Our lab has recently developed computational methods for designing cage-like protein nanomaterials built through the self-assembly of multiple copies of two distinct protein components. This new technology holds great promise for a wide range of applications including targeted drug delivery, vaccine design, and biofuels. Moving towards these real-world applications, one desired but as of yet unrealized feature of our current designs is the ability to produce each protein component independently of one another and then trigger assembly by mixing the purified components in vitro. Obtaining this level of control over the assembly process requires that the individual protein components are stable and soluble in the absence of their designed binding partner. This property is not explicitly considered in our current design protocol. Here we present work aimed at addressing this problem by implementing a new step into our design protocol wherein we redesign the amino acid sequences of the proteins to increase the net positive charge on the surface of one protein subunit and the net negative charge on the surface of the other subunit. This supercharging approach should improve solubility by disfavoring aggregation between identical subunits of individually purified components while favoring the interactions between distinct subunits required for nanocage formation. To supercharge the proteins, we use the molecular modeling software package Rosetta to determine positions on the surface of each protein that are predicted to tolerate mutations to either positively or negatively charged residues. This information is used to automatically generate initial designs, which we then manually inspect and refine.

The Muon g-2 experiment hopes to test the difference between the experimental and theoretical values of the anomalous magnetic moment of the muon (g-2) out to a greater statistical precision than has been previously accomplished. Such a discovery would provide strong evidence of new physics such as dark photons, supersymmetry, or possibly something not yet considered. During the experiment, muons are injected into a large superconducting ring. A muon decays to an electron and the electron curls to the inside of the storage ring and strikes an array of lead fluoride crystals. When an electron hits a crystal, it produces light that is proportional in intensity to the energy of the electron. The energy and the time of its arrival help to indicate the direction of the spin which is used in the calculation of g-2. My research investigates how light is propagated and distributed in these crystals and how different reflective or absorptive wrappings affect this distribution. I measure light yield and pulse width, which correspond to two different extremes of wrapping material. For maximum light yield, I use a special reflective white paper called Millipore, while shorter pulse widths are achieved using an absorptive black Tedlar paper. I have developed a 2x2 array of crystals coupled to light sensitive photomultiplier tubes. This setup, which is configured for easy switching of the wrapping material, captures cosmic ray muons that hit the surface of the earth. Capturing these muons simulates the conditions of the experiment. I analyze the resulting data by fitting the pulses and extracting the functional form of the light distribution. The results of this research will ultimately contribute to the development of more complete theories of the fundamental building blocks of the universe.

The vertebrate body forms through a process called embryogenesis. In my research, I focused on the establishment of body length during embryogenesis of the model organism zebrafish (Danio rerio). Under normal conditions, the cells of the developing body intercalate towards the back, or dorsal side, of the embryo, and extend longitudinally to grow the body axis. One observed defect in this process blocks cell intercalation, thus leading to underdeveloped bodies, which is known as a convergent extension phenotype. In my research, I studied the role of Microtubule-Associated Serine/Threonine kinase-Like (MASTL) RNA in body axis formation. Because of a known role for MASTL in regulating chromosome segregation, I hypothesized that overexpression of MASTL would interfere with normal cell function and lead to a convergent extension phenotype with a shortened body axis in the developing zebrafish embryo. To test this hypothesis, I injected embryos with different concentrations of RNA and allowed them to develop. Overexpression of MASTL shortened body lengths at all but the lowest concentration of RNA. I then chose a low but effective concentration of MASTL RNA, injected embryos and quantified embryo length. Finally, I performed RNA in situ hybridizations, a process by which labeled probes are used to localize specific RNA transcripts in embryos. Using a variety of labeled probes, I found in addition to shortened body length, the distribution of the prospective spinal cord cells and muscle cells were abnormal in MASTL-injected embryos. The results of these experiments clearly show that overexpression of MASTL leads to a convergent extension phenotype. To continue this research, I will look at whether or not MASTL affects body length formation through Protein Phosphatase 2a, a known target of MASTL. The results of my research will lead to a better understanding of the role MASTL in cell intercalation and vertebrate body elongation.

