Almost 3 billion people, coming mostly from low-middle income countries, still rely on unprocessed solid fuels (wood, dung, crop, waste, kerosene, charcoal) as their main source of energy to fulfill daily needs, such as cooking, heating and lighting. The prolonged exposure to this form of pollution is strongly correlated to pneumonia and acute respiratory infections (ARI) in children and to chronic obstructive pulmonary disease (COPD) in women. I began this project by analyzing the correlation between these respiratory infections and high exposure levels to particulate matter and CO present in the heavy smokes produced by wood-powered cookstoves. I then conducted a preliminary field study in Granada, Nicaragua, to evaluate the effectiveness of ongoing projects and to examine the elements involved in adoption levels of alternative cookstoves. Speaking fluent Spanish allowed me to collaborate with a Nicaraguan NGO that manufactures, sells and distributes clean cookstoves. Through this organization I approached local community members from a rural area near Granada and conducted both participant and nonparticipant observations of local cooking practices and fuel uses. These observations were supported by informal semi-structured interviews about costs, logistics and household financial management dynamics involved in fuel purchasing (wood). I also analyzed the implementation of wood for cooking practices and the related cultural elements. As an example, the long-standing cultural tradition of cooking on a big open-air fire using large quantities of wood correlates with prestige and wealth within the community. These early findings have informed a second study that I will carry out in July and August 2017 in Nicaragua, which addresses more specific questions about the cultural appropriateness of different alternative cookstoves and the related adoption levels.

The structure of plastid DNA (ptDNA) provides insight concerning its replication, repair, maintenance, and stability. We are studying DNA molecules and their association with proteins in plastid nucleoids as maize seedlings develop from the newest cells in the basal meristem to the oldest in the leaf tip. When observing unbound ptDNA from light-grown seedlings, we have found that ptDNA copy number and molecular integrity decline from complex-branched forms (Type I) in the meristem, to a mixture of smaller complex and simple forms (Type II) in the stalk, and small simple forms (Type III) in the leaf blade. In contrast, preliminary examination of unbound stalk ptDNA showed that for the dark-grown seedlings ptDNA is maintained at high copy numbers, with large complex forms. Furthermore, DNA fibers connected to these complexes were longer in dark-grown than light-grown stalk. We will now continue to examine unbound ptDNA in the meristem and leaf blade from dark-grown seedlings. Once finished examining all samples of unbound dark-grown ptDNA, we will analyze nucleoid-associated ptDNA and deproteinized ptDNA of dark- and light-grown seedlings by gel electrophoresis and fluorescence microscopy. For the basal meristem of light- and dark-grown seedlings, in nucleoid-associated ptDNA, we expect similar high-integrity Type I ptDNA, since this is where replication is highest. In contrast, we expect major structural differences in the nucleoid-associated leaf blade ptDNA, with small Type III in light-grown and high-integrity complex Type I forms in dark-grown plants. We propose that replication and repair occurs exclusively on nucleoid-associated ptDNA and that unbound ptDNA is susceptible to nuclease degradation. Therefore, we expect complex forms for nucleoid-associated ptDNA and small, degraded molecules for the non-nucleoid fraction. On a larger scale, this research will be important for genetically modifying maize tissue, because we will know more about the structural integrity of the DNA we are targeting.

Most organisms show a roughly 24-hour cycle in their physiological and behavioral processes, called circadian rhythms. Circadian rhythms are generated endogenously through the ~24-hour cyclic expression of genes known as clock genes. The expression of these genes oscillates in the master circadian clock of mammals – the suprachiasmatic nucleus (SCN) - but also in nearly every cell of the body. Typically, circadian clocks and the rhythms they sustain are ‘entrained’ by the 24-hour light-dark (LD) cycle. Our lab has found that fear can also act as an entraining factor; we observed that mice and rats subjected to random foot shocks during the dark phase of the LD cycle in their cages’ foraging area changed from their typically nocturnal foraging pattern to a diurnal one, avoiding the fear stimulus presented during the dark. The goal of my project is to understand the neural circuits and molecular processes that are involved in fear entrainment. I have analyzed the expression of clock genes in animals that are exposed to nighttime fear or in control animals exposed to daytime fear, using a technique called in situ hybridization, which labels mRNA levels. This allowed me to assess the circadian rhythm of expression of clock genes of interest (Per1 and Bmal1) in the SCN and the amygdala, the region of the mammalian brain that encodes fear stimuli. Our findings could not only help elucidate the circuits that are involved in fear entrainment but also help understand the mechanisms that link fear and anxiety disorders to problems with circadian rhythms and sleep.

