, but research has not found human macrophages to recognize flagellin. This immune response is evoked in human macrophages by needle protein, an element of the type three secretion system (T3SS) infection apparatus used by P. aeruginosa. The mechanisms human macrophages use to regulate immune response to P. aeruginosa have not been fully established. We hypothesize that NLRC4 regulates immune response to P. aeruginosa in humans and therefore, NLRC4 deficient macrophages infected with P. aeruginosa will have impaired cell death and IL-1β production compared to normal NLRC4 expressing cells. Small interfering RNA (siRNA) will be used to knock down mRNA and protein expression of NLRC4. Macrophages treated with non-specific siRNA and NLRC4 targeted siRNA will be infected with live P. aeruginosa. We will then assay for IL-1β with ELISA and cell death using a lactate dehydrogenase (LDH) release assay. We expect NLRC4 knockdown cells to release less IL-1β and LDH compared to cells treated with non-specific siRNA thus indicating that in humans, the NLRC4 inflammasome is associated with defense against P. aeruginosa infection. Upon establishing the function of human NLRC4 during P. aeruginosa infection, we will proceed to investigate the roles of NLRC4 inflammasome subunits in ligand recognition and initiation of an immune response. Defining these roles could further our understanding of mechanisms behind immunity or susceptibility to P. aeruginosa and potentially lead to novel treatments for infection.

Online health communities provide networks that enable people to talk about and share their health related experiences. These communities, such as CancerConnect.com, DiabeticConnect.com, Patientslikeme.com, offer a broad base of personal health expertise, but discovering more experienced members, or “peer mentors,” with shared circumstances is difficult. To address this issue, we are designing mentoring profiles that illustrate a user’s health interests based on terms that are extracted from their posts from within the online community. Mentoring profiles can be used to connect users with peer mentors who share similar health interests and could provide personalized advice. In this study we are applying principles of user-centered design to develop mentoring profiles for cancer patients and caregivers who interact within CancerConnect.com. Through an iterative design process, we have developed a set of designs that we are implementing as profile prototypes. This set includes a text-based, tag cloud, and timeline variation of the mentoring profile. In particular, we are examining designs that can scale to variations in users’ posting frequency. Through a user study with cancer patients and caregivers, we are assessing those prototypes for interpretability, usability, and design preferences. Conducting a user study to receive feedback on the designs will enable us to understand how well users comprehend and use the different designs. Findings from this study will inform the future implementation of scalable mentoring profiles in online communities. This research contributes novel insights for optimizing the design of innovative matchmaking technology that connects individuals with helpful, experienced peers in online communities.

Rotavirus is particularly important to viral research because it is known to cause diarrhea in small children, usually leading to death. Viruses that are able to infect mammals have created mechanisms to evade host response by antagonizing cellular antiviral responses such as interferon response. Interferon Regulatory Factors initiate the immune response cascade by detecting the viral presence. Rotavirus NSP1 protein causes the dismantling of certain antiviral Interferon Regulatory factors depending on the particular strain. Antiviral Interferon Regulatory Factors 3 and 7 are important to initiate interferon creation, and post infection NSP1 induces degradation. We compared three strains from bovine, Human and porcine sources and used in vitro Site Directed Mutagenesis to try and investigate what about the protein causes such specificity in its selection of which antiviral Interferon Regulatory factor to break down. The overall test was to determine if mutagenizing NSP1 from one strain to another would change the substrate the NSP1 selects to degrade. This leaves the question, what does the transition from OSU to W161 mean? Site directed Mutagenesis changes nucleotides to create a mutation. These amino acids they code for are thought to be important to protein function. With such a detrimental virus, more research is necessary to discover more about the virus’ actions.

 Transition-metal ions promote a wide range of vital enzymatic biological and chemical processes involving dioxygen (O₂), including photosynthetic H₂O splitting, biosynthesis of neurotransmitters, and DNA replication and repair. These are some of the most fundamental processes of life, and yet they are also some of the most poorly understood. A complete grasp of how these processes function at the chemical level is therefore of critical importance to our development of technology that improves our lives and the world around us. For example, understanding how to efficiently oxidize H₂O to O₂ is essential to the development of renewable solar fuels and fuel cells. The challenge for research lies in that these transition-metal containing enzymes are inherently difficult to probe by traditional spectroscopy methods because of their structural complexity. The difficulty in characterizing these species and their reactions, coupled with their importance in the natural world, has made them a subject of intense research across all scientific disciplines. The Kovacs research group studies the structure and reactivity of these transition-metal containing enzymes, or metalloenzymes. Our research method involves modeling a metal ion’s local environment with a ligand scaffold and implementing changes to these so-called biomimetic models that may elucidate how reactivity corresponds with various structural and electronic modifications. Spectroscopy is more diagnostic in these simpler models because the complex proteins that obscure the active site in the actual enzyme are not incorporated into the model. By comparing similarities in reactivity between the biomimetic model and the actual enzyme, we may help elucidate properties and chemistry of the enzyme itself. Preliminary work has been undertaken with a novel series of thiolate ligated, coordinatively unsaturated Co(II) complexes; the reactivity of these complexes with O₂ and O₂^- is currently being probed.

The study of defects (nitrogen-vacancy centers) in diamond may be instrumental in developing better biological sensors that optically detect tagged biological molecules without interfering with natural processes. Using nitrogen-vacancy (NV) centers, our goal is to develop highly sensitive microscopes that detect magnetic fields produced by magnetic nanotags. Since the NV center photoluminescence intensity depends on its spin state, a decrease in photoluminescence will occur when a magnetic field is applied to the NV center. We employ this property in the detection of nanotags. The applications of this research include the study of biological systems, and faster, cheaper and earlier detection of diseases. While performing optically-detected magnetic resonance in NV centers results in high-sensitivity detection of magnetic fields, a reduction in signal occurs when the NV center symmetry axis is misaligned from the instrument’s externally applied magnetic field. Helmholtz coils, which produce near uniform magnetic fields, may be used to align the magnetic field to the proper orientation. Two Helmholtz coils, placed on the horizontal plane of our sample, will allow for clearer detection of photoluminescence dips as the magnetic field will be aligned to one of the NV center axis. In this work, we integrate and test Helmholtz coils in our NV based magneto-optical microscopes. This includes the development of electrostatic simulations and a search algorithm for the alignment of the magnetic field, as well as the fabrication of the coils.

Proteins typically adopt a three dimensional (3D) structure that allows for their specific activity and function. While proteins’ 3D structures are usually stable, they can be damaged by physical and/or chemical stresses. Such damage results in loss of structure, more commonly known as misfolding, which is often accompanied by exposure of hydrophobic residues normally buried in the interior of the protein. Exposed hydrophobicity can lead to the aggregation of misfolded proteins, and protein aggregation is associated with many prominent neurodegenerative diseases such as Alzheimer's and Parkinson's disease. To avoid these deleterious consequences, cells have evolved protein quality control (PQC) systems that degrade misfolded proteins. In eukaryotes, PQC utilizes ubiquitin ligases to flag proteins for proteosome degradation. How these PQC ligases recognize their misfolded substrates remains an open question. We have been addressing this question by studying the yeast PQC ubiquitin San1, which destroys misfolded proteins in the nucleus. We discovered that San1 recognizes exposed hydrophobicity minimally equal to 5 contiguous hydrophobic residues. Interestingly, we found that there is another PQC degradation system that recognizes exposed hydrophobicity at a lower threshold than San1. In this study we are trying to understand the mechanism by which this new pathway functions. By using degradation assays and western blots, we will take multiple constructs of varying degrees of hydrophocity to define how broad the pathway is. This allows us to determine whether the constructs are San1 dependent or independent, and if the San1-independent pathway is in fact a ubiquitin ligase. We know that a couple of the constructs are San1 independent, although they possess the hydrophibicity threshold required for San1 degradation. By exploring the mechanisms that ensure misfolded protein degradation, we hope to gain a better understanding of the PQC degradation systems within the cell and how they prevent severe pathologies caused by protein aggregation.

In most eukaryotic organisms, sexual reproduction is a critical component of the life cycle. Through meiosis, diploid cells containing two copies of each chromosome divide to produce genetically diverse haploid gametes (e.g. sperm) that contain one copy of each chromosome. This process is fundamental in sexual reproduction. Generally, meiosis results in a 50:50 transmission of heterozygous alleles into gametes. For example, human males make approximately 50% sperm with an X chromosome and 50% with a Y chromosome. Because only one copy of each chromosome can contribute to a given offspring, there is an evolutionary pressure on alleles to compete for preferential inclusion in gametes. Sometimes alleles can “cheat” to gain a greater than 50% transmission through meiosis. This is known as meiotic drive. Meiotic drive could have significant implications: altering fertility, speciation, and the evolution of chromosomes in eukaryotic organisms. For example, competition between alleles may be driving the rapid evolution of chromosome segregation factors acting during meiosis and mitosis, potentially driving chromosome segregation away from a theoretical ideal. Though meiotic drive has not been well understood due to limited model systems, we have developed a novel system utilizing genetically tractable fission yeast S. pombe and S. kambucha, to investigate meiotic conflicts between competing alleles. In S. pombe, heterozygous alleles are widely thought to segregate fairly in meiosis, suggesting that active meiotic drive loci are not present in this organism. We have found that silenced meiotic drivers can be de-repressed in aged germ cells, leading to greater than 90% transmission of certain alleles through meiosis. This provides experimental evidence in support of the evolution of cheating in meiotic processes that could ultimately result in decreased fertility amongst eukaryotic organisms, including humans. 