Solar power is an increasingly significant resource as we look to move from fossil fuels to a cheap and sustainable source of energy. Solar cells can be manufactured using organic semiconductors by using cheap roll-to-roll printing techniques, however, the efficiency of such organic photovoltaic (OPV) devices is currently too low for cost-effective implementation. These devices utilize a built-in electric field, resulting from the difference in potential energy between the device’s electrodes, to collect photogenerated charges at the electrodes as current. Fine-tuning of the electrode energy levels also dictates the amount of voltage that can be generated by the device. As a result, the ability to chemically alter the electrode energy levels can greatly influence the overall power output of the solar cell. Phosphonic acid self-assembled monolayers (SAMs) are molecules that can be chemically attached to a variety of electrode interfaces and have been shown to alter the energy levels of certain electrodes. I am investigating the effect that attaching SAMs to the zinc oxide surface of an OPV electrode will have on both the built-in voltage and power conversion efficiency of an OPV cell. In order to test the hypothesis that the SAMs can affect OPV performance, I have fabricated solar cells with SAMs in order to compare them with solar cells without SAMs. Experiments that measure the built-in voltage of the device and investigate the lifetime of photogenerated charges will help to clarify the specific role that the SAMs play in affecting OPV device performance. If this hypothesis is correct, we have the potential to achieve higher power conversion efficiency than what is currently reported in these devices and this may lead to a greater emphasis towards understanding the role of interfaces in developing sustainable solar technology.

Transforming growth factor-beta (TGF-β) signaling in smooth muscle cells (SMC) contributes to normal vascular development but may also play a significant role in the progression of aortic disease. Accordingly, TGF-β signaling is emerging as a therapeutic target, with strategies to block TGF-β signaling proposed as a means of preventing human aortic disease, including aortic aneurysms and dissections. However, because TGF-β signaling contributes to normal vascular development, blockade of TGF-β signaling could be harmful. We hypothesized that elimination of TGF-β signaling in aortic SMC of adult mice would cause aortic disease. We used inducible Cre-lox recombination technology to knock out Tgfbr2 in adult murine SMC. Tgfbr2 encodes TGF-β receptor II, believed to be essential for all TGF-β signaling. Mice were euthanized 4-14 weeks after SMC Tgfbr2 knockout, and effects of Tgfbr2 knockout (compared to control mice) were assessed by gross observation of aortas as well as sectioning and staining of aortic tissue. We found intramural hematomas, penetrating aortic ulcers, aortic dissection, and aortic dilation as well as significant thickening of the medial wall and adventitia in the knockout mice. Our data strongly suggest that loss of TGF-β signaling in SMC has a negative effect on aortic health and support a cautious approach in pursuing blockade of TGF-β signaling as a therapeutic strategy.

 When students study abroad, some find it harder than others to integrate within the culture of their host country. Scholars have created models mapping students’ process of acculturation over time. A stage of crisis, or “culture shock,” is identified as a fundamental phase of the abroad experience. This study investigates four elements influencing students’ ability to overcome this crisis stage: students’ interaction with the host community, confidence using the host language, previous experience traveling abroad, and the disparity between students’ home and host culture. This study aims to determine the role communication plays in the navigation of the crisis phase; I do so specifically by examining these four elements, and how the connection between these factors and student adjustment manifests. Past studies have flagged these elements as major predictors of a students’ achieved assimilation while abroad. This study aims to determine if and how a connection exists between these factors and students’ cultural adjustment. The qualitative method of semi-structured interviews was used to collect responses via Skype from four students in Quito, Ecuador and four students in Rome, Italy during their Winter quarter abroad. The eight students' narratives provide insight on the modern impact of the four key elements on intercultural development—and an understanding of why: why some students overcome the crisis phase and achieve healthy cultural adjustment while others remain culturally stagnant for the entirety of their exchange.

Songbirds are a model organism of interest to neurobiologists as they are one of a few groups of organisms that display sensorimotor vocal learning, akin to that of humans. Once learned, birdsong is highly stereotyped and while slightly variable, has not been thought to vary systematically from instance to instance. However, biological processes separate from learning mediate the qualities of a bird’s song. It is well known that the amount of singing varies throughout the day with the light cycle, but is not well understood how the acoustics of birdsong vary with light. Recent work has shown that pitch and loudness vary in a consistent manner throughout the day, rising through the morning and early afternoon and falling through the afternoon and evening. Whether these changes in song depend on an internal circadian rhythm or external light cues remains unknown. My project focuses on determining whether finch song is endogenously driven by an internal clock. I collected song data from zebra finches housed under typical light patterns (14 hours of light, 10 hours of dark) and constant light conditions. The fundamental frequency of a sample syllable was analyzed for each bout of singing and then plotted across the span of weeks. Preliminary results suggest that birds under constant light conditions take a few days to unentrain from the light cycle, then sing throughout each 24-hour day, with FF fluctuation around a baseline frequency. I hypothesize that if birdsong acoustics vary according to a circadian cycle, FF fluctuations that occur during constant light conditions are part of a regular pattern that is regulated by an internal clock. Finding that finch song is circadian has implications for understanding how a clock might connect to song system nuclei and demonstrating how complex motor behavior and learning can be influenced by time of day.