As of 2015, approximately 36.7 million people worldwide are living with HIV/AIDS, of which almost 15 million receive antiretroviral therapy (ART). These patients require regular HIV-1 viral load (VL) tests to monitor ART effectiveness and compliance. However, the majority of affected people live in low-resource settings, where accurate diagnosis and disease monitoring through lab-based instrumentation systems, such as nucleic acid amplification tests (NAATs), are inaccessible. Thus, there is an increasing need for accurate, affordable HIV-1 VL tests at the point-of-care (POC). We have leveraged an electrokinetic separation and preconcentration technique, isotachophoresis, and an isothermal nucleic acid amplification method to develop a fully integrated POC NAAT for extraction, amplification, and detection of HIV-1 nucleic acids in whole blood. We have demonstrated the ability to extract these nucleic acids from human serum and quantify the nucleic acid amplification with fluorescent detection. The fluorescent read-out is correlated to the initial sample concentration, providing semi-quantitative VL information. Our device operates in a single step with limited equipment in 15 minutes, consequently cutting the time, cost, and complexity of NAATs for infectious disease diagnosis, especially in low-resource settings. Our POC NAAT has the potential to improve patient care, and we are currently investigating applying this technology to diagnose other infectious diseases.

Currently, the only two treatments available to people with chronic kidney disease are organ transplants and dialysis. With such a high demand for kidneys, regenerative medicine is needed to create new therapies. Human pluripotent stem cells (hPSCs) can be used to generate immunocompatible tissues on-demand, which can be used to study human disease processes or potentially transplanted back into the original patient as a tissue replacement therapy. New protocols allow the differentiation of pluripotent stem cells into small, kidney-like organoids that contain the major proximal structures of the nephron, the functional subunit of the kidney. These organoids include podocytes, proximal tubules, distal tubules, and endothelial cells, but they lack a collecting duct system, a crucial component of the kidney. To address this need, we have developed a protocol to differentiate hPSCs into ureteric bud (UB) cells, which are the precursors of collecting duct cells. Undifferentiated hPSCs were treated with a growth factor, glial cell line-derived neurotropic factor (GDNF), that signals the outgrowth of the UB in the human kidney, and a differentiation factor, CHIR 99021, at different concentrations. Immunofluorescence was used to characterize the resulting cells, which were stained with Dolichos biflorus agglutinin (DBA), a collecting duct marker, DAPI, a DNA marker, and GATA 4, a transcription factor that is important for kidney development. We found that both DBA and GATA 4 were co-expressed together in isolated patches of cells under one of the conditions tested. This staining pattern suggested that these were UB cells. This is the first time UB cells have been differentiated from hPSCs. The development of this differentiation protocol will lead to the growth of the collecting duct system in the kidney organoids, which will optimize them for kidney disease modeling, high-throughput drug screening, and regenerative medicine approaches to reduce the need for kidney transplants.