The U.S. goal of producing 16 billion gallons of cellulosic ethanol by 2022 will be possible only by carefully making use of all available feedstock sources. Forest residues, including material remaining after logging or thinning, are an inexpensive and widely available feedstock, with estimated potentials between 30 to 120 million dry tons a year on the source. However, their heterogeneous nature and relatively low cellulose content renders them a challenge to current pretreatment and bioconversion methods. Forest residues typically consist of branches, needles/leaves, bark and wood in varying proportions and particle sizes, so some degree of homogenization prior to pretreatment is essential for consistent results. Our objective in this research was to develop methods to utilize this material for production of ethanol via SO₂ catalyzed steam explosion pretreatment, enzymatic hydrolysis and fermentation. To achieve this, we first homogenized biomass consisting of Douglas-fir and Hemlock residues. We pretreated this material at temperatures ranging from 195°C to 205°C, with residence times from 5 to 20 minutes to fractionate the cellulose and hemicellulose from lignin. We subsequently quantified the sugars remaining after pretreatment, in both the liquid and solid phases using gravimetric and HPLC analysis to determine which pretreatment resulted in the best sugar recovery from the original raw biomass. This data was then used to determine which pretreated solids were the best candidates for enzymatic hydrolysis, which we then conducted to convert all of the insoluble cellulose to soluble, fermentable glucose. From our data we compared sugar recovery, enzymatic hydrolysability and other factors to determine the best pretreatment conditions to use to increase the overall production of ethanol from heterogeneous forest residues.

Impulsivity is often found to be related to alcohol use (Pearson and Henson, 2012; Simons, 2003). Additionally, a correlate of alcohol expectancies is increased alcohol use (Hull and Bond, 1986). However, no research to date has looked directly into the association of impulsivity, alcohol expectancies, and alcohol use. Therefore, the present study looks into impulsivity, alcohol use, and alcohol expectancies in high school seniors (N = 3,352, 56.6% Female) from Washington and Sweden. Participants completed a baseline online survey as part of a larger trial that assessed peak blood alcohol content (BAC) in the past 12 months, impulsivity as measured by the sensation seeking scale (Zuckerman, 1964), and alcohol expectancies as measured by the brief Comprehensive Effects of Alcohol questionnaire which consists of positive (sociability, tension-reduction, liquid courage, and sexuality) and negative (cognitive/behavioral impairment, risk/aggression, and self-perception) classifications (CEOA; Fromme, 1993). Regression analyses revealed a significant main effect of impulsivity, cognitive/behavioral impairment, sociability, risk/aggression, and self-perception onto peak BAC (p < .05). However, significant interactions were only found for impulsivity onto risk/aggression and self-perception. A significant moderating effect of risk/aggression onto the relationship between impulsivity and peak BAC (β = .002, F (3, 2913) = 114.71, p < .05) was found such that those with lower impulsivity and lower risk aggression scores (alcohol seen positively) have higher peak BAC. A significant moderating effect of self-perception onto the relationship between impulsivity and peak BAC was also found (β = .001, F (3, 2908) = 132.04, p < .05) suggesting that individuals with lower self-perception scores (alcohol seen positively) and higher impulsivity have higher peak BAC. These results suggest that individuals who are both highly impulsive and have higher positive expectancies for alcohol drink more. Future research should continue to investigate the relationship between alcohol expectancies and impulsivity in vivo.

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.

Two 5-membered rings with a nitrogen at one of the fusion sites is the core structure for a variety of natural products known as pyrrolizidine alkaloids. Many of these compounds are produced by plants as a defense mechanism due to their hepatotoxicity. Other varieties of these compounds, however, have been shown to possess antiviral, anticancer, antidiabetic, and antiobesity drugs. Because of their inherent biological activity and the relative costliness of extracting these compounds from natural sources, they have become an important synthetic target. Previously in the Michael group, a palladium catalyst was developed to facilitate the addition of an N-H bond across a C=C bond. This transformation is known as hydroamination. When the protected amine and alkene are present in the same molecule, cyclization occurs, forming 5- and 6-membered nitrogen containing heterocycles. This most recent work sought to extend the capabilities of this reaction by replacing the amine protecting group with a highly electron deficient aldehyde. Thus after the initial addition of the N-H bond across the C=C bond occurs, the C=C bond will then perform a nucleophilic attack on the aldehyde forming a second ring fused to the first. This reaction sets 3 new stereocenters, forms 2 new rings, and generates a C-N and C-C bond in a single, diastereoselective step. The substrate synthesized in order to test this reaction was a 7 carbon terminal diene amine coupled with dimethyltartrate and subsequently oxidized to generate the desired diene-amide-aldehyde functionality. This substrate when submitted to the hydroamination conditions resulted in the diastereoselective formation of the tri-substituted pyrollizidine in 35% yield. Further work in this project will include application of these methods to the synthesis of several natural pyrrolizidine alkaloids. It is hoped that further manipulation of the substrate will allow expansion of the range of attainable targets to that of indolizidines and quinolizidines.

Alcohol use consistently correlates with negative physical and psychological consequences like dependence, memory loss, impaired judgment, etc. Studies also link the use of sleep medications with similar negative consequences. Yet, no study examines the interaction of sleep medication and alcohol use onto these negative consequences. Therefore the present study investigated the relationship between peak blood alcohol content (BAC) in the past 6 months, negative consequences of alcohol use in the last 12 months as measured by the Rutgers Alcohol Problem Index, and lifetime, 3- and 12-month non-prescribed sleep medication use in Washington state and Swedish high school seniors participating in a larger study (N = 3,352, 56.6% Female). Regression analyses revealed a significant main effect of BAC and lifetime use of sleep medication onto negative consequences (β = 18.69, F (3, 2875) = 115.86, p < .05) such that individuals with a higher BAC and who had a lifetime incidence of sleep medication usage experienced significantly greater negative consequences than those who only drank or used sleep medications. There was no significant moderating effect of frequency of sleep medication use in the last 3 or 12 months onto BAC and negative consequences (p >.05). While these results suggest that use of sleep medication and alcohol interact to contribute to increased risk for negative consequences, there are limitations. Lifetime use of sleep medication may reflect a tendency to act impulsively or self-medicate (e.g. for sleep/depression/anxiety), not a direct effect of the medication per se. Additionally, analyses did not control for other drug usage or mood disturbances, and all sleep medication drugs were grouped under one survey item. Given our observations, additional research testing combined effect of sleep mediation and alcohol use as related to negative consequences is warranted.

French films about World War II tend to fall into either the category of heritage film, which promotes a narrative of reconciliation in relation to the collaboration, or another group of films that serves to implicitly or explicitly question that narrative of reconciliation. Films of the latter category have probed the memory of the Vichy collaboration since the end of the war despite Charles de Gaulle’s efforts to repress narratives that challenged the idea of a country united against Germany. In the 1980’s, however, heritage film became the prevalent genre and offered a Gaullist view of World War II in France. This thesis asks, why, given the plethora of films dealing with the realities of the French role in World War II dating from the late 1940’s, a rubric of heritage film emerged as one way of classifying filmic discourse on the Vichy collaboration. I hypothesize that the heritage cinema rubric, specifically the Vichy cinema rubric, asserts a legibility of French identity, which was compromised by France’s collaboration with Nazi Germany, and attempts to thwart filmic narratives of identity loss. Through tropes of the uncanny and characters that experience repressed trauma and blocked mourning, films such as Le Silence de la Mer (1949), Lacombe, Lucien (1974), Mr. Klein (1976), and Le Dernier Metro (1980) reflect the French collective experience of loss during and in the aftermath of the collaboration. WWII films produced in the decades leading up to the emergence of this genre in essence dramatize a troubling disappearance of French cultural identity, which the heritage rubric sought to control.

Free available chlorine (FAC – generally comprising of hypochlorous acid and hypochlorite in aqueous solution at circumneutral pH) is currently the most common disinfectant in drinking water facilities around the world. Although the use of FAC is inexpensive and generally effective, some microorganisms that may be present in drinking water are recalcitrant to inactivation by the use of FAC alone. We are investigating the use of FAC photolysis by sunlight as a novel approach to enhancing the inactivation of such microorganisms. This process is known to generate atomic oxygen, which may react with dissolved oxygen to generate ozone -a much more effective disinfectant than FAC toward many microorganisms. Our studies have already indicated that the photolysis of FAC can enhance inactivation of highly chlorine-resistant B. subtilis spores (common surrogates for chlorine-resistant waterborne pathogens) through the generation of ozone. The main focus of the research to be presented addresses development of a method for in situ quantification of ozone formation during chlorine photolysis and its effects on the inactivation of B. subtilis spores. We are currently investigating the use of various probe molecules for this purpose, including cinnamic acid and vinyl phosphoric acid – each of which reacts selectively with ozone via Criegee ozonolysis; yielding stoichiometric quantities of benzaldehyde and phosphate, respectively, which may be quantified as measures of ozone exposure. Once validated, the use of such an approach would enable direct quantification of the ozone levels to which microorganisms are actually exposed during FAC photolysis, in turn facilitating the prediction of pathogen inactivation rates in treated waters.

Horseradish peroxidase (HRP) is part of a family of heme-containing metalloenzymes that reduce the O-O bond of peroxides while oxidizing a wide variety of substrates; in the case of HRP, hydrogen peroxide is reduced to water. In the presence of an oxidant and the absence of a substrate to oxidize, hydrogen peroxide coordinates to the iron cofactor of HRP, forming a ferric-hydroperoxide intermediate known as Compound 0, and subsequent O-O bond cleavage releases water and a very oxidizing ferryl-oxo porphyrin radical cation intermediate, known as Compound I. The goal of this project is to probe the potential reversibility of Compound I formation, a step traditionally thought to be irreversible. A key experiment will react HRP with unlabeled H₂O₂ in ¹⁸O-labeled water, and determine whether mixed-labeled H₂O₂ is formed. The presence of mixed-labeled H₂O₂ indicates that there is reversibility of the O-O bond cleavage that forms Compound I and its existence will be determined by addition of a water soluble phosphine that is too bulky to fit into the active site of HRP. In this case, the phosphine cannot be oxidized by Compound I, but can readily react with free hydrogen peroxide in solution to give the corresponding phosphine oxide. ³¹P-NMR will be used to determine whether the phosphine oxide is labeled because the NMR shifts of ¹⁶OPR3 and ¹⁸OPR3 are distinct. If this reaction proves to be reversible, this knowledge could then be used to describe other enzymes that catalyze oxidations with similar intermediates. This would not only be important to enzymatic systems, but possibly catalytic chemical processes.