Microbial growth can be monitored by changes in the optical density of a culture. In the past, these measurements were generally made by hand, one at a time at regular intervals, in order to produce a growth curve. Currently no such device is suitable for monitoring the growth of fastidious anaerobes. This presented problems for both efficiency and resolution of experiments requiring analysis of multiple cell lines and different growth conditions. For growth periods greater than a work day, researchers needed to take alternating shifts in order to capture data. This is a very tedious process which is draining on the tester. The result of such a worker demand was infrequent measurements to make the minimal curve, resulting in inherent tester bias. Naturally, the desire to automate the process arose. I began development on the ROGR system began in order to implement a microcontroller operated test setup using LEDs and phototransistors to replace the manual measurements made with a spectrophotometer. Previous work has shown that LEDs of these wavelengths do not interfere with cell growth. As this is an ongoing experiment, it is imperative that the method used to measure growth does not change the testing environment. For example, the anaerobic cultures have long been incubated with constant shaking with the culture tubes oriented in a horizontal plane, but measurements were made in the upright position in order to create an accurate reading. The ROGR is a multi-disciplinary project that addresses these project needs. The electronic design ensures system isolation and achieves high precision and dynamic range. Design also ensures mechanical stability, reducing error in LED phototransistor pairs readout originating from changes in their physical orientation. Presently the system is undergoing the physical implementation phase with its circuitry at a working state. This reproducable system will allow for data colection across multiple teams to be connected with confidence of small error.

Composites of conjugated polymers with semiconducting nanocrystals (NCs) are of interest for low-cost, solution- processable photovoltaics. However, compared to typical polymer/fullerene composites, relatively little is known about factors influencing charge generation. Without understanding these factors, design principles for improving solar cell applications remain elusive. Previous studies of blends of the polymer poly(3-hexylthiophene-2,5-diyl) (P3HT) with PbS nanocrystal quantum dots showed the excited NC undergoing charge transfer by hole transfer from the NCs to the polymer. To further investigate this hole transfer mechanism, quasi-steady-state PhotoInduced Absorption (PIA) was performed on blends of P3HT with PbS nanocrystal quantum dots as a function of excitation energy. As the energy exciting the PbS quantum dots was increased, the yield, per photon absorbed, of positive charge carriers on the organic host polymer increased. This occurred even at excitation wavelengths that only the NCs could absorb at. Our result provide direct evidence that holes generated on the NC retain some of the additional energy from excitation, e.g. hot holes. Further understanding of these results could provide even more insight to the mechanisms of organic/inorganic photovoltaics.

Thin-film solar cells can address the cost barrier of solar power by virtue of less material usage and more efficient manufacturing processes like roll-to-roll printing. The thin-film material Cu/Zn/Sn/Se (CZTS) is of particular interest because it is composed of earth-abundant elements; however, the current record for CZTS device efficiency is 12.6%, which is far from the 20% efficiency that is needed to be commercially competitive. Low CZTS device efficiencies have been linked to low open-circuit voltages (Voc), which are likely due to high charge-carrier recombination. Recombination can be increased by irregularities in crystal structure and composition known as defects. In this project, different microscopies will be used to study the defects present at the interfaces of a working CZTS photovoltaic device in relation to the device's V_oc. The interfaces of concern include those between different material layers and those between CZTS crystal grains, i.e. grain boundaries. A variety of scanning probe microscopies (SPM) will be used to map local electronic properties at these interfaces, relating position on a device cross-section to current and voltage characteristics. Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) will be used to correlate these electronic properties with CZTS device structure and composition. In making these correlations between SEM, EDS, and SPM data we hope to better understand the origins of Voc loss in CZTS devices and, in turn, inform more effective CZTS cell fabrication. Thus far, we have acquired SPM, SEM, and EDS images that display the distinct layers of the device cross-section. Further optimization of sample preparation is required to achieve both resolution of grain boundaries and a sample surface that is smooth enough for SPM to obtain reliable electronic data.

