For the first time we are able to non-invasively measure levels of a molecule that indicates the ageing and disease state of an individual. This molecule called nictotinamide adenine dinucleotide (NAD) and its other forms (NAD, NAD+, NADH) are related to aging and muscle function. High levels of the form NADH is found in individuals with disease and old age while high levels of NAD+ is found in active and younger individuals. Through exercise on elderly subjects we show that ageing effects on muscle can be reversed and levels of NAD+ and NADH can be restored to those found in adults. The form NAD+ is an index of mitochondrial capacity for making energy. With aging the oxidative capacity of the mitochondria declines, the amount of mitochondria declines and the levels of NAD+ declines. Through exercise training this effect can be reversed to yield higher muscle capacity in elderly. Our measurements were made on a 1.5 Tesla Magnet. We acquired Phosphorus spectra on the FDI (First Dorsal Interosseous) muscle and analyzed the data using software called jMRUI. From the Phosphorous spectra we are able to extract and analyze the NAD+ and NADH peaks.

This study examines the effects of administering hydrogen sulfide (H2S) to ancient plant species to examine the organisms’ response to the stress. While sulfur is an essential macronutrient required by several organisms for growth and productivity, there are many toxic variants of the element that have detrimental effects and produce physiological stress. It is speculated that the accumulation of H2S, a lethal gas, may have existed as a major contributing factor in past mass extinction events, where the environment was fairly anoxic with fluctuating temperatures. The potential of this toxic variant to exist as an environmental stressor suggests that certain organisms may have adapted to survive these periods of mass extinctions. It is hypothesized that due to the abundant presence of hydrogen sulfide in the past, ancient land plants may have an adaptive advantage that allowed them to survive and thrive. In the study, species of bryophytes, specifically Hypnum and Dicranum are exposed to specific concentrations of aqueous H2S over a 7 day period and measured for their photosynthetic capacity at timed intervals using a FluorCam. Studying the effects of this toxic gas on ancient plants is imperative to our understanding of sulfur’s varying biological roles, and provides insight on the evolutionary phenotypic variations amongst plants and stress responses in order to survive mass extinctions. Current results indicate that bryophytes are able to tolerate significant quantities of the toxic substance and show resilience through increased photosynthetic capacity over a period of exposure, indicating a genetic and phenotypic legacy response. The results also imply that H2S may have the potential to act as a plant regulator due to its diffusive properties and influence in minute quantities.

Deadly and leaving patients with little time to live, pancreatic cancer is the third largest cause of deaths due to cancer in the US. Early detection and research of the disease beyond current capabilities are crucial and could be achieved by imaging fully intact pancreatic tissue core biopsies (TCBs) in three-dimensions (3D). Hence, The Human Photonics Laboratory is developing a microfluidic device to process fully intact TCBs (L=0.5-2.0 cm, d=200-1200 µm) in an all-in-one approach. Simultaneously, the device transports the TCB towards a 3D imaging platform for the end goal of enhancing cancer diagnosis. To achieve this goal, each step must be fit and performed in the small device. Typically, microfluidic devices are microfabricated with rectangular cross-sectional channels and used with no larger than cells or cell slurries, but never TCBs. To prepare TCBs for a pathologist in a microfluidic device, transportation is done in curved, circular cross-sectioned channels (CCCs)—both of which have never been achieved before this project. The channels’ cross-sections are circular to produce an intuitive Poiseuille flow profile. TCBs require a smooth directional change due to larger length and diameter of a TCB. Therefore, curved channels are necessary for direction changes and an optimized device footprint. Traditional microfabrication methods are not practical due to the time and precision required for mircofabricatation of a smooth CCC. Therefore, a new simplified method to fabricate microfluidic devices was developed to allow for CCCs and transport availability for TCBs. This new method allows for microfluidic devices containing CCCs to be fabricated in less than one hour and can provide channels that curve in 3D. Transportation of TCBs through CCCs is demonstrated for planar and multi-dimensional direction changes. Minimized channel lengths required to successfully change a TCB’s direction by 90 and 180 degrees were determined to reduce the microfluidic device’s footprint.

