Bismuth nanoparticle (Bi NPs) have wide-ranging applications in both biomedical imaging and nanotechnology. These applications are hampered by the absence of a sized-controlled, uniform synthesis using conventional laboratory techniques. The current synthesis of such particles is arduous and requires specialized techniques and equipment, specifically air-free techniques, which are not readily available to all. By using air stable and cost effective reactants, we hope to develop and characterize a synthesis that is reliable, aerobic, free of lithium based organic bases, and potentially offers size control between 30 nm and 70 nm. This synthesis would produce high quality Bi NPs that are available to a wider range of researchers.

The Coalition for Reform of Undergraduate STEM (science, technology, engineering & math) Education and other prominent agencies have called for increased integration of STEM content, particularly in the first two years of a student’s post-secondary education. Interdisciplinary learning in the humanities has been shown to foster a more complete understanding of core concepts and provide a method for incorporating complex thinking and heightened cognitive demands into daily classroom activities. Despite the compelling evidence for this approach, interdisciplinary curriculum is still rare in introductory STEM courses. Even more rare are studies that focus on identifying the impact interdisciplinary thinking activities have on student learning, student motivation and retention in STEM disciplines. In this study, a recently designed RISE (Research-based Interdisciplinary Science Education) curriculum was implemented in the general chemistry series at North Seattle College and student learning was investigated through a multi-modal approach. Student drawings and video of student working groups were analyzed and coded as a means to capture the thinking that occurs during an interdisciplinary investigation. Exam questions were collected from a traditional classroom and from an interdisciplinary classroom and assessed for understanding of hydrogen bonding (a threshold concept). Preliminary analysis of student pre- and post- challenge drawings indicates that there is an increasing trend in the ability to demonstrate integration of disciplines, in the overall complexity of the thinking that occurs, and in creativity. In addition, students in the interdisciplinary course scored higher on the hydrogen bonding exam question than the students in the traditional course. The results of this study demonstrate that students benefit from an interdisciplinary approach to learning chemistry and provide a pathway forward as more educators seek to transform how we teach introductory STEM courses.

Algae have been shown to be a promising renewable source of biofuel due to the ease of large scale growth, significant lipid content (30% by dry weight) and the low impact on global food supply. Current studies have provided foundational information to aid producing and extracting oil from a variety of algae species, yet there remains a need to establish species specific and local protocols. Here, an investigation focused on developing Pacific Northwest algal growing conditions and combining this with an optimal oil extraction technique is presented. Chlorella vulgaris, Botrycoccus braunii, and Nitzschia sp. were used as model organisms to establish growing, culturing and oil extraction parameters. Crucial for efficient extraction of the lipid content in algae cell walls was the use of silver nanoparticles (AgNP), which help break down cell walls and generally increase oil extraction yields. Based on preliminary experiments, it is anticipated that ideal conditions for growing algae will be around 5% moisture content and 55°C incubation temperature. Oil extraction yields were highest when AgNP were utilized, and with a 6:1 solvent-to-solid ratio (1% diethyl-ether and 10% methylene chloride in n-hexane solvent). This study, in combination with other similar investigations, will contribute to the establishment of ideal growing conditions for algae biofuel production and ultimately provide a best practice for producing biofuel from algae in the Pacific Northwest.