Solar technology is a potential way to help meet the growing demand for clean, renewable energy. Hybrid composites of inorganic quantum dots, with organic semiconducting polymers, offer a potential means of producing low-cost, solution-processable photovoltaics. Photovoltaics can convert sunlight directly into electricity to capture solar energy. The synthesis of quantum dots typically involves the use of large surfactant molecules to facilitate particle growth and solubility. These native ligands act as electrical insulators that impede charge transport in photovoltaic devices. Therefore, it is necessary to exchange these large ligands with small molecules to achieve efficient charge carrier photogeneration and transport. In this research, we examine bulk heterojunction blends of low band gap PbS quantum dots with the conjugated polymer poly((4,8-bis(octyloxy)benzo(1,2-b:4,5-b')dithiophene-2,6-diyl)(2-((dodecyloxy)carbonyl)thieno(3,4-b)thiophenediyl)) (PTB1) treated with different ligands including halide ions and organic crosslinkers. By using photoinduced absorption and transient photovoltage techniques, we study the long-lived charge generation dynamics and recombination kinetics of our devices. We find that the ligand treatments exhibiting higher device performance correlate to longer carrier recombination lifetimes in both spectroscopic and photovoltaic device measurements.

Physicochemical DNA research contributes to cutting edge medical applications such as single nucleotide polymorphism (SNP) detection and drug delivery. DNA can be modified with azobenzene moieties to form azobenzene DNA. I used UV light to induce a trans to cis isomerization reaction of the azobenzene moiety which causes double stranded azobenzene DNA (dsDNA) to split into a single stranded DNA (ssDNA). We study the effects of UV light on the azobenzene photoisomerization of perfectly matched and mismatched DNA. For the experiments, a glass substrate is functionalized with amino-silane molecules and single stranded azobenzene DNA. I hybridized the azobenzene DNA with either a perfectly matched or a mismatched strand to form double stranded DNA. I destabilize the DNA duplex by adding UV light to the hybridized DNA sample. Since the mismatched strand has a single base mismatch, it is less intrinsically stable than a perfectly matched strand due to less contributing intermolecular forces. Consequently, the melting temperature is lower for the mismatched strand. The mismatched DNA strand returns a less intense fluorescent signal in my detection. Experimental parameters such as hybridization time, UV exposure time and temperature applied to the experimental set-up are varied in order to study changes in ssDNA vs. dsDNA fluorescence. Consistent reproduction of these results is vital for a reliable DNA chip assay that can detect single nucleotide polymorphisms (SNP) in fragments of DNA.

Thaumarchaeota are widespread in marine and terrestrial environments. Many of them are thought to carry genes coding for a putative ammonia monooxygenase (AMO), suggesting a capacity for ammonia oxidation. However, strong evidence from isotopic studies and molecular surveys, as well as direct physiological characterization shows some populations of ammonia-oxidizing archaea (AOA) have a capacity to use alternative fixed carbon for biosynthesis, and suggests their mixotrophic lifestyle. In this study, we investigate the influence of organic substrates on growth and activity of the only two available isolated representatives of marine AOA, Nitrosopumilus maritimus strain SCM1 and strain HCA1. The certain intermediates of the TCA cycle are supplemented into AOA pure cultures, and the growth response of each strain to different organic substrates are monitored by measuring nitrite production and cell counts. The calculated specific growth rate, doubling time and specific cell yields are compared between organic carbon amended cultures and organic carbon-free controls. The SCM1 and HCA1 growth rate and cell yield were greater in cultures supplemented with alpha ketoglutaric acid (from 93.4 to 112.6 x 1012 cells mol-1 NH4+ for SCM1, and no growth to 80.8 x 1012 cells mol-1 NH4+ for HCA1), which suggest that marine AOA may prefer a mixotrophic lifestyle. This data expands our knowledge of thaumarchaeal metabolic capabilities and can provide information on the competition between AOA and other microbial groups to improve our understanding of microbial ecology.

Modern frogs and toads (Anura) are environmentally sensitive vertebrates that are often viewed as bellwether taxa for the modern biodiversity crisis. As such, one would expect that they did not fare well in the Cretaceous-Paleogene (K-Pg) mass extinction event (ca. 66 million years ago) that killed off the non-avian dinosaurs. However, relative to other taxa, little is known about Late Cretaceous and early Paleogene anurans or how they fared in the K-Pg event. I have documented temporal changes in anuran diversity leading up to and across the K-Pg boundary, based on the vertebrate microfossil assemblages in the Hell Creek and Tullock formations in Garfield County, Montana. These geologic formations span approximately the last two million years of the Cretaceous and the first one million years of the Paleogene. I have identified known anuran species and distinct morphotypes, which likely represent unnamed species, from more than 200 isolated fossil anuran jaws and hip bones. Initial results, based on maxillae, show an increase in anuran species richness from 9 morphospecies in the lower Hell Creek Formation to 17 morphospecies in the middle of the formation. Then, species richness drops before the K-Pg boundary down to 9 morphotypes in the upper Hell Creek Formation. Anuran fossils are relatively scarce after the K-Pg boundary: the lower middle Tullock Formation includes one species that locally survived the mass extinction and one apparent immigrant taxon. Later in the middle Tullock Formation, I document only one unnamed immigrant taxon. These results parallel pre-K-Pg declines in some vertebrate taxa (e.g., mammals) from this study area but contrast with the high survivorship of more fully aquatic lissamphibians (e.g., salamanders). I hypothesize that the high levels of extinction suffered by anurans at the K-Pg boundary may be due to the greater vulnerability of their more terrestrial life habit.  My research aims to bring a greater understanding to the timing and mechanisims of one of the most important events in the history of terrestrial life (the K-Pg mass extinction), and possibly give insite into the current biodiversity crisis effecting extant amphibians.