The goal of this research is the generation of induced pluripotent stem (iPS) cells and derived kidney organoids from urinary cells. iPS cells are somatic cells reprogrammed to an undifferentiated state, making them an exciting advancement in regenerative medicine as they could model an in-vitro progression of many disorders and show great promise for replacing or repairing tissue damaged due to disease or injury. To generate new kidney tissues, adult somatic cells were isolated from a patient’s urine sample. This method of sample collection is advantageous over the traditional skin biopsy because it does not require a physician and is easier for the patient to supply. This protocol was further optimized to accommodate remotely collected urine samples, showing the ability to isolate somatic cells from patients located across the globe. We successfully isolated somatic cells with various morphologies, ranging from epithelial to mesenchymal-like cells, from 25 different patients. By introducing into these cells RNA encoding four specific transcription factors, a safer alternative to introducing DNA as it eliminates the risk of inserting a mutation into the patient’s genome, we successfully reprogrammed urinary cells of an international patient with autosomal recessive polycystic kidney disease into iPS cells. These iPS cells were further differentiated into kidney organoids, which have the capacity to become a powerful tool in improving current therapies for kidney disease as an in-vitro model. Thus, for the first time, we have generated patient-specific kidney tissue from a urine sample. This will increase our understanding of the disease and improve current methods for the screening of investigational therapeutics. The ability to create new kidney tissues in the lab also raises the possibility of transplanting these tissues back into their original patients, where they could potentially function without the need for immunosuppression.

Cross-flow turbines are a promising approach for extracting renewable energy from tidal and river channels. These turbines consist of a set of blades rotating about an axis perpendicular to the water flow direction. While blade mounting geometry has implications for parasitic drag, lift-induced drag, and blade lift generation, which strongly influence turbine performance, little research has been published on this topic. The impact of blade mounting geometry on turbine performance was evaluated by comparing the power conversion efficiency of ten two-bladed turbines with varying mounting geometries in a recirculating water flume. Each turbine was also tested without blades to evaluate interactions between blades and mounting geometry. A servomotor rotated the test turbine at constant angular velocity and two six-axis load cells were used to measure the torque produced by the turbine rotor. Six tests were performed with connecting struts at each end of the blades. Cross sectional geometry (rectangular, rounded, and foil) and thickness were varied (chord length held equal to the blade). Three tests were also performed with solid disks of varying radii mounted to each end of the blade. Finally, one test was performed with a single foil strut mounted at the center of blade span. Complete performance curves at four Reynolds numbers were generated for each mounting geometry by varying the free stream velocity. At the highest Reynolds number, the thin foil strut performed with greatest efficiency, followed by the thin rounded strut and thick foil strut. The smallest disk, thin rectangular strut, and thick rounded strut performed similarly. Strut and disk drag was analytically modeled and compared with experimental data to characterize power loss from each mounting geometry. These results offer insight into cross-flow turbine design for optimal efficiency and encourage investigation of additional mounting geometries such as winglets or curved mounting interfaces.

Oxidative stress arises from an imbalance between reactive oxygen species and an organism’s ability to avoid or repair the damage caused by these reactive molecules. The fruit fly, Drosophila melanogaster, demonstrates varying degrees of resistance to oxidative stress. To understand the genetic basis of this variation, our lab analyzed a collection of inbred, fully sequenced Drosophila lines, the Drosophila Genetic Reference Panel (DGRP), and tested the associations between resistance and each of more than two million single-nucleotide polymorphism (SNP) markers throughout the genome. We found a particular SNP at the second chromosome located within the gene u-shaped (ush), had the strongest statistical relationship with oxidaive stress resistance. We then hypothesized that this gene contributes to the degree of resistance in the flies. For my research, I am evaluating this hypothesis by comparing the peroxide resistance of two sets of genotypes to confirm if the SNP at 2L:531863 bp contributes to the trait. For this comparison, I used two groups of lines, one with the ‘resistant’ and the other with the ‘sensitive’ version of the SNP, and distributed mated females from each line into vials with or without peroxide added into the food. I counted the number of dead flies at regular intervals and used these data to calculate the mean lifespan for each line, where lifespan is a measure of peroxide resistance. If both groups show similar lifespans, it suggests that this particular SNP does not affect peroxide resistance and I may test other SNPs at nearby loci with a similar procedure. However, if the two groups show significant differences in resistance, this result would support the connection between the SNP and peroxide resistance. I would then go on to manipulate the expression of ush using RNA interference to test whether changing mRNA levels impacts peroxide resistance.