Chemical modifications to histones, such as acetylation, phosphorylation, methylation and SUMOlyation, play a major role in dictating gene expression by changing the structure of chromatin. In particular, the modification of histone H4 by SUMO (called SUMOylation) was reported to be associated with gene repression. However, preliminary results from our lab show that the structure of SUMOylated chromatin resembles transcriptionally active, rather than silent chromatin. In order to comprehend how SUMO achieves gene silencing, biochemical studies with nucleosomes containing uniformly and site-specifically SUMOylated histones are necessary. Here we present a synthetic strategy to produce histone H4 modified by SUMO at Lysine 12. This strategy requires two native chemical ligation reactions. The first is between two fragments of H4- one synthetic and one recombinant. The second is a ligation between the SUMO-3 isoform and H4. In order to achieve the ligation of SUMO-3 to the side-chain of Lys in H4, we have developed a new ligation auxiliary. The synthesis of this auxiliary will be presented along with its application toward the total synthesis of SUMOylated histone H4. By understanding how SUMO-H4 controls gene function we hope to identify novel means to change cellular processes that result in diseases such as cancer.

Auxin is the plant hormone, guiding phototropism, gravitropism, cell elongation, cell division, cell determination, and a host of other processes. Plant cells respond to auxin by turning on certain genes. When auxin enters the cell, it binds to a receptor protein; this receptor then binds to a repressor protein which typically prevents expression of auxin genes. This interaction triggers repressor breakdown and gene expression. We are investigating auxin-induced interactions between the receptor and repressor proteins (IAA’s). In our model plant, Arabidopsis, there are 29 IAA repressor proteins and 6 receptor proteins. Some combinations of repressors and receptors are known to result in differential degradation of the repressor; other interactions do not seem to lead to degradation. Our data suggests that receptors AFB4 and AFB5 cannot degrade repressors. This could be because AFB4/5 do not interact with the repressors (hypothesis 1) or because their interaction with the repressors does not trigger degradation (hypothesis 2). We are testing these hypotheses by examining repressor/receptor interactions in Arabidopsis proteins expressed in budding yeast cells. To address hypothesis 1, we are using a yeast two- hybrid assay to investigate the strength of interaction between several IAA repressors and the AFB4/5 receptors in the presence of auxin. To address hypothesis 2, we will determine whether IAA: AFB interactions identified at the yeast two-hybrid assay lead to repressor degradation. We have tagged the IAAs with yellow florescent protein and will co-express them with AFB4/5 in yeast. Flow cytometry will be used to measure changes in florescence levels upon addition of auxin. The next step will be to confirm these findings in plants. Understanding the variation in auxin repressor/receptor interactions brings us a step closer to understanding how auxin drives so many different plant behaviors and responses.

Characterizing the migratory behavior of tumor cells is essential in understanding cancer progression and the invasive capacity of the cells that could lead to tumor metastasis. Previous research has established a correlation between the signalling protein known as stromal cell-derived factor-1 (CXCL12) and its corresponding CXCR4 receptor, which are expressed by a subset of cancerous glioma stem cells. Glioma cells expressing the CXCR4 receptor will move toward areas of higher CXCL12 concentration. This signalling pathway plays a significant role in glioma development by enhancing cell proliferation and motility. Thus, there is strong therapeutic potential by neutralizing this pathway through the use of CXCR4-inhibiting drugs. Microfluidics offers a suitable platform to optically characterize the migration of glioma stem cells under the influence of these inhibitory drugs while in the presence of controlled CXCL12 gradients. We present a microfluidic device integrated into a porous membrane to create an air-fluid interface that allows for organotypic slice culture and a fluidic chamber below the culture membrane that enables the formation of stable gradients. The integration of a 96-well plate allows for the simple addition of reagents using a standard pipette and circumvents the need for excess tubing and gels necessary for conventional microfluidic gradient assays. This device offers a spatial control of the biochemical gradient in an organotypic slice culture model to study chemotactic migration of glioma cells in a physiological-relevant manner. This model gives insight into the signaling processes related to the invasiveness and proliferation of glioma stem cells, and could potentially lead to new therapeutic strategies that target the CXCL12/CXCR4 pathway.

Because African immigrants are often lumped into the same category as African Americans, there has been very little research conducted on the health outcomes of African immigrants. This has resulted in an ideology of promoting preventative medicine versus treatment.  This study, based on self-reported information, will ask the question: what are the differences in susceptibility to hypertension and other cardiovascular disease-related risk factors among different African immigrant groups in the Pacific Northwest, specifically those from Somalia, Nigeria, and Ethiopia. The hypothesis of this study is that Ethiopian and Nigerian immigrants are at a lower risk of hypertension and other cardiovascular diseases as compared with Somali immigrants because of differences among their country of origin's health care systems. To be able to answer this question, I examined and evaluated the recent historical backgrounds of these three countries by conducting literature review. I also used surveys to collect data and critically analyzed and grouped the responses according to key words and comment phrases to test the correlation of the responses to the main research topic. I expect that the results will paint a mixed picture about African immigrants’ health outcomes, specifically rates of hypertension and cardiovascular disease, as region of origin confers major health effects on immigrants. This research will shed light on the differences among African immigrant groups separate from the African American population.

Burma is now undergoing an incredible transition toward peace, stability and democratic governance – a transition that we found requires a fundamental re-thinking of U.S. policy relations. While we discovered Burma’s commitment to reform to be genuine, its challenges are numerous. Instability and underdevelopment continue to dominate the Burmese landscape, defined by a weak constitutional government, repressed civil society, unreformed military, ethnic violence and an underdeveloped economy. This past year marked President Obama as the first American president to visit Burma, extending a hand of friendship, committed to strengthening political and economic ties between the United States and Burma. As well as a real commitment by the United States to reduce economic sanctions, aid in the democratization process, and lift the Burmese economy out of poverty -  elements we proved to be essential in ensuring a successful transition in Burma. Our task force recommends greater engagement, aid, and targeted policy consultation to help the country emerge from decades of military-junta rule and rejoin the international community. Ultimately, Burma offers a new roadmap in the United States in re-thinking the reform process of developing countries, one that now requires trading the tools of isolation and international pressure for those of integration and international cooperation. The world is now watching Burma as it prepares to chair the ASEAN council in 2014 and hold democratic elections in 2015. No one knows how long this moment for reform will last, but the time for action is now.

A recent discovery in reproductive endocrinology, the kisspeptin system has been found to play a pivotal role in the regulation of the brain-pituitary-gonadal axis. Kisspeptin signaling in the brain is vital to the onset of puberty, control and regulation of sex steroid production, and regulation of fertility and reproductive function in mammals. However, the importance of the kisspeptin system in other vertebrates, such as fish, is not as clear. The goal of the current study was to clone the kisspeptin genes (kiss1, kiss2, and the receptor kiss1ra) for the first time in the marine teleost Anoplopoma fimbria (sablefish), and explore their potential roles in gonadal development. Previously, expression of the kiss2 gene was demonstrated in the gonads of sablefish. Partial kiss mRNA sequences were obtained by PCR and rapid amplification of cDNA ends (RACE) cloning techniques. Semi-quantitative PCR assays developed for each gene allowed us to measure kiss1, kiss2 and kiss1ra mRNA levels in a variety of adult tissues. We found the highest abundance of these mRNAs in the gonads, brain, and pituitary. In particular, kiss2 mRNA was abundant in the ovary and appeared to be the most highly expressed of the kiss genes. We are currently analyzing the expression of the kiss genes in gonads of fish undergoing sexual differentiation. Preliminary data suggest that there is sexually dimorphic expression of all three genes early in life. The ultimate goal of this project is to determine if gonadal kiss gene expression plays an important role in sexual differentiation and/or gametogenesis in fish.

The lift-to-drag ratio (L/D) of a wing or propeller is the most important measure of its performance, as a higher ratio defines more efficient designs. The goal of my group’s project is to show that sufficiently strong and persistent vortices along the top of a wing will positively benefit its aerodynamic efficiency. Previous results in the UW Fluid Dynamics Lab show that a wavy wall modeled after the Kelvin-Stuart Separatrix shape -  which intends to more accurately outline the vortice's flow - further minimize boundary layer turbulence compared to previous wavy wall shapes. We believe that modeling the upper half of our wing along the Kevlin-Stuart Separatrix will keep the boundary layer attached even at very high angles of attack, with relatively low drag coefficients. Two model wings are being tested in the wind tunnel, a control wing with a familiar smooth top half and our experimental model outfitted with vortex generators and the top half shaped to match the Kelvin-Stuart separatrix. Further research may include attaching surface tufts to both models, which qualitatively aid in visualizing flow. The models are mounted to a force balance system, which returns the lift and drag. Our experiments test various angles of attack and vortex generator configurations to compare the wings in different situations, such as take-off and cruise. Achieving the desired L/D will have broad implications for both wing and wind turbine propeller design. More efficient wings could drastically lower fuel costs for airliners, and more effective wind turbines would return more energy at lower cost.