Dental caries is the breakdown of tooth enamel that causes cavities and is the most common chronic disease in children and adolescents between the ages of 6 and 19 years. Therefore, much work is being done to combat caries. We believe that the scanning fiber endoscope (SFE) developed in our lab can be used to improve the detection of early caries in children. The SFE is a small flexible probe that scans low-powered RGB laser light. It performs high-contrast high-resolution imaging and dual laser fluorescence spectroscopy with 405- and 532-nm laser excitation on both in vitro and in vivo teeth. My project focuses on the development of a calibration standard to use with the SFE in an upcoming clinical trial to be conducted at the UW Center for Pediatric Dentistry. A photostable calibration standard was created to match the fluorescence pattern of dental enamel. Spectral measurements of the standard will be taken before and after collecting data from patients' teeth with the SFE to monitor the instrument's behavior. This will allow us to account for drift in measurements and ensure that we can draw accurate conclusions from our data. 

Childhood caries, also known as tooth decay, is on the rise worldwide and has increased the demand for portable technologies that can quickly and accurately detect and diagnose early stage carious lesions. These lesions, if identified at an early stage, can be reversed with remineralization treatments, education, and improvements in home care. A multi-modal optical prototype was previously developed at the University of Washington’s Human Photonics Laboratory for the detection and diagnosis of early occlusal caries demineralization in vivo. The device uses a 405-nm laser as an illumination source to obtain high-resolution images that can better guide the operator to identify a suspicious enamel region. When the suspicious region is located, the device can be switched to perform dual laser fluorescence spectroscopy using 405-nm and 532-nm laser excitations generating an auto-fluorescence (AF) ratio with the main goal of diagnosing the start of early caries. Recent research had the device test in vivo on 7 children’s teeth with clinically diagnosed carious, non-cavitated lesions. Using the 405-nm scanned light source, lesion depth was visually estimated by the clinician. Within a month, the clinician assessed the maximum drill depth after the tooth had been drilled and the spectroscopic data acquired. The researcher and clinicians were masked from previous measurements in a blinded study protocol. Using the drill depth as the gold standard, the researcher assessed the preliminary results of the 7 patients to see if the severity or depth of the lesion could be compared quantitatively by using the device. Preliminary results indicate that the quantitative AF spectrum of a tooth correlates with the severity of demineralization as assessed by the clinician after drilling.

Low oxygen concentrations in oceans exist at mid-latitudes where water circulation occurs sluggishly, causing oxygen minimum zones (OMZ’s) to form. Recent trends suggest that OMZ’s are expanding, which could decrease biodiversity, disrupt food chains, and alter seafood industries. The low oxygen content causes OMZ’s to host denitrification, a process in which organisms use nitrate rather than oxygen in cellular respiration. Understanding these rates of denitrification will allow us to predict the consequences of climate change on nutrient cycling and ocean productivity. Autonomous floats with chemical sensors are currently being designed to quantify the rates of denitrification. To ensure that future oxygen measurements will be highly accurate, I calibrated the oxygen sensor using the Winkler method, a chemical procedure. A new amperometric titration Winkler kit was used on the R/V Oceanus to analyze saltwater and freshwater samples near the Columbia River. The data showed that as the temperature and salinity of the samples increased, oxygen content decreased. These samples had oxygen concentrations in the range of 2.4 to 6.0 mL O₂/L H₂O with approximately 1% precision. For optimization of oxygen measurements in OMZ’s, past publications suggested diluting chemicals as well as adding sodium azide. I conducted a dilution experiment involving the Winkler titrant, sodium thiosulfate. OMZ water was created in the lab by adding sucrose to seawater and measuring the oxygen content with an optode, which reported around 2% O₂ saturation. This sample was analyzed using Winkler chemicals with a tenth of the baseline concentration. However, this experiment was limited by the titration system parameters and significant data could not be collected. Revision of the system programming is necessary to finish the experiment. Additionally, further research with the sodium azide modification will increase the accuracy of oxygen measurements in OMZ's and therefore greatly advance the calibration of the oxygen sensor.