At the Cretaceous-Paleogene (K-Pg) boundary, approximately 66 million years ago, a major mass extinction event eliminated up to 75% of all species on the planet. Considerable debate surrounds the cause of this extinction. Was the extraterrestrial bolide impact at the K-Pg boundary the sole cause? Or were ecosystems already deteriorating prior to the impact? In non-marine ecosystems, dinosaurs (except birds), lizards, metatherian mammals, and sharks and rays (Chondrichthyes) suffered the greatest levels of extinction. Chondrichthyans have received little research attention relative to other taxonomic groups. Here, I investigated the fossil record of freshwater chondrichthyans through the Hell Creek Formation in eastern Montana. The formation is ~90 meters thick, divided into lower, upper and middle parts, and spans the last two million years of the Cretaceous. I examined 12 vertebrate microfossil assemblages through the section, identifying teeth, dermal denticles, and vertebrae of freshwater sharks and rays to the genus level. I used this dataset to estimate generic richness and relative abundance trends leading up to the K-Pg boundary. My richness results show that one genus of ray occurs through the entire section. From the lower to middle parts of the section, sharks increase from one to five genera and then drop back down to one genus in the upper part of the section. This pattern temporally correlates with a minor rise and fall, respectively, in sea level of the Western Interior Seaway. Alternatively, this pattern may reflect differences in sampling intensity and site availability through the Hell Creek Formation. I will test the latter hypothesis using rarefaction analysis, which compensates for differences in sampling intensity. Results will help me determine whether these trends are a true glimpse into the shifting biogeographic ranges of non-marine cartilaginous fish communities of the latest Cretaceous or an artifact of variable sampling through the section.

Ammonia-oxidizing archaea (AOA) are essential to the nitrogen cycle in the environment.  By processing ammonia into nitrite, AOA impact plant growth and other environmental factors such as greenhouse gas emissions and groundwater chemistry- mainly leaching of mobile nitrite and nitrate.  To optimize agricultural practices and minimize ecological impacts, a better understanding of the role of these microorganisms in the environment is needed.  AOA have been identified as the dominant nitrifying group at long-term agricultural study sites in eastern Washington.  Enrichment cultures of AOA have been established from these sites.  However, the relationship of the enriched populations to other AOA remains unknown. The goal of my research is to scale-up existing AOA enrichment cultures for nucleic acids extraction and molecular analyses.  Isolated DNA will be PCR amplified for phylogenetic and functional markers, mainly the 16S rRNA and ammonia monooxygenase subunit A gene, respectively.  These PCR products will then be cloned, sequenced, and compared to other AOA in public databases to assess their relationships with other populations.  These data will greatly aid in the design and interpretation of further physiology and environmental genomic surveys of AOA in the environment.

Chitosan and alginate are widely used natural polymeric biomaterials that are biocompatible, biodegradable, and non-toxic. Chitosan is cationic as well as hydrophobic in nature whereas alginate is anionic and hydrophilic in nature. Ionic interaction between the amine and carboxyl group causes the formation of polyelectrolyte complex in the scaffold and thus enhancing the mechanical properties of the scaffold. Mechanical properties of the scaffolds are dependent on the fabrication process, cross-linking process, and sterilization process. Similarly, porosity plays a crucial role in determining the mechanical strength of the scaffold which relates to the lyophilization process and molecular weight of the polymer used. Chitosan-Alginate (CA) scaffolds are prepared by mixing equal wt% amounts of each polymer and due to chitosan's hydrophobic nature, acetic acid was added. The solution was mixed using a Thinky mixer at 2000 rpm for five minutes. After obtaining a homogeneous mixture, pH was tested and solution  casted into desired well plates. The solution was then refrigerated at 4 degree Celsius for 12 h at 20 degree Celsius overnight, and lyophilized for 24 hours. Scaffolds were then cross-linked with 70% CaCl2, and sterilized with Phosphate-buffered saline (PBS). Scanning electron microscope (SEM) is used in to image pore morphology of the scaffold. High mechanical strength and porous structure is presented in pore size ranges of 100-500 micrometers which is required for bone tissue engineering. Mechanical strength of the scaffold is obtained by performing compressive test. Samples are compressed at 4.00 mm/min using micro-tensile tester with 1N load cell. Dry samples were found to have a young's modulus (YM) of 1-2 MPa, whereas wet samples were found to have a YM of 1-2 KPa.  CA scaffolds were found to be mechanically strong with good porosity which was directly impacted by fabrication process. Mechanically strong CA scaffolds with proper porosity were achieved with the fabrication process used. Further work will allow an optimization of CA scaffolds for specific applications and cell lines.  