Software systems are often difficult to comprehend. When a system behaves in an unexpected manner, or when developers must modify the code written by someone else, they have the challenge of understanding the system behavior. A typical way developers use to gain insight into a system is logging. Logging is a method that captures program activities and states during an execution into a log file (a text file), which developers then inspect. Unfortunately, the size and complexity of logs are often too large for manual inspection. Synoptic is a tool that supports the task of inspecting execution logs. Synoptic takes a log as input and infers a finite state machine model of the process that generated the log. The model that Synoptic produces is compact and captures essential behavior traits of the subject system. However, there is a missing piece of information. Synoptic is only interested in system activities, thus the models it infers are activity-based models. System states, which are also often captured in a log, are ignored. This project presents an approach to utilize state in model inference. We extend Synoptic to model data values in a subject system by annotating states in the inferred model with conditions on data values. We use Daikon, an implementation of dynamic detection of likely invariants, to determine those conditions from an input log that captures data values at each state of the system. We argue that this new model helps developers reason about their systems better.

Studies using electroencephalography (EEG) have supported the theory that mu rhythms, oscillating bands in the 8-13 Hz range, are related to sensorimotor processing. At rest, EEG mu rhythms are continuous but become suppressed during the observation and execution of biological motion. Individuals with autism spectrum disorder (ASD), a disorder characterized by social and communication impairments, display atypical mu activity. Individuals with ASD exhibit typical mu suppression when executing self-made movements but significantly less suppression when observing the same movements. Individuals with ASD also show consistent impairments in imitative ability. One theory is that dysfunction in the observation/execution system contributes to this impaired imitation ability. To this point, studies have primarily focused on mu attenuation during the observation of hand or body movements, but few have focused on mu in response to facial movement. Since facial processing is integral to social learning, understanding perception deficits for facial movements may be important for understanding social impairment in individuals with ASD. This study will investigate whether sensorimotor processing of faces correlates to imitative ability by analyzing the relationship between mu attenuation of facial movement and facial imitation in children with ASD. For our study EEG was recorded while children and adolescents with and without ASD observed computer generated faces performing biologically possible and impossible movements. Participants then completed a battery of imitation tasks which included imitation of facial gestures using the mouth, eyes, or tongue. We hypothesize that the level of mu attenuation during observation of biologically possible facial movements will correlate with facial imitation ability in children with autism. Furthermore, decreased mu attenuation to biologically possible faces will be related to an increased number of imitation errors. Results of this study may help to improve understanding of social impairments of ASD children as related to deficits in sensorimotor perception of biological motion.

 Washington State is home to many diverse prairie ecosystems, which have severely been degraded or converted to agriculture/farmland. Due to the impact over the last several decades, it is crucial to research Washington’s native prairie ecosystems and work towards preservation, conservation, and restoration of these areas. There are many ongoing projects on these prairies, in efforts to gain more knowledge about the growth, development, and interactions of rare prairie species. It is very important to determine underground processes because these processes could affect the long-term success of restoration. I compared microbial metabolism and nitrogen mineralization among three restoration treatments: burning, herbicide, or solarization, at Glacial Heritage Preserve in Olympia, WA. Plots were seeded with a diverse mix of native prairie species 2, 3, or 4 years before this study. The goal of the project is to determine if soil processes differ among plots, and if it is a result of treatment or year seeded. Soil samples were collected from replicates of each treatment plot from each year, and extracted and inoculated into Biolog Ecoplates^TM. The amount of Nitrogen available for mineralization was also measured. Preliminary results display that there is some difference between treatments and years, and available Nitrogen varies greatly among treatment plots. The results of each experiment are being further investigated.

Glioblastomas (GBMs) are grade four astrocytomas, composed of heterogeneous cells with unique molecular features. Using brain tumor and tumor stem cell lines, we are developing ex vivo methods to use on-chip, single cell characterization and mass spectrometry to better understand brain tumor heterogeneity in order to personalize drug therapy and optimize treatment of GBMs. We first performed an on-chip, single cell culture, using a biocompatible poly dimethyl-siloxane (PDMS) microchip that contains 50-100 nm subnanoliter wells. A diluted solution of cultured cells was loaded onto the microchip while the chip was submerged in DMEM, and the cells settled into the wells by gravity. The PDMS microchip was then subjected to hybridization conditions, to simulate the technique of assaying cytokines. Cell viability was monitored using a live/dead fluorescence assay. To pursue the goal of analyzing protein arrays using single cell mass spectrometry, we loaded a PDMS microchip; first with bovine serum albumin digest, then with hemoglobin, as controls to obtain a MALDI (Matrix-Assisted Laser Desorption Ionization) spectrum. We found that a solution of approximately 7.14x103 cells/mL ensured that one cell was delivered per well. The neuroblastoma cells did not exhibit typical dendritic morphology within the microchip. They remained round due to the non-adherent properties of PDMS. When the loaded microchip remained submerged in DMEM and incubated, the cells grew to confluence and remained viable up to seven days. Under hybridization conditions, the cells remained viable up to five days. The MALDI controls were assessed, but did not produce a spectrum. The soft and thick PDMS surface may inhibit the MALDI laser’s accuracy to pinpoint proteins. Also, PDMS is not electrically conductive and impedes analyte ionization. Therefore, the project has been continuing to optimize the surface for analysis. This work is still ongoing with promising results.

Today, plants are facing increasing environmentally stressful conditions due to several factors attributed to climate change such as heat, drought and water salinity. These factors hinder most plant growth and, in response, there is often an increase in the use of chemical fertilizers. These fertilizers can become expensive and also have adverse effects on the environment. A possible alternative to fertilizers is the use of nitrogen fixing endophytes and abiotic stress reducing fungal endophytes. Endophytes are bacteria or fungi that live within a plant, establishing a mutualistic relationship in which they provide essential nutrients or abiotic stress relief for the plant in return for nutrients and protection. Conifers are an important part of the forest industry and are extensively planted for timber production. Thus, it would prove beneficial for plantations and the environment alike to decrease the use of fertilizers. In my research I have inoculated Cedar, Pine and Douglas Fir seeds with different strains of nitrogen fixing endophytes and stress reducing fungal endophytes. Because confirmation of inoculation is essential, these endophytes have been tagged with a florescent marker to confirm inoculation using fluorescent microscopy. Once inoculation was confirmed, I introduced the conifers to various stressful growing conditions such as; low nitrogen levels, high heat, water scarcity and increased water salinity. Nitrogen levels, growth, and response to stress between inoculated and uninoculated conifers were then measured. These fungal and bacterial endophytes have been shown to increase plant metabolic efficiency, reduce water and nutrient use, and produce greater biomass and yields in crop plants. Thus, similar results are predicted in conifers inoculated with these endophytes. If these results are confirmed, the use of these endophytes could become a sustainable alternative to chemical fertilizers and, in turn, aid in the mitigation of climate change on forestry ecosystems and the environment.

Because of its unique immune characteristics, the vaginal tract is highly susceptible to attacks from sexually transmitted infections (STIs) and other pathogens. Therefore, there is a need for vaccines that can be delivered directly to the vaginal mucosa and locally protect against STIs. Due to the high vascularization of the vaginal tract, free drug may traffic to other parts of the body before it has the ability to noticeably impact the mucosal immune environment. To address these challenges, we propose utilizing a vaccine encapsulating, lipid nanoparticle that is capable of storing drug, that can be retained in the vaginal tract until it directly interacts with local immune cells capable of mounting a strong local immune response. The Woodrow Lab (at the University of Washington Department of Bioengineering) has been working on developing a protocol to synthesize a stable, biocompatible lipid nanoparticle that can encapsulate a multi-component vaccine. The particles are synthesized by cross-linking together concentric polar-lipid bilayers to form stable nanoparticles. The multi-bilayer cross-linked nature of these nanoparticles increases stability and allows them to be retained within the tissue longer than simple single-bilayer particle systems. Here we show that our particles exhibit better material efficiency and have the potential to encapsulate greater levels of drug than similar lipid particle platforms. Through electron microscopy we have demonstrated that our protocol successfully produces particles with the characteristic uniform multi-bilayer morphology that we expected. We are currently working on optimizing our production process for scale-up production of the particles for larger trials. In future experiments we plan to encapsulate vaccine in the particles and use in vivo mouse models to demonstrate that our particle-vaccine system creates a more potent and longer lasting immune response than free vaccine alone. This method of vaccine delivery may prove effective at preventing several STIs including herpes and HIV.

Glioblastoma Multiforme, the most prevalent form of primary brain tumors, is characterized by its aggressive, invasive nature and short patient lifespan post-diagnosis - approximately 12 months. Through gene analysis of RNA sequencing data, we have found two miRNAs, miR-149 and miR-181, that target gene products whose increased expression has been shown to promote invasion and migration of U-87 human glioblastoma cells. miRNAs are short, non-coding RNAs that bind to messenger RNAs (mRNAs) - the body’s method of transferring genetic information, and target them to be degraded or prevent them from being translated into protein. As the miRNAs and the genes are inversely related, treatment of U-87 cells with the miRNAs would cause a decrease in migration, and blocking the miRNAs would cause an increase in migration. To quantify migration, we have employed a scratch test or wound healing assay, in which glioblastoma cells are grown on a dish and the middle of the plate is scratched, leaving space for the cells to migrate. The cells are treated with the miRNAs, and the percent of the gap filled after 24 hours is measured. We expect changes in miRNAs to affect the amount of migration in the gap compared to control - providing evidence that these miRNAs inhibit migration. miRNAs have been investigated as markers for disease, and the presence or absence of this miRNA in a patient’s glioblastoma may have implications as to the severity of their case. Additionally, characterizing these miRNAs adds to the database of knowledge concerning glioblastoma. The more that is known about this disease, the closer scientists are to finding a practical treatment.