 The population of surfers has grown rapidly over the past 50 years and the surfing industry has expanded with it. The surfing industry was calculated in 2005 to be worth $7 billion annually and has grown since then. With this increase in popularity the ‘perfect’ surf spots around the world have become overcrowed (Scarfe, 2009). Hence there is a desire for Artificial Surf reefs (ASR) that take into account coastal, ecological and surf enhancement. In order to build successful Artificial Surf Reefs (ASR) there are many parameters that need to be taken into consideration. Three important ones are breaking wave height, wave peel angle and underlying bathymetry. Combining these three parameters produces a simple ranking system for the Surfablilty of both the wave and the location. I collected data at 3 different locations in the southern hemisphere each with a different ranking to allow a good range in data (New Zealand; Te Arai & Shipwreck bay, Tahiti; Vairao). Data was collected on underlying bathymetry, wave height and wave peel angle. I hypothesized that there was a relationship between bathymetry and the surfability of waves specifically; a sharper change in bathymetry into shallower water depth regions would produce a higher ranked wave.  Wave height mesurements were collected using pressure sensors located at the seabed, for peel angle video recordings were analyzed. For bathymetry I surved each location using a depth sounder and single beam transducer.

---still working on results

The Carbon IV emission line in quasar spectra is well-known to show systematic shifting toward smaller wavelengths often attributed winds flowing off the accretion discs along the line of sight. We investigate variability in the shift of the C IV line in two ways, using spectra observed multiple times: through correlation of the shift with changes in the intensity of the rest of the quasar, as well as through the effects of this variability on host supermassive black hole mass estimates. Our results show weak to no correlation between changes in the shift with quasar light flux between observations, potentially indicating near-constant outflow speeds. We additionally investigate biases in existing SMBH mass estimates by comparing our multiple-observation estimated masses with masses derived from single observations. Our results indicate that the previously observed correlation between C IV shift and SMBH mass can be empirically corrected thanks to the lack of variability in C IV shifts, leading to less biased SMBH masses. This will help pave the way for much more accurate methods of calculating masses for extremely distant black holes, and helps constrain physical structure models of active galaxies.

Physical limitations have forced computer hardware designers into providing multiple processors rather than faster ones. To take advantage of this, software engineers must structure their programs in a parallel fashion. Separately, programming languages with commands similar to those of hardware, known as "systems languages", have long been criticized for their lack of safety. This means that programs written in these languages are easily incorrect and difficult to reason about. Rust, a new systems language from Mozilla, seeks to address both parallelism and safety by using modern language concepts. To those ends, Rust’s concurrency primitive is the task, which can be thought of as a lightweight thread that does not share memory with any other task. Message passing is used between these tasks in order to coordinate their work. While this primitive is sophisticated and suited to parallelism, the development community has expressed a desire for an Actor library. At a high level, the Actor model is a concurrent object-oriented programming model, where each Actor maintains state and takes actions based on the messages they receive from other Actors. In this investigation, the suitability of the Rust language for an Actor library is evaluated by writing and refining an implementation. It is expected that the implementation will be backed by the task primitive and its associated structures. Various criteria will be evaluated throughout development, including overall throughput, library overhead, and ease of use. The success of the Actor model within the Rust language would continue its goal of bringing straightforward parallelism and safety to the systems language space.

Trypanosoma cruzi is a protozoan parasite prevalent in South and Central America that causes life-long infection in humans. Approximately 30% of infected individuals develop a condition called Chagas disease which usually manifests as life-threatening cardiomyopathy or pathologies in the gastrointestinal system. Over 8 million people are believed to be infected. Unfortunately, current drugs for treating Chagas disease have low antiparasitic activity, are expensive, and are known to cause harmful side effects. As a result, research needs to focus on discovering new drug targets in Trypanosoma cruzi to help lead to improved drugs. Most eukaryotic organisms synthesize sterols such as cholesterol and ergosterol for essential biological functions that can lead to cell death if these sterols were made incorrectly. This research is investigating the effects of blocking the synthesis of ergosterol by deactivating an enzyme called sterol 14-demethylase. This enzyme catalyzes one of the intermediate of 20 steps of ergosterol synthesis. Inhibitors of sterol 14-demethylase, known as azoles, have been shown to be extremely active on T.cruzi in vitro and are now the center of research regarding anti T.cruzi drug discovery. Further evidence shows that blocking sterol 14-demethylase can lead to an accumulation of sterol intermediates that are converted into toxic agents in the presence of another enzyme, ERG3 enzyme, which is normally active in a later step in the biosynthesis of ergosterol. The objective of the research project I am presenting is to analyze the ERG3 homologs of T.cruzi in the context of the ergosterol synthesis. The putative homologs have been cloned and sequenced. The research now is focused on engineering genetic knockouts of the ERG3 genes to test the hypothesis that the knockout parasites may be resistant to treatment with azole drugs.

