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.

 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.