Skeletal muscle myosin can use a variety of nucleotides to varying effectiveness as substrates for contraction. We previously demonstrated that complete replacement of ATP with 2 deoxy-ATP (dATP) in activation solutions increases contraction of demembranated rabbit skeletal muscle and that enhanced performance results from increased myosin binding and cycling kinetics, especially during sub-maximal calcium activation. Here we report on a transgenic mouse (Tg-RR) that overexpresses the enzyme, ribonucleotide reductase, which converts ADP to dADP, which is rapidly phosphorylated to dATP. This results in a 10-fold increase in dATP content of skeletal muscle, which still constitutes less than 2% of the adenosine triphosphate nucleotide pool. We are examining the contractile and metabolic properties of skeletal muscle in this transgenic model. Preliminary data indicates the phosphocreatine to ATP (PCr:ATP) ratio of Tg-RR mice is significantly elevated relative to wild type mice, suggesting the Tg-RR mice may have an energetic advantage due to increased high energy phosphate reserves. Furthermore, the Tg-RR mice ran for a longer period of time at increased speed and for longer distances in a graded exercise test. Direct in situ stimulation of the gastrocnemius muscle indicates improved resistance to fatigue, consistent with both the exercise and the metabolic (PCr:ATP) findings. Interestingly, our preliminary experiments suggest the Tg-RR mice produce significantly less peak force and show signs of atrophy. Ongoing experiments are examining changes in mitochondrial content and function as well as contractile properties of isolated muscles to determine the mechanisms underlying the increased fatigue resistance of the Tg-RR mice. These results suggest a new direction for developing interventions to improve exercise tolerance in human patients.

Heart disease is often responsible for morbidity and mortality in the United States. Current treatments aim to slow the progression of the disease by modifying calcium handling in the heart, but do not restore function to healthy levels. Endogenous troponin C (TnC) is the calcium binding subunit of troponin, a complex that regulates contraction in muscle. The amino acid substitution of leucine for glutamine at position 48, L48Q, in cTnC has been shown to increase calcium binding affinity of the troponin complex. We hypothesize that the expression of engineered protein L48Q-cTnC will increase calcium sensitivity of a failing heart and restore function without disrupting calcium handling. The aim of this study is to determine the expression pattern of this engineered protein in vivo in healthy rat hearts after various dosages of adeno-associated viral vectors of serotype 6 (AAV6) containing L48Q-cTnC. In addition, we aim to determine the correlation between vector dosage and changes heart function. We will achieve these goals by administering our AAV6 virus to healthy rats and monitoring whole heart function over the next four weeks. Heart function will be assessed in vivo with non-invasive echocardiography ex vivo on a Neely whole heart apparatus four weeks post-injection. The Neely apparatus allows us to control the environment of the heart while measuring function, which is not possible in vivo. The expression of L48Q-cTnC will be determined by immunohistochemistry and Western blotting. From this data, we will be able to calculate the dosage necessary to achieve maximum expression and increased heart function in our healthy heart model. This initial study it vital to characterizing the expression and efficacy of a potential heart failure therapeutic and will directly impact the design of future studies in disease models.

Does a genetic predisposition towards excessive alcohol consumption influence the acquisition of habits? The study of alcohol dependency and its effects on learning and behavior continues to be a subject of growing importance. With an animal model of alcoholism, we sought to uncover some of these potential behavioral consequences. We focused on the development of both flexible and habitual spatial behaviors in rats selectively bred for High Alcohol Drinking (HAD) and Low Alcohol Drinking (LAD). Using a standard plus-maze task, rats were trained to run to an unchanging reward arm. We hypothesized that HAD rats would rely more on response learning strategies than spatial strategies, and would develop a habit more quickly than LAD rats. Initially, visual cues were placed around the room, and two probes were conducted to assess the progress of habitual behaviors in each animal. Later, cues were removed and one last probe was conducted. Preliminary data analysis revealed some unexpected findings, the most striking of which was seen in the LAD rats’ behavior. We expected both groups of animals to form a habitual response by the end of testing, however the percentage of LAD rats using spatial strategies increased with each successive probe. HAD rats, on the other hand, did appear to increasingly rely on response strategies over time, suggesting that they may be more prone towards developing a habitual behavior under these conditions. Additionally, a large number of HAD rats consistently omitted trials and demonstrated increased latencies when compared to LAD rats, possibly indicating that receiving a reward was not enough of an incentive to run the task. These preliminary findings may suggest that genetic preferences for alcohol consumption could influence the learning strategies employed in solving simple behavioral tasks.

Ischemic preconditioning (IPC) refers to the neuroprotective phenomenon in which a brief ischemic exposure in the brain increases resistance to injurious effects of subsequent prolonged ischemia. Ischemia is the restriction of blood to tissues. Interferons (IFNs) are cytokines released during ischemic exposure to activate the Interferon alpha receptor (IFNAR)  complex and trigger the transcription of interferon stimulated genes (ISG). ISG gene products may contribute to IPC-mediated neuroprotection. However, IFN receptors are expressed on myeloid cells as well as neural cells; the cell type-specific contribution of IFN signaling in IPC is not established. Microglia are the resident immune cells in the brain and are central in the inflammatory responses to stroke. Data from our lab strongly suggest that microglia exposed to ischemia both in vitro or in vivo demonstrate a robust expression of ISGs. We hypothesize that expression of IFNAR1 and intact IFN signaling on microglia is required for IPC mediated neuroprotection. Thus, the administration of IFNβ prior to stroke will mimic IPC in the wild type (WT) but not in myeloid cell targeted IFNAR1 (Cre/loxP) knockdown mice. We obtained IFNAR1fl/fl strains through collaboration and purchased LysM-Cre. We then used selective breeding and PCR genomic strategies to generate mice that are combined for homozygous IFNAR1fl/fl and heterozygous for LysM-Cre and wild type (WT) genes. We are also currently optimizing our method to administer IFN β using a stereotactic injection system through the lateral ventricle of the brain. We have recently confirmed the appropriate positioning of the intracerebroventicular (ICV) injection and plan to carry out ICV injection of IFN β on the WT and IFNAR1fl/fl /LysM-Cre strains. After inducing stroke, we will quantify and compare the infarct volume and neurobehavioral outcomes. This study will demonstrate the cell-type specific contribution of microglia in IFN-mediated neuroprotection. If successful, our findings can be used to develop pharmaceutical therapies for stroke and gene therapy remedies.

Essentially all growth and developmental processes in plants require action of the hormone auxin. Auxin regulates gene expression by mediating the degradation of repressor proteins called IAAs. A subset of IAA proteins are involved in the initiation of new lateral roots, which are important for overall root architecture and function. Members of the Nemhauser lab have shown that proteins within this subset from Arabidopsis thaliana have varying auxin-induced degradation rates, using degradation assays in a heterologous yeast system. I hypothesize that differences observed between the degradation rates of the IAAs contribute to the specificity and diversity of auxin responses. When one of these lateral-root expressed IAA genes, IAA28, is mutated so that it is completely unable to be degraded, the plant does not form lateral roots. I predict that small changes to the degradation rate of IAA28 will lead to differences in plant phenotype, specifically the developmental stages and numbers of lateral roots that develop, as is suggested by preliminary data from work with IAA14. Currently, I am engineering and testing modified versions of IAA28 with the goal of obtaining a range of degradation rates. It is difficult using plant-based system to measure the degradation rates of modified IAA28 proteins due to feedback and the inability to control auxin. To test the degradation rates of modified versions of IAA28, I am expressing them in the yeast, Saccharomyces cerevisiae. Once I have obtained a cohort of modified IAA28 proteins that display a range of degradation rates, I will transform these proteins into plants. I predict that my observations of plant phenotypes will allow me to determine whether the precision of IAA28 degradation rates matters. The results of my research may partially explain the importance of the differences between IAA degradation rates in determining specificity of auxin response phenotypes in the plant.

Auxin is one of the most important plant hormones that regulates almost every developmental process during plant life cycle. Auxin response factors (ARFs), are DNA-binding transcription factors that regulate auxin-mediated transcription by activation or repression. In Arabidopsis, ARFs are encoded by a family of 28 genes, and each member is thought to play a central role in various auxin-mediated developmental processes. ARF2 has shown to be a transcriptional repressor. Nemhauser lab uses Arabidopsis seedling hypocotyl, the embryonic stem, as a model to study growth. Loss of ARF2 alters hypocotyl growth in Arabidopsis seedlings. arf2-9 is a single point mutation in DNA-binding domain that results in loss of ARF2 function. I have analyzed the hypocotyl growth in arf2-9 mutant in presence and absence of exogenously applied auxin. The results indicate that arf2-9 exhibits longer hypocotyl in comparison with wild type seedlings not only in control untreated condition but also in response to auxin application. This data suggests that ARF2 is a negative regulator of hypocotyl elongation. To investigate the mechanisms how ARF2 regulates hypocotyl growth and elongation, I have crossed arf2-9 mutant with a reporter line P_PKS4::GUS that marks actively growing hypocotyl region. I have used dCAP genotyping method for arf2-9 single point mutation and Basta resistance selection for P_PKS4::GUS transgene to isolate double homozygous arf2-9 X P_PKS4::GUS plants. My hypothesis is that long hypocotyl in arf2-9 is a consequence of indeterminate growth. Given that, my prediction is that GUS staining pattern of P_PKS4::GUS will be different in arf2-9 genetic background compared to the original line. This includes possibility of having GUS staining in different regions of hypocotyl or extended staining pattern over the time when in wild type background P_PKS4::GUS marker is no longer active. Those differences will help us understand the function of ARF2 better.