The treatment of cancer with proton beams makes use of beams’ favorable dose profile, which allows for the delivery of more energy to a targeted tumor than to surrounding healthy tissue. Recent investments into proton therapy centers have spurred interest in developing laboratory tools to support preclinical proton beam research. A crucial portion of this work is the accurate determination of parameters which describe these beams by comparing models to experimental results, so that the beams can then be accurately replicated in research simulations. Modeling of the research beams was done with TOPAS, (Tool for Particle Simulation) a program which incorporates the popular Monte Carlo simulation toolkit Geant4. Characterization of two beams at different energies was to be done by matching dose profiles of experimental data with those resulting from the simulation software. Experimental measurements were taken with a parallel plate ion chamber. Parameters to be determined by simulation were precise base energy, beam energy spread, and beam angular spread. Simulations with different combinations of parameter values were run and accurate values for these parameters were determined by distance-to-curve metric minimization. The energy of the low energy beam was found to be 28.9 MeV with an energy spread of 1.5% and an angular spread of 0.003 radians. These values for the high energy beam were found to be 50.0 MeV, 1.25%, and 0.022 radians. The results of this process have given an insight into the effects of each simulation parameter on beam profiles, as well as provide a procedure for determining beam parameters in the future. This work will allow any future research simulation to accurately match the results of experimental measurements were they do be done using these beams.

Biofuels are a promising means towards the goal of creating cleaner and more sustainable energy sources, but current biofuels are hampered by incompatibility with current infrastructure and difficulties with using food crops for production. This project addresses these problems with a novel approach to consolidated bioprocessing: genetically engineering a synthetic organism to digest low cost feedstock and produce alkanes, the main constituent of diesel fuel, which can serve as a drop in replacement to traditional petroleum. One challenge of consolidated bioprocessing is that cellulose, the primary feedstock, is far too large for bacteria cells to digest, so engineered strains must be able to break down the cellulose extracellularly. To alleviate this problem, the Klavins lab has implemented a cellular “division of labor” in E. coli which serves to generate specialized cell states: the consumer state and the altruist state. In the consumer state, the cell consumes nutrients and proliferates; in the altruist state, the cells produce large quantities of cellulase and a lysis protein to release the enzyme payload to the culture environment. Cellulase hydrolyzes cellulose into cellobiose, a molecule small enough to be imported and digested by both consumer and altruist cells. This project is focused on the addition of two key genes, previously shown to produce alkanes, into the “division of labor” strain. We extracted these genes using PCR and integrated them into the cellulolytic “division of labor” strain with Gibson assembly. We then identified and quantified alkane output using GCMS (gas chromatography / mass spectroscopy). We also tested the growth dynamics of our strains using cellulose as its sole carbon source through time-lapse cell counts. The results of this project can invite opportunities for industrial scale-up and can lead the way in producing other relevant high value biochemicals such as drugs, chemical intermediates, or other fine chemicals.