Beavers create complex modifications to the physical and biological components of stream ecosystems through their creation of dams and wetland complexes. Beaver impoundments alter the movement of surface and subsurface waters through riparian systems, which substantially affects biotic and abiotic ecosystem processes that together make up habitats for different species. Although there is general agreement that beaver impoundments modify stream temperature, there has been very little focus on their effect at larger spatial scales. In prior research, traditional temperature assessment methods have failed to capture the full spatial breadth of riverine systems. The use of thermal infrared remote sensing (TIR) is an effective tool for measuring surface stream temperature variability at landscape scales. To better understand the role of beavers in the regulation of stream temperature, we evaluated TIR imagery collected within the Snoqualmie and Stillaguamish River basins in Washington State. Our objective was to identify whether TIR imagery could be used to identify areas where beaver dam complexes have measurable effects in decreasing downstream water temperature due to the upwelling of groundwater. We used an intrinsic potential beaver habitat model to identify areas within the TIR flight paths where beavers were likely present and surveyed these areas to confirm presence of beaver wetland complexes. We compared upstream and downstream surface temperature, thermal complexity, and heterogeneity within these areas using a suite of spatial statistics. Our results have the potential to demonstrate that the use of TIR is a novel approach for monitoring the effects of beaver on riparian systems. Its use provides for assessments at scales and breadth not previously possible using traditional approaches.

Development of antibody-based diagnostics, biomolecular sensors, and biomaterial with reactive surface requires the ability to control the adsorption of bioactive protein at a surface. All medical devices exposed in an in vivo biological environment are immediately coated with a layer of adsorbed protein that plays an important role in the body’s behaviors towards the devices and the effectiveness of the devices themselves. In this study, we use a multi-technique approach to characterize the effect of protein G B1 (6 kDa) orientation on multi-layer antibody binding. By conjugating a cysteine group at different positions along the protein’s surface, we can immobilize five variants of the protein (V21C, D35C, E42C, T11C, and WT) onto maleimide and bare gold surfaces. We have confirmed through X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry experiments that the proteins adsorb onto these surfaces. In addition, we introduced primary IgG and secondary F(ab’)2 antibodies to investigate if the protein’s orientation will affect the binding of the antibodies. The adsorbed mass are measured using quartz crystal microbalance with dissipation monitoring. We have demonstrated that the cysteine mutants orient better on maleimide surfaces and encourage more IgG binding. We will also study the effect of temperature on adsorption mass and extend the experiments to amine and carboxyl functionalized surfaces. Fully developed techniques to accurately characterize the orientation and quantify the amount of protein and secondary adsorption will significantly contribute to understanding the interactions of biomaterials with the biological environment.

Although questionnaires are helpful for identifying employee retention targets in research contexts, such surveys do not benefit employers in small, real-world organizations due to the inevitability of gathering biased data (e.g., due to social desirability or lack of anonymity). The purpose of the current study was to test a tool employers could use to more accurately identify turnover risk and targets for individualized retention strategies. This tool focuses on employee engagement, which research has shown to be related to employee turnover (i.e. high engagement is related to lower employee turnover intentions). This tool enabled us to determine employee level of engagement and predict employee group identification (i.e. long-term retention vs turnover). We hypothesized that higher levels of engagement will predict employee long-term group identity, whereas, low levels of engagement will predict turnover group identity. We conducted a qualitative study using a between-subjects design to compare semistructured phone interviews with 25 community mental health clinicians (13 long-term, 12 turnover), in which we asked clinicians what “ideal changes” they would like to implement in their agency. Using the Q12 Engagement Measurement Model, derived from Maslow’s Hierarchy of Needs, we determined clinician engagement levels, which we then used to predict their group identification. We predict our analyses will reveal that clinicians who suggest more personal-level changes are more likely to be in the turnover group, while clinicians who suggest more altruistic-level changes are more likely to be in the long-term retention group. The literature indicates that employees with higher engagement levels are less likely to turnover; therefore, if we achieve our predicted results, future implications of this study could be that employers could use this tool in their annual review process to more accurately assess employee turnover risk and improve employee retention strategies.