Manganism, a movement disorder characterized by neuronal death in the basal ganglia and symptomatology similar to Parkinson’s disease, has been shown to involve manganese-mediated increase of toxic reactive oxygen species (ROS) production in mitochondria. The protein alpha-synuclein, a product of the SNCA gene, has been implicated in glial and neuronal degeneration in Parkinson’s disease, and preliminary data from our laboratory suggests that removal of alpha-synuclein from astrocytes confers some protection against ROS toxicity. Astrocytic hydrogen peroxide metabolism has been shown to produce ROS capable of damaging a variety of cellular macromolecules. The present study seeks to compare the effectiveness of toxicant attenuation in SNCA knock-out and wild type astrocytes in response to hydrogen peroxide after gradated 24-hour manganese pre-treatments. Calcium imaging with fluo-4 dye will be performed to measure astrocytic calcium by quantifying the amplitudes and durations of induced calcium waves, known to form an important mechanism of astrocytic communication, after addition of hydrogen peroxide. Based on previous studies, we predict that increased manganese dosages will correlate with elevated calcium wave amplitudes in both SNCA knock-out and control cultures. We further predict significantly lower calcium wave amplitudes in wild type cultures after each manganese treatment.

Borderline personality disorder (BPD) and alcohol use disorders (AUDs) frequently co-occur and are each associated with an increased risk of suicide. However, little research has been conducted comparing individuals with comorbid BPD and AUDs to individuals with BPD only. The present study sought to determine whether individuals with comorbid BPD and AUDs exhibit more non-suicidal self-injury (NSSI), suicidal behaviors, and emotion dysregulation than individuals with BPD and no history of AUDs. Data from previously conducted research studies were analyzed via multivariate analysis of variance (MANOVA). It was expected that the group with comorbid BPD and AUDs would more frequently engage in NSSI and suicidal behaviors, and would have more emotion dysregulation, on average, than the group with BPD and no AUDs. The results of this study have implications for the treatment of individuals with comorbid BPD and AUDs. In addition, results may provide evidence for increasing AUD prevention efforts among those with BPD in order to improve the likelihood of positive future outcomes for these individuals.

The Learn and Serve Environmental Anthropology Field (LEAF) School at Edmonds Community College has partnered with Nikkei Manor and the Wing Luke Museum to develop the Nikkei Concerns Oral History Project. Through this project, students have the opportunity to serve as both mentors and mentees. Eight students have undergone training, interviewed and recorded the experiences of Japanese Americans who were interned during World War II. From these survivors we are learning more than the easy to record factual history, we are learning about experience, about the diversity of coping strategies, adaptive solutions, and emotional struggles these Americans lived. We found unexpected stories relating to aspects of control and freedom within the camps, as well as humor, acceptance, bitterness and forgiveness. The hope in a project such as this is to develop a greater understanding of the experience of these people, to record their stories and create awareness of what they faced. This is a unique opportunity for students to hear firsthand accounts from internment survivors and participating students will continue to learn about the internment era while transcribing the stories shared by Nikkei residents, and mentoring their classmates in the transcription process. While there has been much research elsewhere, the story of internment in the Northwest has received less attention. This project has allowed for student engagement and development as well meeting the needs of the Nikkei residents who wished to share their stories. Recordings will be kept both by the Wing Luke museum and Edmonds community college.

Pancreatic cancer is the fourth most common cause of cancer-related deaths in the United States. The overall survival rate is low because current therapies are ineffective. However there is growing research that High-Intensity Focused Ultrasound (HIFU) can be used to significantly improve current therapies for treating pancreatic cancer. HIFU works by accumulating ultrasound waves (i.e. energy), which generates heat causing tissue damage (i.e. hyperthermia). The thermal damage caused is measured by using thermal dose. Thermal dose describes the temperature the tissue is treated at and how long that treatment occurs. The theory of thermal dose has been tied to hyperthermia of cancer cells since at least the 1980s and there is a lot of strong mathematical background behind the time-temperature relationship. However with the rapid development of clinical hyperthermia for cancer therapy there is a need for accuracy when it comes to predicting thermal dose. My research focuses on whether or not equivalent thermal doses actually mean equivalent cell death (with cell types Panc-1 and MIA PaCa) and if this can be translated into a clinical setting where higher acoustic power can be used (i.e. using higher energy with HIFU to get lower time of treatment). I simulate HIFU by treating pancreatic cancer cells in water baths at equivalent thermal doses and measure cell survival with spectroscopy. My initial results suggest that the thermal dose relationship is accurate in relating amounts of cell death. An early conclusion from this is that thermal dose can be used to provide better calculations in a clinical setting and help evaluate data. Patients may even be able to experience shorter treatment times through using higher acoustic power with HIFU resulting in a more comfortable experience.

According to the American Cancer Society, pancreatic cancer has a five-year survival rate of less than 15 percent and is the fourth leading cause of death from cancer in the United States. Present-day chemotherapy treatments attempt to lengthen the lives of patients but are often unsuccessful due to the nature of the disease. Doxorubicin and Gemcitabine are already commonly prescribed as pancreatic cancer treatments while Thermodox®, a lysolipid thermally-sensitive liposome (LTSL) technology, is still undergoing clinical trials. LTSLs encapsulate Doxorubicin releasing the drug when the agent is exposed to temperatures greater than 40 degrees C. The goal of this study is to compare the in vitro cytotoxicity of four chemotherapeutic conditions: unheated Thermodox®, heated Thermodox®, Doxorubicin, and Gemcitabine. Varying concentrations of each drug condition are applied to LMP, PANC-1, and Mia-PaCa cell lines, which originate from mouse and human pancreas carcinoma. The remaining post-treatment viable cells and relevent controls are then stained with a fluorescent dye (Hoechst 33342) and analyzed by a fluorescent plate reader, from which cell death can be determined. Preliminary results show that that unheated Thermodox is not toxic to cells, and heated Thermodox®, Doxorubicin, and Gemcitabine is toxic to cells. The results of this experiment can lead to greater knowledge of the cytotoxicity of current chemotherapy agents as well as new technologies in cancer treatment.

Chitin is the second most abundant polysaccharide extracted from the cuticles of insects and crustaceans. Chitin is known for its biocompatibility and superior mechanical strength. Silk fibroin is produced by the Bombyx mori silkworm and is known for its biocompatibility and superior toughness. Composites of chitin and silk fibroin would possess each biomaterial's unmatched mechanical strength and toughness, making them ideal candidates for biomedical applications requiring such mechanical properties. Here, squid pen β-chitin and Bombyx mori silkworm cocoon silk were blended in Hexafluoroisopropanol (HFIP) solution in specified ratios to synthesize the nanocomposites. As a result, chitin/silk hybrid nanocomposite thin films were produced to test the mechanical properties. Studies of both X-Ray Diffraction (XRD) and Fourier Transform-Infrared (FTIR) Spectroscopy revealed a successful effective interaction between the chitin nanofibers and the silk proteins matrix, which was most likely caused by hydrogen bonding. Results of the self-assembled chitin nanofibers embedded in the silk matrix showed a noticeable enhancement in mechanical properties. The tensile strength, flexibility, and toughness of the biomaterial nanocomposites were enhanced compared to just pure chitin or pure silk fibroin. Due to the improvement in mechanical properties, these chitin/silk engineered nanocomposites can be utilized in biomedical applications such as hybrid scaffolds for bone tissue engineering or microneedles to serve as a new methodology for drug delivery systems.

Nitrogen is a commonly limiting nutrient for Douglas-fir growth in commercial stands throughout the Pacific Northwest. Fertilizer nitrogen is applied to mitigate these deficiencies though some studies estimate that only about 30% of the nitrogen is actually taken up by the trees. Knowing where the rest of the nitrogen goes will help determine the best way to fertilize trees and reduce excess use of nitrogen fertilizers. This is beneficial both to stand managers looking to reduce costs associated with fertilization and to the environment as excessive fertilization can lead to increased nitrates in water. For this study, fertilizers labeled with 15N were used to track to the movement of nitrogen within a commercial Douglas-fir ecosystem. Nitrogen-15 is a stable isotope of nitrogen and is often used to trace nitrogen compounds in the environment. Three enhanced fertilizers designed to increase fertilizer N uptake by reducing volatile loss of N and one industry standard fertilizer were used on ten different commercial Douglas–fir stands throughout the Pacific Northwest. The fertilizers were applied at same rate over a period of one year. For the purpose of this study we will be looking specifically at two of the stands. Mineral soil (up to 60cm depth) and foliage samples were collected prior to fertilization to determine the background abundance levels of 15N. Following fertilization, mineral soil and foliage samples were collected at regular intervals to quantify the amount of N15 recovered over time and final sampling was conducted one year after fertilization. Mineral soil and foliage samples were prepared and analyzed for N15. To quantify the amount of N15 taken up by each component, background abundance of N15 was subtracted from the amount of N15 recovered in both the mineral soil fractions and the foliage. We anticipate that the enhanced fertilizers will increase N uptake.

Heart disease and stroke are the first and third leading causes of death for men and women in the U.S. and together account for more than a third of all U.S. deaths. It has been shown there is a correlation between decreased intercellular forces in the endothelial cells in the artery and higher permeability of the arterial lining. This can lead to atherosclerosis, a disease defined by hardening of the arteries, a major underlying cause of heart attacks and strokes. The focus of this research was to determine how different blood flow rates affect the intercellular forces of the endothelial cells that line the arterial wall. The first step was to fabricate microposts. Negative molds of the posts were cast off of a master array of microposts for which the spacing, height and diameter of the microposts were known. A positive mold was then cast off of the negative mold to produce a substrate on which the cells could be cultured. Next a flow chamber was designed for these experiments. Human Pulmonary Artery Endothelial Cells (HPAECs) were then cultured and transferred onto the microposts which were stamped with fibronectin, a protein that would help the cells bind to the tips of the microposts. The stamping portion of the experiment required much trouble shooting and ultimately the experiments were halted here. If the research had continued the experiments would have run for 14 hours, after which the cells would be fixed in place, stained with a fluorescent dye and imaged using microscopy. These images would then be transferred to a custom written Matlab code that would calculate the deflection of the microposts and the forces induced by cells. This data could then be used to determine the effect of varying blood flow rates on the permeability of the arterial wall.