Glucose and glutamine are primary carbon sources for cells to produce biomolecules and energy. Increased aerobic glycolysis, de novo fatty acid synthesis (FAS), and glutaminolysis are hallmarks of cancer cell metabolism and many tumors are dependent upon these for their survival. Likewise, virally infected cells also have considerably altered metabolic requirements. Our lab studies Kaposi’s Sarcoma Herpes Virus (KSHV), the etiologic agent of Kaposi’s Sarcoma (KS). KS is the most common tumor found in AIDS patients. Previous work from our lab showed that latent KSHV infected endothelial cells also require increased aerobic glycolysis and FAS. Our lab also published a global metabolic analysis of latent KSHV infection, revealing that latent KSHV infection also induces increased glutamine levels. Exogenous glutamine is metabolized through Glutaminolysis; glutamine is converted to glutamate and alpha-ketoglurarate (αKG) through two key enzymes, glutaminase (GLS) and glutamate dehydrogenase (GDH). Further, αKG can enter the tricarboxylic acid cycle (TCA) and support ATP production. Cancer cells utilize glutamine to replenish the TCA cycle when glucose is primarily used to produce lactate and fatty acids. Preliminary data shows that exogenous glutamine is required for latent KSHV infection through starving infected endothelial cells of glutamine. While glutamine starvation does not reduce viability of mock-infected endothelial cells, the absence of glutamine leads to significant cell death in KSHV infected cells. I hypothesize that glutamine is required during latent KSHV infection to replenish the TCA cycle. To study this, I inhibited glutaminolysis using a compound called BPTES, an inhibitor of GLS. Inhibiting glutaminolysis also leads to significant cell death of KSHV infected cells. This data indicates that glutamine is required during latent KSHV infection to replenish the TCA cycle. In the future I will add back TCA cycle intermediates to rescue cell death. Through our metabolomics studies of latent KSHV infection, we are identifying potential targets for KS therapy.

Exoplanets are planets outside our solar system. The discovery and characterization of exoplanet systems informs us of the types of worlds found in nature and whether they might support life. There have been an unprecedented number of exoplanet detections in recent years spurred in large part by the advanced observing capabilities of NASA’s Kepler Space Telescope. The Kepler Spacecraft continuously monitors a large, fixed set of stars and collects time series photometric data that are unparalleled in both quantity and precision. Plots of stellar brightness against time called lightcurves are the canonical representation of Kepler data. The goal of our research is to detect exoplanets from the characteristic features they produce in these lightcurves known as transits. A transit occurs when an orbiting planet partially eclipses its stellar host, reducing the brightness observed by the telescope and recorded in the lightcurve. To detect transits, we use the Quasi-Periodic Automated Transit Search Algorithm (QATS). As an automated tool, QATS is instrumental in reducing the effective size of the Kepler dataset. However, QATS is sensitive to variability of the target star and to systematic artifacts of the telescope just as it is to transits. Thus optimal detection efficiency requires removal of all non-transit signals prior to applying the algorithm. Determining the best way to do this is the present focus of our work because it bounds our ability to detect planets whose size, composition, and orbital parameters are conducive to life.

 At typical interstellar distances, the separation between a moon and its planet is too small to resolve; the separate spectra of the two objects combine and appear like one object. At most wavelengths the planet will outshine its moon, heavily skewing the center of light towards the planet. However at certain wavelengths a moon may outshine its planet, skewing that center of light towards the moon instead. Spectro-astrometric methods would find exomoons by detecting a shift in the center of light at a moon-dominated wavelength versus a planet-dominated wavelength. Once a moon is detected, tracking the motion of the center of light would also provide information about the masses and separation of the moon and planet. This research project is examining two systems: the Moon around the Earth, and an Earth-like moon around a warm Jupiter. Modeled spectra of the three objects were then used to model the detection of the two types of systems at a distance of 10 parsecs. For the Earth-Moon system, the strongest Moon dominated wavelength was found to be 2.83 microns. For the Jovian-Earth system, the strongest Earth dominated wavelength was found to be 2.44 microns. Further work will explore the signal to noise ratios and thus determine how large of a telescope is necessary to feasibly use this technique in the future.