Jack London's American Naturalist novel is one of the greatest works the author had written, and along with "The Call of the Wild," appeared on newly formed American Bestseller lists for American novels after its publication in the early 1900's. While a popular novel, the Sea-Wolf is also a major literary achievement. As one of Jack London's early pieces, The Sea-Wolf exhibits London's efforts at responding to major philosophical and political ideas of the era's international climate: London explores in his novel, ideas of Social Darwinism, alongside the need of altruism in an age of the consolidation of the Capitalist system and its economic policies. These two ideas owe the attention given to them to the genre of Naturalism and Realism, whose division is controversially blurred, which was a literary movement occuring at the time of the novel's writing. These two ideas are accompanied by the Naturalist's focus on the individual in the city, and is foregrounded by an antecedent Romantic literary tradition, which put concerns of the individual in front of societal ones. In the Naturalist tradition, the opposite occurs, and the individual must survive in the face of an antagonistic and often mechanistic environment. My research will entail close-reading passages of the Sea-Wolf to show that it cannot be strictly one genre, and that its hybrid form is in fact an emergent one in which new methods are found to say new things. I will be using as my theoretical framework, and supplementary texts, Donald Pizer's definition of Naturalism and also Donna M. Campbell's examination of Naturalism under the lens of gender in her "Resisting Regionalism". My aim in this presentation is to show The Sea-Wolf trafficks in Naturalist themes, such as the inability to escape the forces of environment, and the transformative, and even the degenerative effect it can have on the human encased within it. I want to show London's novel deals with the grittier aspects of life, as many Naturalist novels do, but it also utilizes the strategies of a more feminine literary tradition in the creation of the displaced domestic space existing between the novel's Humphrey Van Weyden and Maud Brewster. I want to show that by the end, Maud's last line as she spots a search boat, "Rescue us from ourselves," has implications equivallently significant both for a Naturalist reading and a Local Color one.

Rotavirus is the main cause of viral diarrhea among young children. There exist licensed vaccines, but rotavirus still kills nearly half a million children yearly. Live, attenuated vaccines quickly lose potency at room temperature. My host lab is assisting a Seattle NGO callednPATH and the Bill and Melinda Gates Foundation with developing temperature-stabilized, inexpensive vaccines. The current industry standard assay for measuring vaccine potency is time-consuming, subjective, and low throughput. My goal is to develop an objective, inexpensive, automatable, and high-throughput assay of the potency of candidate, next-generation, temperature-stable rotavirus vaccines. To develop this assay, susceptible cells are infected with candidate vaccine-strain viruses manufactured in India and undergoing development at PATH. After cell membrane modification to allow antibody access to cytoplasmic viral proteins, an anti-rotavirus antibody and a fluorescent secondary antibody are used to detect viral protein within infected cells. Experimental samples undergo single-cell flow cytometry analysis of individual cells to differentiate infected and uninfected cells. The vaccine industry-standard potency assay is performed in parallel to directly compare assays. Comparison and titration of polyclonal and monoclonal rotavirus-specific antibodies identified the optimal antibody and concentration for specific intracellular recognition of rotavirus protein. Testing of secondary antibodies and fluorochromes resulted in cell fluorescence characteristics yielding a favorable signal-to-noise ratio without causing false-positive results. A direct relationship between the amount of live rotavirus input and the percentage of infected cells with good linearity and acceptable coefficients of variation was observed. Future work is focused on reducing material costs, comparing the flow cytometry assay to the industry standard, and adapting the assay to additional rotavirus vaccine strains. Based on the above results, flow cytometry is a promising candidate assay for quantifying rotavirus vaccine potency to assist the development of novel live rotavirus vaccine formulations and storage formats.