This study used the Hamilton Search and Set Breaking tasks to assess working memory, cognitive flexibility and inhibition control of a variety of domestic dog breeds. This memory task has been tested on pigtail macaque monkeys, as well as, other species. The Hamilton Search task consisted of 4 containers which the dog was allowed to search to find a randomly placed food reward. The latency and search patterns were recorded to determine performance of the task. After 5 sessions of Hamilton Search testing, the dogs moved on to the Set Breaking protocol. Set Breaking consisted of placing the reward in the same container every trial until the dog reached criterion by searching the correct location first 5 times consecutively. The container that the food reward was placed in was determined by calculating the container which was visited the least often during the pervious Hamilton Search trials. The results from this study will be compared across species and within the species of domestic dogs. Enough dogs of very diverse breeds were tested to allow us to analyze for similarities and differences between breeds. The results showed that dogs approached this task much differently than the pigtailed macaque monkeys. Current data suggests that the dogs, not dependent on breed, are approaching this task in a very systematic manner starting a search pattern at one end and working towards the other end. With this approach, rechecking a container is very rare, demonstrating perfect memory, but in fact the dogs are using a systematic approach that is less flexible, but efficient alternative to the use of memory. Results of this study can be applied to help refine training techniques and to establish the validity of these tasks for studying canine spatial learning and memory.

Breast cancer continues to be a major threat to women’s health. For survivors, breast cancer often recurs 2 to 15 years after the initial treatment, either locally in the same or contralateral breast or at a distant recurrent site (metastasis). Currently, the performance of traditional methods, such as mammography and genetic markers, for screening breast cancer recurrence is limited, and alternative methods is strongly desired. Because of their sensitivity to biological status, metabolite markers may provide better diagnostic accuracy and earlier detection. Therefore, metabolomics, which focuses on the parallel measurement of hundreds of small molecule metabolites, has emerged as a powerful methodology for understanding biochemical systems and metabolic changes due to cancer. The Raftery group identified 11 metabolites (lactate, glutamic acid, etc.) that are useful for breast cancer recurrence detection. This model provides an earlier and more accurate approach to detect recurrence compared to the current standard, cancer antigen 27.29 (a FDA-approved blood immunoassay for breast cancer treatment monitoring). Recently, they have developed a targeted liquid chromatography mass spectrometry method (LC-MS), which enables us to simultaneously monitor changes of more than 150 metabolites in 25 different metabolic pathways (such as Glycolysis and the TCA cycle) that are of potential significance to breast cancer. In our present work we are validating their previous results and expanding this research to investigate a more comprehensive metabolic profile. Our specific task in this project is to prepare the samples and analyze them using the LC-MS platform. Using commercial serum samples from recurrent and non-recurrent breast cancer patients, we anticipate additional metabolite biomarkers that will be very valuable for developing a better diagnostic test for detecting breast cancer recurrence. In the long run, these markers should be useful for improving survival rates of breast cancer patients and thus for reducing social and financial burden. 

Many individuals with severe depression experience chronic symptoms despite the aid of pharmacological interventions. This population may benefit from further interventions such as deep brain stimulation (DBS). Indeed, promising studies have illustrated the antidepressant effects of DBS on brain reward centers in patients with severe depression. However, these studies are limited by a lack of regulation of DBS delivery. We proposed that stimulation may be more effective if it is not delivered continuously, but only when needed. For example, DBS could be given when some physiological measure of depression increases. Therefore, we sought to provide proof-of-concept that an animal could learn to volitionally modulate a specific neural signal in order to receive DBS. To accomplish this goal, we used an operant conditioning paradigm wherein a rat gradually learns to volitionally modulate brain activity recorded from the surface of the cerebral cortex. The reinforcement consists of electrical stimulation in an area of the brain known as the medial forebrain bundle (MFB). When activated by electrical stimulation, neurons coursing through the MFB release dopamine, creating a strong reward sensation. We associated activity-dependent stimulation with a green light which served as an external cue to the rat. DBS was delivered only during times in which this light was on. Initially, results revealed that the production of the intended behavior was not correlated with the external cue. Over the course of repeated experimental sessions however, the desired neural activity became dramatically elevated during periods of activity-dependent stimulation. This finding suggests that through repetitive conditioning, the animal did in fact learn to volitionally control the neural signal to receive MFB stimulation. Despite these encouraging early findings, future work to determine the long term effects of DBS, as well as the optimal stimulation parameters, will be critical to the ultimate clinical implementation of this technology.

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.

Deficiencies of monoamine neurotransmitters including serotonin, norepinephrine, and/or dopamine have been associated with many CNS diseases including depression, schizophrenia, ADHD, autism, Parkinson’s disease, and Tourette syndrome. The intensity and duration of monoamine signaling is mainly determined by the clearance of the released transmitters through uptake1 and uptake2 transporters. The recently discovered uptake2 transporters including the plasma membrane monoamine transporter (PMAT) and organic cation tansporter3 (OCT3) have been less studied partly due to the lack of specific inhibitors. Recently, inhibition of PMAT and/or OCT3 has been proposed as a strategy for the development of antidepressant drugs with improved efficacy and faster onset of actions. The goal of this project is to characterize specific chemical inhibitors for PMAT and OCT3. Previously, 10-40 small molecule inhibitors for PMAT and OCT3 were identified from high throughput screening. We will choose two to three inhibitors for PMAT and OCT3 obtained from the initial screen and further analyze their potency, specificity and mechanism of inhibition towards PMAT and OCT3. We first will determine their inhibition potencies (IC50) towards PMAT and OCT3 using radiotracer uptake assays in cell lines stably expressing these transporters. Preliminary data showed that two compounds inhibited OCT3 with IC50 values of 1.97 ± 0.766 and 2.86 ± 0.532 µM. One compound inhibited PMAT with an IC50 value of 2.15 ± 0.537 µM. Further studies will be performed to determine the specificity and mechanism of inhibition for these inhibitors. The proposed studies may lead to novel, highly specific and potent PMAT and OCT3 inhibitors that could be further evaluated and developed in preclinical models for the treatment of depression and other monoamine-related diseases.

My research focuses on the ethnobotanical uses of plants by the Monte Salvado native community of the Las Piedras River in the Amazon Rainforest of Peru. One-fourth of all Western pharmaceuticals, including very common drugs such as aspirin, have their origin in rainforests. Much of the knowledge surrounding the use of these plants comes from indigenous communities living in the Amazon. Many of these communities, such as the Monte Salvado, use plants as both medicine for anything from stomach aches to snack bites, as well as a spiritual agent.  My research centers on the medicinal and spiritual purposes of plants such as Curare, Suma, and Clavillia. My research questions include, “What are the main purposes of and beliefs about plants in this community? How are these plants used and what are their effects? How do women use plants to aid in maternal and menstrual health? The prepatory work leading up to my community immersion this summer is creating a research proposal, as well as learning and practicing Anthropological research methods. 

Trichloroethylene (TCE) is a widely found pollutant that is carcinogenic and hazardous to humans. Phytoremediation, which uses plants to degrade toxins, offers a more economical solution to removing toxic wastes from the environment. The Doty lab has isolated an endophyte (microbes living inside plants) from hybrid poplar that can degrade TCE, an enterobacter sp. strain PDN3. In this study, we are determining its potential to colonize the plants, protect against TCE toxicity and its ability to help plants degrade TCE. Colonization was studied using fluorescent microscopy, using plant tissues of S365 (Salix discolor) inoculated with fluorescent PDN3 through either foliar spray or root inoculation. The presence of the inoculated endophyte in roots of root-inoculated plants suggests that PDN3’s can colonize roots. We also tagged PD1(a phenanthrene degrader) with a red fluorescent marker to study co-colonization by both PD1 and PDN3 in S365 so that we can understand their preferred methods of colonization and their potential use in remediating sites contaminated with multiple pollutants. Because PDN3 is known to degrade TCE, we want to see whether it enhances the plants’ capacity to degrade TCE. In the next study we inoculated S365 with PDN3 and exposed them to TCE in hydroponics solution to monitor TCE removal and chloride release (metabolite of TCE). In a previous study, clone SX64 (Salix miyabeana) showed decreased biomass when exposed to TCE. So we want to examine if adding PDN3 would help the plants against the toxic effects of TCE. We are also screening other willows and poplar clones for phytotoxicity to TCE and the data generated in these studies will help us in developing a cost effective clean up technology.

 The Washington State TF-CBT and CBT+ Initiative offers training in Cognitive Behavioral Therapy (CBT) for clinicians working in public mental health across Washington State. While CBT has been shown in previous studies to be an effective therapy for anxiety, depression, behavior problems, and Post-Traumatic Stress Disorder, its use is limited in public mental health because of the lack of available training. The purpose of the study is to examine whether clinician orientation affects clinician learning of CBT for anxiety, depression, behavior problems, and PTSD. Our first hypothesis is that clinicians with a cognitive behavioral orientation will have a higher skill level before training in comparison to clinician of other orientations. The second hypothesis is that clinicians of other orientations will have a higher skill level after the training than those clinicians with a cognitive behavioral orientation due to a higher rate of improvement. Clinician self-reported efficacy in elements of CBT for anxiety, depression, behavior problems, and PTSD, was assessed before the three-day training, after training, and after 6 months of bi-weekly phone consultations with a CBT expert, and will be compared for each theoretical orientation. The participating clinicians in this study vary in theoretical orientation, including integrative/eclectic, biological, interpersonal, cognitive behavioral, psychodynamic/analytic, systems, humanistic/existential and others. We plan to integrate the results of the study implications for future clinician training of CBT. If our hypotheses are supported, it will suggest that clinicians with a cognitive behavioral orientation may need specialized training in CBT that takes into account their previous knowledge and offers more advanced training options for those with past experience in CBT.