Forests in the Appalachian Mountains are downwind from coal-fired power plants and other industrial sources in the Midwest, thus forests are exposed to relatively high concentrations of metal pollutants. Despite heightened public concern and tighter regulations to reduce anthropogenic pollutants, spruce-fir forests in the Appalachian Mountains are declining. In previous work at our study site, elevated phytochelatins were correlated with red spruce (Picea rubens) forest decline by altitude. Phytochelatins are metal-binding peptides synthesized from glutathione, from the precursor cysteine. Phytochelatins provide a sensitive and direct indication of metals stress at the cellular level. Metal analysis in soils can reflect historical metals deposition, while lichens can reflect recent deposition. In previous research, we studied metal stress and phytochelatin production in P. rubens from six mountains in the Appalachian Mountains. We found that patterns of foliar uptake and PC production are complex, but appear consistent with induction by Zn and Cd in organic soil. Our current purpose was to collect data along an elevational gradient on Whiteface Mountain, New York to supplement the east-west transect data collected previously and to characterize mycorrhizal fungus communities on sapling and mature tree roots. In recent work, we i) explored elevation patterns in the dominance of mycorrhizal species using PCR, DNA sequencing, and BLAST search; ii) measured Cd, Cu, Hg, Pb, and Zn in organic soil, mineral soil and metals uptake in P. rubens foliage and lichens; and iii) assessed P. rubens phytochelatin, glutathione, and cysteine production in order to monitor ongoing changes in red spruce response to metals deposition. While there was a consistent trend of increasing Pb and Zn with elevation in mineral soils, reflecting historic deposition patterns, there was no evidence for any systematic variation in metals content of foliage with elevation. Phytochelatin and mycorrhizal analyses are ongoing.

After removing the Elwha dam, sediments were exposed which pose a challenge for revegetation, due to lack of nutrients and organic matter. Ectomycorrhizal fungi (EMF) are known to facilitate uptake of nutrients and water and therefore effective colonization of seedlings would be expected to promote seedling establishment, survival and growth. Seedlings were planted in nursery potting soil mixed with local forest soil, compared to potting soil alone, to determine whether this practice will promote colonization and diversity of EMF on Abies grandis (grand fir) and Pseudotsuga menziesii (Douglas-fir) seedlings before outplanting at the Elwha restoration site. EMF species will be identified by examining morphology and then, for each morphotype, DNA extraction, PCR using ITS-1F and ITS-4 primers, and sequence analysis will be used to compare sequences to published fungal sequences using the BLAST algorithm. While molecular analyses are not yet complete, preliminary analysis of morphotypes revealed a consistent trend of increased diversity due to inoculation, based on an increased number of unique morphotypes per seedling, from 3.8 to 4.5 for grand fir, which was not significant, and from 4.2 to 5.6 for Douglas-fir, which was weakly significant (2 tailed t-test, p=0.093). This suggests that pre-inoculation with forest soil may promote colonization and therefore aid in restoration efforts.

The incidence of esophageal adenocarcinoma (EAC) is increasing world-wide. Early and specific detection of EAC and its precursor non-cancerous stages, particularly Barrett’s esophagus (BE), is crucial to treatment and long-term survival. Currently, endoscopy coupled with serial biopsy is the gold standard for diagnosis. Standard endoscopy lacks specificity. BE presents as a salmon-reddish discoloration against the pale-pink color of healthy esophageal tissue. Cardia intestinal metaplasia (CIM) arising from gastric tissue is visually similar; both develop in response to prolonged acid reflux and are often confused during histological analysis. However, CIM is unrelated or even negatively correlated to EAC development. Distinguishing between BE and CIM at the gastroesophageal junction is crucial to minimizing the use of invasive biopsy. Targeting molecular markers specific to disease state can enhance specificity. Fluorescent dyes attached to targeting molecules are used to label these biomarkers. Instrumentation designed for in-vivo fluorescent imaging, like the ultrathin, flexible, multimodal scanning fiber endoscope (SFE) can then excite and image the fluorescent dyes, effectively red-flagging abnormal regions for both enhanced biopsy capture and point-of-care diagnosis. Reliance upon one biomarker alone is ineffective due to patient heterogeneity, as expression varies between patients due to factors including genetic background and lifestyle. Multiple biomarkers are necessary for specificity. The purpose of our work is to first demonstrate that the three cell surface biomarkers, EGFR, C-MET, and ERBB2, well established in other cancers, are appropriate for early EAC or BE diagnosis. We plan to demonstrate the simultaneous application of all three biomarkers to provide added specificity and sensitivity for diagnosis and ultimately prognosis. These goals will be achieved through immunofluorescent staining of cell lines and paraffinized tissue samples excised from patients. The efficacy of using multiple markers will be assessed through image processing and statistical analysis.