The current approach for FDA drug approval requires an exorbitant amount of time and money, often spent in pre-clinical animal studies, and has a high late-stage failure rate.  This is partly due to the fact that pharmaceutical companies develop drugs to treat the average patient with any given disease.  A personalized medicine approach would increase the effectiveness of a new drug for each patient and decrease the need for animal testing.  Induced pluripotent stem cells (iPSCs) are becoming the next-generation tool to study disease and discover new drugs.  Patient-specific somatic cells are reprogrammed to a stem-like state, and then differentiated in culture into the cell type most likely to manifest features of a particular disease.  High-throughput drug screens can be designed to find new compounds that can correct the disease defect in the culture dish.  Using this technology, we extracted somatic cells from the urine of patients with autistic syndrome disorder (ASD).  In addition to their cognitive deficiencies, these young patients suffer from chronic and debilitating constipation, which profoundly impacts their quality of life.  This lack of gut motility is suspected to be caused by a defect in enteric neuron function.  Our hypothesis is that the same synaptic malfunction causing cognitive deficits in ASD children also causes the enteric neuron defect.  Preliminary data also suggests that neural crest cells - an intermediate cell type in the enteric neuron lineage - can be generated using a combination of growth factors.  Therefore, I am conducting growth factor dose-response experiments on iPSCs to optimize neural crest cell differentiation.  I will phenotype the resultant cells via immunocytochemistry and qPCR analysis.  If successful, this project will lead to the discovery of new compounds able to relieve ASD patients of their constipation and further validate iPSCs as a tool for disease modeling and drug discovery.

Gene therapy for monogenic diseases is undergoing an exciting resurgence in the last few years. Patients with hereditary blindness, blood cancers and hemophilia have been cured with minimal to non-existent long-term side effects. Our lab has been studying X-linked myotubular myopathy—a rare disease of young boys that causes profound weakness in all skeletal muscles including the diaphragm. An adeno-associated viral (AAV) vector gene therapy has been proposed to treat the X-linked muscle degenerative disease. However, the viral dose required is in excess of 10^13 viral particles per kilogram. The goal of this proposed project is to find ways to manipulate muscle cells to increase virus uptake and enhance activity of the muscle protein expression. Artificially induced mechanical stretch and contraction have been shown to stimulate intracellular signaling and gene expression. Thus, we hypothesize that preconditioning muscle cells by in vitro stretching will increase both virus uptake and gene expression. This study aims to test the influence of mechanical stretch on muscles cells treated with the AAV mediated gene therapy by inducing stretching motions with the FlexCell machine before vector treatment. The FlexCell produces cellular stretching through regulated fluctuations in air pressure. Skeletal myocytes were cultured on a clathrin coated bioflex plate and underwent cycles of equibiaxial cyclical stretch for a set time period. Cell cultures were then treated with the proposed myotubular myopathy AAV vector therapy. Transgene expression was compared using immunofluorescence between cultures that underwent mechanical stretch and cultures that did not to determine the effects of stretch in virus uptake and transgene expression. Increases in both will enable future attempts of gene therapy in animals or humans to use lower doses, which should be accompanied with lower toxicity and fewer and less severe adverse side effects.

Currently, kinases are thought to function primarily in signal transduction cascades by phosphorylating target proteins. Based on emerging evidence, a novel role for kinases is promising: direct kinase binding of DNA and chromatin. To analyze the role of kinases in the nucleus and investigate whether direct kinase binding of DNA or chromatin takes place, I will generate genome-wide histone phosphorylation and kinase localization maps for multiple kinases in human embryonic stem cells. We first verified the presence of specific kinases in the nucleus of H1 human embryonic stem cells using mass spectroscopy. To visualize protein presence in the nucleus, I stained whole cells and whole nuclei using immunofluorescence. I then studied specific kinase-chromatin interactions and histone phosphorylation sites using ChIP-seq (chromatin immunoprecipitation coupled with high-throughput sequencing). Sequencing data are expected to show genomic locations where direct kinase binding of DNA and chromatin occurs. We predict we will see kinases present at promoter and enhancer elements, as phosphorylation of proteins in the complexes that bind these elements or the surrounding nucleosomes may be important for gene regulation.