Research has made tremendous gains in therapy efficacy for children and adolescents, yet the therapies have not effectively been implemented into usual care settings. The mental health field has recently initiated a huge drive to disseminate Evidence Supported Therapies, or ESTs, in order to better serve our children. Do clinicians trained in ESTs deliver the clinically relevant components to their clients and utilize important structural elements of EST such as agenda setting, assigning/reviewing homework? If clinicians are not utilizing the components and structure supported for the specific clinical target, then children may not be receiving efficacious therapy. Clinicians across Washington State were invited to participate in the Washington State Initiative and receive components based Cognitive Behavioral Therapy, or CBT Plus. The training included components for Anxiety, Depression, Behavioral Problems and PTSD. Each clinician involved in the study entered assessment data on his/her cases as well as what components and structural elements were delivered. Research has shown that clinicians are reluctant to deliver exposure.  For instance, if a child has PTSD and has a panic attack each time he hears an ambulance, exposure to ambulances reduces the related anxiety.  It is predicted that clinicians delivering CBT Plus with a clinical target of anxiety or PTSD will be less likely to deliver the adequate components due to this exposure reluctance. The preliminary data shows that many clinicians did not consistently provide the necessary components and structure for their clients’ clinical presentations. If clinicians delivering EST are not utilizing the relevant components, then the children are not receiving the best care possible and the ESTs are not being effectively disseminated. Training for ESTs would thus need to focus more strongly on combating clinicians’ negative perceptions of exposure. This may be achieved through more thorough psychoeducation and theory explanation during EST training.

Ischemic Preconditioning (IPC) is a neuroprotective phenomenon in which a brief ischemic exposure increases resistance to serious injury from subsequent ischemia. Understanding the basic mechanisms of IPC and taking advantage of this knowledge may provide better treatment options for those at high risk for stroke. We hypothesize that ischemia induces brain tissue release of endogenous Toll-like receptor (TLRs) agonists (sometimes referred to as damage association molecular patterns (DAMPS)) that stimulate microglia Toll-like receptors, thus releasing interferon (IFN) α/β to trigger the transcription of neuroprotective interferon stimulated genes (ISG). Our objective was to culture mouse microglial cells and stimulate the various microglia TLR receptors with their respective agonists to identify the receptors that mediate ISG release. We also wanted to demonstrate TLR-agonist induced release of IFN α/β, especially because IFNs are already effective FDA-approved treatments for Multiple Sclerosis and could serve to increase ischemic tolerance for patients at high risk for strokes. Due to results from previous experimental paradigms strongly suggesting that IPC-mediated neuroprotection is TLR4-dependent, we proposed that stimulation of TLR4 generates release of IFNs that in turn induce transcription of ISGs. Our preliminary results revealed that activation of multiple TLRs (TLR 3, 4, and 9) could mediate the release of ISG chemokines, with notable ISG release following TLR3 and TLR4 activation. IFN release was not detected through TLR4 but instead TLR3. We are currently exploring the possibility of a synergistic relationship between TLR3 and TLR4 in regulating IPC-mediated neuroprotection.

Ischemic preconditioning (IPC) is a neuroprotective phenomenon in which a short ischemic episode confers resistance to prolonged subsequent ischemic attacks, and is dependent upon functional Toll-Like Receptor 4 (TLR4) and possibly downstream interferon (IFN)-related signaling. Understanding this mechanism and being able to mimic it pharmaceutically may lead to better stroke therapy and treatment. In our current study, we use the optic nerve—a pure axonal tract—as our model in determining the effects of stroke in white matter. Roughly half the human brain is composed of white matter—the myelinated axons and supporting cellular milieu that connect neurons into networks. In our experimental paradigm, we induce IPC in vivo by performing common carotid artery occlusion (CCAO) and then expose ipsilateral (IPC-induced) and contralateral (control) optic nerves to ex vivo oxygen-glucose deprivation (OGD) (in order to model ischemic or “stroke” related injury). We then characterize electrophysiological properties and axonal integrity of the isolated optic nerves. In addition, we characterize glutamate receptor isoform expression and interferon stimulated gene induction. IPC-exposed optic nerves showed improved compound action potential (CAP) recovery following OGD in wild-type versus TLR4-/- mice. Using immunohistochemical techniques we show that oligodendrocytes are protected from OGD-induced death. Likewise, axonal integrity is maintained better in IPC-treated optic nerves as shown by immunohistochemistry of phosphorylated neurofilaments. Glutamate receptor (GluR) isoform variants flip and flop can exhibit different channel opening kinetics by affecting rates of receptor desensitization—possibly influencing how CAPs respond following IPC. However, our data indicated little regulation of GluR mRNA transcript isoforms. We did however observe upregulation of several interferon-stimulated genes (ISGs) following IPC in optic nerve, which may contribute to the mechanism by which IPC can mitigate the injurious effects of stroke. Studying IPC and stroke pathophysiology in the optic nerve may lead to potential therapeutics specific to white matter.

The late Oligocene-early Miocene fossil record (30-18.8 million years ago) of North America includes an abundant fauna of entoptychine gophers (Rodentia: Geomyidae: Entoptychinae). These burrowers make up as much as 20% of the mammalian individuals of the faunas they are part of, and can be involved in habitat partitioning or competition with other burrowing herbivores. The wide geographic range of these animals and their use in constraining the age of the fossil assemblages of the Cabbage Patch beds (western Montana) and John Day Formation (eastern Oregon) makes them critical to understanding the biogeographic relationships among these fossil faunas. To understand these spatiotemporal patterns, I am evaluating the taxonomic richness of entoptychine gophers. Previous studies have discriminated among these gopher species by using morphological features of skulls and dentaries. However, anatomical elements are rarely preserved in fossil assemblages, and isolated teeth are commonly the only material available for identification. Since the occlusal surfaces of the cheek teeth are usually worn down as a consequence of feeding, they lack discrete features for species identification. Instead, some researchers have hypothesized that the more continuous variation in the enamel outline of select cheek teeth could be used for discrimination. Here, I use semi landmark-based geometric morphometrics (an arithmetical comparison of geometric coordinates) on the upper last premolar and molar. Applying this method to 98 modern geomyine gopher specimens (Geomyidae: Geomyinae), representing 10 taxa, helps quantify intra- and interspecific tooth shape variation and offers guidelines for recognizing fossil gopher species. Preliminary analyses indicate that tooth shape is species-specific in modern gophers, supporting the use of this method to differentiate fossil gopher taxa. Applied to 96 specimens of fossil gophers, I found a strong overlap of fossil taxa in the morphospace, suggesting that previous researchers may have overestimated the species diversity of some fossil gophers.

Cigarette smoking is the leading cause of preventable death in the United States. The adverse health effects from tobacco use account for about one of every five deaths each year in the United States. Moreover, smokers with depressive symptoms have lower cessation rates and more difficulties while quitting than the general population of smokers. Treating smokers with depressive symptoms effectively is challenging and new smoking cessation interventions are always needed. In order to determine how recent smoking cessation treatments are benefiting smokers with depressive symptoms, the present study examines an existing web-based treatment using the innovative Acceptance and Commitment Therapy (ACT). ACT provides mindfulness-based techniques that help smokers dealing with the urges, thoughts and negative feelings associated with smoking while quitting. The study aimed to determine the users: (1) willingness to experience smoking cues, and (2) 3-months post randomization smoking status. Adult participants were recruited nationally into the double-blind randomized controlled pilot trial which compared web-based ACT for smoking cessation (webquit.org) with smokefree.gov—the national standard for web-based smoking cessation interventions. The present study will report the outcomes for smokers with depressive symptoms. Data analysis is ongoing. Based on previous research, we expect the web-based ACT intervention to be more effective than the standard treatment (smokefree.gov). This trial will provide insights for ongoing research in smoking cessation interventions to help smokers with depressive symptoms to quit smoking.

Tissue engineered skin is a candidate for skin therapies since detrimental wounds require replacement of both the epidermal and dermal layers. For successful replacements, it is important to mimic the skin structure consisting of two layers: a top epidermis layer with a dermis layer beneath, which is made up of keratinocytes and fibroblasts respectively. In the present study, we developed the skin graft by seeding the two types of skin cells, keratinocytes (HeCat) and fibroblasts (hFF), on opposite sides of a porous, biodegradable, and biocompatible 3D natural polymer-based scaffold. The skin graft was investigated by using Alamar Blue Assay, Histology, and SEM. The results showed that (1) the HeCat and hFF proliferated better in co-culture on the scaffold, (2) HeCat forms differentiate into 5 layers, compared with control. This infers that the 3D structure of the scaffold not only acts as a mechanically sufficient substrate for cells to grow and receive nutrients, but also creates a good environment for signaling between the two types of skin cells. These findings indicate that the integration of a natural polymer-based scaffold with co-cultured HeCat and hFF in vitro can be used for a living skin graft in vivo.

Dopamine (DA) modulation is important in treating neurological disorders such as Parkinson's disease. Available methods fail to deliver DA into the brain due to its inability to cross the blood brain barrier (BBB). Attempts have been made to overcome this limitation, such as the development of L-DOPA. Despite its ability to cross the BBB, the pro-drug still causes many unwanted side effects. The demand for new DA delivery led to the development of my DA nanoprobe. This newly designed nanoprobe is modified to cross the BBB and can be safely uptake into the brain. The addition of a fluorophore allows for in vitro detection while the iron oxide core allows for in vivo real-time magnetic resonance (MR) imaging. The ability for the DA nanoprobe to cross the BBB and to be detected through optical/MR imaging provides new applications to many diagnostics and therapeutics of central nervous system disorder.