Mutations have a large range of effects on an organism’s observable characteristics, from neutral (no effect) to deleterious (lower an organism’s fitness) to beneficial (increase an organism’s fitness). I engineered three mutations into the virus phi-6 and evaluated their effects on viral thermotolerance and growth. Because these mutations were found in a population that had experienced high temperature heat shocks, I hypothesized that the mutant viruses would have a higher thermotolerance than the ancestral virus. To test the thermotolerance of these mutations, I heat shocked bacteria-free lysates of mutant virus and ancestral virus at various temperatures and then calculated the percent survival of the lysate at each temperature. The mutants were also evaluated for their fitness through growth competitions with a common competitor. I found that only one mutation conferred a higher thermotolerance than the ancestor. However, fitness competitions showed that all mutations and combinations of mutations did increase viral growth. This suggested that additional selective pressures may have been present in the evolution experiment. Specifically, the viruses had been grown at low temperatures between heat shocks, which may have resulted in the evolution of higher growth rates, even at the cost of thermotolerance. My research shows that all selective pressures must be taken into consideration when studying evolutionary trajectories. We cannot accurately predict how organisms will evolve in response to one selective pressure such as increased environmental heat without taking in to account the other selective pressures they experience.

Plasmids are small circular pieces of DNA that replicate separately from the chromosome. Plasmids often contain antibiotic resistance genes that allow their host bacteria to grow in environments containing that antibiotic. They can be transmitted both vertically during cell division, and horizontally via conjugation to new bacterial hosts, and thus spread antibiotic resistance through populations. Plasmids are often costly to their bacterial hosts when not in the presence of antibiotics, but the coevolution of a bacteria and plasmid can reduce these costs through compensatory mutations. Typically a reduction or an amelioration of plasmid cost is seen during plasmid/host coevolution, but not a fitness benefit. However, preliminary data has shown evidence of a fitness advantage to containing a plasmid for the bacterial host Pseudomonas sp. nov. H2 after it was evolved with the plasmid RP4. In this project, I determined whether the fitness benefit seen in the aforementioned plasmid/host pair also occurs in three other bacterial species with different plasmid types. For each of these pairs the plasmids initially conferred a cost to the host. We hypothesized that compensatory mutations (on either the host chromosome or the plasmid) would result in the reduction, amelioration or reversal of the initial high cost of maintaining a plasmid for all three pairs. To test this, we evolved three plasmid/host pairs in antibiotic-containing media for several hundred generations, selecting for antibiotic resistance genes on the plasmids. We ran competitions between an evolved host/evolved plasmid pair and an evolved host with the plasmid removed, to determine whether there remained a cost to containing a plasmid, or whether that cost has been reduced, ameliorated, or reversed. Our experiments will help explain why plasmid-conferred antibiotic resistance persists in bacterial populations.

Human-associated bacteria are important for human health, but little is known about how these bacteria interact with each other. Bacteroides fragilis (B. fragilis) is a Gram-negative bacteria that is found in the human gut and assists in nutrition. B. fragilis genomes encode the type VI secretion system (T6SS), a needle-like structure that transports toxins into nearby competitor cells. T6SS is used to gain advantage over competitors, yet our lab has observed that B. fragilis strains in adults are four times more likely to lack T6SS than strains in infants, suggesting B. fragilis may lose this mechanism over time. Why would T6SS be lost if it is used to gain an advantage over competitors? I hypothesize that B. fragilis inactivates T6SS because it is no longer required. To test this, I passaged B. fragilis strains with T6SS under competition conditions in which the recipient is either susceptible or immune to the effects of T6SS-delivered toxins. I predicted that recipients with immunity will cause B. fragilis to inactivate its T6SS mechanism, while predicting recipients that are susceptible to be killed off by the toxins delivered via T6SS in B. fragilis. Competitions proceeded for 24 hours, followed with the selection of the donor strain by addition of antibiotics. After selection of donor strains, each were archived in glycerol for evolutionary documentation. Subsequent passages were commenced with newly selected donors. Every 5 generations, competitions proceeded to evaluate competitive fitness of T6SS; whether or not B. fragilis has inactivated its T6SS. Whole genome sequencing revealed the mechanism by which T6SS was inactivated at the level of DNA mutations. By using experimental evolution to study interbacterial interactions within the human gut, we will have a better understanding of the long-term dynamics of the human microbiome and its role in influencing human health.