Street signs, traffic lights, and maps are the predominant navigational cues in society, each being inherently visual. When navigating through pedestrian areas that only provide these cues, people with visual impairments can experience disability. This study proposes the design of a drone for navigation, or NavDrone, a pocket-size, pilotless, and autonomous aircraft that will provide visual description of navigational cues. By creating access to visual information in the environment, the proposed NavDrone can assist people with visual impairments in navigating unfamiliar areas. The NavDrone can be used in place of or in addition to common navigation aids, such as guide dogs and white canes. We conducted a study using Value-Sensitive Design methodology, a technique that incorporates the analysis of human values throughout the design process. The purpose of this study was to investigate how the NavDrone can support the human values of autonomy, access, and safety by providing GPS navigation, auditory feedback and control, visual descriptions, and fine-grained obstacle detection. We conducted a stakeholder analysis and considered the values at stake for users and bystanders of a NavDrone in a public place. We conducted a survey with visually impaired stakeholders to gain an understanding of the value tensions that might arise when relying on a NavDrone to navigate in an unfamiliar public place. In our survey, we prompted stakeholders to reflect on a value scenario and to describe the types of information and strategies that they would use to navigate in an unfamiliar environment. We analyzed the data to find themes and underlying values that informed the technical specifications for the design of a NavDrone. Finally, we reflected on how the values of autonomy, access, and safety are implicated in the design of a NavDrone and how they might be implicated in other assistive drones for people who experience disability.

This project investigates how to create a website system that will store service information, strengthen usability, and improve the website’s function to improve community service needs. One important aspect of creating a helpful website is reinforcing the database management, web development, and information analysis techniques. The organization, Chinese Baptist Church (CBC), hopes to improve their user-friendly website. So the CBC has decided to redesign their website. I investigated community members’ user experiences to improve the old version of the website. I reviewed the written code and analyzed the user feedback for future improvements, utilizing previous skills involving HTML, JavaScript (Ajax, jQuery, etc.), PHP, and CSS programming languages. This includes driving down the bug count before deploying the final product to CBC’s operations and teaching their team members how to use the website and its database efficiently. Currently, I have conducted research and constructed a suitable platform for the new CBC website and database. Our group is investigating the design of the website’s structure. Our goal is to improve the user experience and to make an interesting and significant website serve the local community.

Roux-en-Y gastric bypass (RYGB) surgery, which promotes major, sustained weight loss, is the most commonly performed bariatric operation in the United States. A ramification of RYGB is the remission of type 2 diabetes mellitus (T2DM) in patients. With 25.8 million people in the U.S. suffering from diabetes and 90-95% of adult diabetes being T2DM, we hope to understand the underlying mechanisms of RYGB on glycemic improvement in T2DM patients. The two leading hypotheses for the impact of RYGB on T2DM resolution are the lower intestinal hypothesis and the upper intestinal hypothesis. The lower intestinal hypothesis postulates that increase in nutrient delivery to the distal intestine stimulates glucagon-like peptide-1 and increased insulin secretion. The upper intestinal hypothesis postulates that elimination of contact between the ingested nutrients and the proximal small intestine exerts direct anti-diabetic effects. To test the two hypotheses, we use a porcine model and perform various bariatric gastrointestinal (GI) operations to assess the effects of each operation on glucose homeostasis. Ossabaw pigs are used for their similarities in gastrointestinal anatomy to humans. We feed them a high-fat diet to induce obesity and insulin-resistance resembling those seen in T2DM patients. The four GI operations - Gastrojejunostomy (GJ), GJ with Duodenal Exclusion, Roux-en-Y Gastric Bypass (RYGB), and RYGB with Vagotomy - rearrange various parts of the stomach and small intestine. To assess the effects of surgery on glucose homeostasis, we perform two types of blood tests: intravenous glucose tolerance test and meal tolerance test. The monitoring of the post-operative changes in insulin and gut peptide hormone levels provides insight into the mechanism of T2DM remission. Although more data is needed to support either hypothesis, the results of this study will help identify new targets for surgical and medical interventions for T2DM patients.

 Embryonic stem cells can be used to study development and diseases. Mouse embryonic stem cells (mESC) are used as a model in developmental research and display several pluripotent properties not seen in the current human embryonic stem cell (hESC) lines. A newly derived line of naïve hESCs is thought to recapitulate an earlier stage of development, and resemble mESCs. To understand how the naïve hESCs differ from typical hESCs we are investigating DNA methylation, a form of epigenetic regulation, on a global scale at nucleotide resolution. The underlying genomes of cells are highly similar, yet their epigenomes change with cellular differentiation and development. Epigenetic modifications are important factors controlling gene regulatory elements. To analyze whole genome DNA methylation of the naïve hESCs, I am using MethPipe, a computational tool, to determine how the naïve hESC line differs from a later stage hESC line, and if the naïve line is more similar to mESCs at the epigenetic level. I will compare global methylation levels, hypo/hyper-methylated regions, and partially methylated domains. I will determine which regulatory elements, such as promoters, enhancers and CpG islands, show similarities and differences. We also expect to determine differential non-CG methylation (a characteristic of ESC). This characterization of naïve hESCs will provide a foundation for studying the earliest stages of human development.