Organisms face a range of stressors over the course of their lifespans. As with most traits, we see significant variation in response to stress even between individuals within species. In our attempt to explain differences in stress responses between individuals, the Promislow lab studied the metabolome, the total profile of all small molecules in an organism, and found variation in stress response is correlated with variation in contents of the metabolome. Indeed, in a study of peroxide resistance in the fruit fly, Drosophila melanogaster, we found resistant flies tended to have increased galactose levels, compared to sensitive flies. This observation led to the novel hypothesis that flies fed supplemental galactose will have increased peroxide resistance compared to control flies. For this experiment, we chose flies among the most sensitive and resistant lines examined previously because the most sensitive flies, with low galactose levels, may show increased effects of supplemental galactose on lifespan. In this experiment, flies feed on food with varied galactose concentrations, including a negative control without galactose. We then place the flies into media with peroxide or water, the control. We record the number of flies that die in each condition multiple times a day, and the death rate provides a quantitative measure of peroxide resistance. By comparing the death rate of flies within a line kept in varying concentrations of galactose exposure, we can test the effect of galactose on resistance. If our hypothesis is correct, the flies receiving supplemental galactose should die at a slower rate than those without additional galactose. However, it is possible flies receiving supplemental galactose will die at faster rates, suggesting galactose makes flies more sensitive. Determining whether galactose makes flies more resistant or sensitive to peroxide may prove to be important in counteracting effects of oxidative stress in other organisms.

Our goal is to quantify the fraction of H-α emitting B stars in M31. A small subset of massive stars emits strongly in H-α, which indicates the presence of hotter gas than is typically associated with such stars. These stars’ line emission may be related to stellar rotation, but it is unclear what causes the rotation or if there are other possible causes for the line emission. In order to shed light on this problem, we’ve begun a project to identify a homogeneous sample of these stars in M31. Our data are comprised of imaging from six fields in M31. Using resolved photometry of over two million stars, we have found 50 candidates. Using a ratio of these candidates to ordinary stars of the same temperature that are not emitting in the H-α, we can put observational constraints on models of the formation of stars in this mass range, as any viable model must be able to reproduce the observational statistics of these rare stars. Therefore, despite their rarity, they provide a key diagnostic of stellar evolution models fundamental to many fields of astrophysics.

Supernovae enrich the interstellar medium, allowing it to create fascinating and exciting objects such as stars, planets, and people. However, supernovae are difficult to study because, most of the time, we do not observe these supernovae directly, since the whole event can occur in a very short time. Fortunately, they leave behind bright remnants that can give us information about the supernova and even the stars that existed before. We have observed one such supernova remnant in the nearby spiral galaxy NGC 300, which is approximately three million light years away from Earth. We have acquired multi-wavelength data of this source from Chandra X-ray Observatory, XMM-Newton X-ray Observatory, and Hubble Space Telescope. Using this variety of telescopes, we can understand how this supernova remnant behaves in both the x-ray and optical energy bands which gives us a more complete picture of our source. From our Hubble data, we gain detailed measurements of the size and shape of the source. From our Chandra and XMM-Newton data, we can shed light on the properties of this supernova remnant by performing a spectral analysis on the x-ray data. This spectral analysis gives us information about the temperature and metallicity of the source, as well as the density of the surrounding gas. With these quantities, we can better understand this supernova remnant and supernova remnants like it which, in turn, let us better understand the life of the star that created this source.