Urban watersheds are uniquely vulnerable to toxic runoff. Rain carries carcinogenic polycyclic aromatic hydrocarbons (PAH) from motor oil into stormwater control measures where they accumulate in the sediment and biosphere. Mushrooms have recently proven to be effective in remediating oil-contaminated sites by processes similar to extracellular digestion, whereby enzymes degrade large recalcitrant compounds into smaller, more bioavailable compounds. Identifying fungal species indigenous to the Pacific Northwest for use in stormwater mycoremediation systems is a critical first step in creating an ecologically responsible, reproducible installation. In this study, thirteen fungi species collected from local, contaminated sites were cultured on agar containing used motor oil to assess for PAH tolerance. Oil amount (% volume) was increased at each stage to select for higher motor-oil tolerance. To quantify PAH degradation of a given fungi species, an agar plate contaminated with oil was inoculated with mycelium, grown for fourteen days and analyzed by GC-MS to determine PAH concentrations. By comparing the original PAH concentrations in the motor oil to the PAHs present in the myceliated agar, each species’ relative effectiveness at reducing large PAHs was determined. Initial results show that ligninolytic fungi such as Hypsizygus ulmarius, Trametes versicolor, and Pleurotis pulmonarius are the most successful at growing in increasing concentrations of oil. Using this method of concurrent fungal selection and testing, the mycoremediation potential of previously unexamined species (of which there are hundreds) can now be easily assessed and characterized. Identifying fungi effective at breaking down PAHs provides a natural and economical means of bioremediation. This research will contribute toward providing organisms and systems capable of bioremediation on larger scales.

Traditional antibiotics are known to wipe out many species of bacteria, but often leave several resistant microbes to dominate. These resistant bacteria are a huge concern for human health. Medical researchers are on the hunt for new approaches to combat bacterial infections on a large scale. It has recently been discovered that bacteria communicate through Quorum Sensing (QS), which if inhibited, presents a promising strategy to control these pathogenic bacteria. Instead of eliminating the weakest strain of bacteria resulting in a proliferation of antibiotic resistance, inhibition of quorum sensing silences gene expression, virulence and pathogenicity. A variety of marine organisms have demonstrated quorum sensing inhibition (QSI) potential, however there are many marine organisms that have yet to be screened for QSI compounds. In this study, three species of marine algae native to the Pacific Northwest were investigated for quorum sensing inhibition (QSI) capabilities. Ulva Lactuca, Chondracanthus exasperatus, and Iridaea Cordata were subjected to QSI capability testing that involved use of the indicator strain Chromobacterium violaceum Bergonzini (ATC 12472). The indicator bacterium uses the QS signal N-acyl-homoserine lactone (AHL) to produce a purple pigment. Each species of algae was overlaid with agar inoculated with Chromobacterium violaceum and incubated overnight. Absence of pigment in the indicator strain suggested the presence of a QSI compound. Preliminary evidence indicated that two of the three species of algae tested demonstrated QSI activity. The next step of this research involves identification of the specific compounds involved in QSI, thereby contributing to the expanding catalogue of known QSI molecules from marine environments. Ultimately this work aims to make a contribution to the fight against antibiotic resistant pathogens.

Tissue engineering has provided medical researchers with the ability to grow transplantable tissues, simple organs, and complex organ tissues. Despite the tremendous progress in tissue engineering, there is an ongoing emphasis on optimizing cell growth parameters. A lack of sufficient blood flow, vascularization, and the need for oxygen delivery prior to angiogenesis are central problems that need to be overcome when growing cells in vitro. Hyperbaric oxygen (HBO) treatment utilizes elevated pressure and elevated oxygen concentrations. Multiple studies have demonstrated that HBO contributes to increased proliferation and differentiation of cell growth, stimulation of growth factors, bacterial defense, and improved oxygen delivery to cells. In this study, Saccharomyces cerevisiae was used as a model organism for eukaryotic cell growth to gain a better understanding of the effects hyperbaric oxygen has on simple cellular metabolism. This was accomplished with the reduction of a resazurin-based reagent to track cellular metabolism. The samples were evaluated with a spectroscope and absorbance was used to calculate the amount of resazurin reduced by NADH, proving an indicator for metabolic activity. Preliminary evidence indicates that hyperbaric oxygen stimulus increases rate of metabolism. Future experiments will provide optimal pressures and oxygen concentrations for HBO growth protocols. This study indicates that HBO bioreactors provide a useful tool for controlling growth in cell cultures, and provides early evidence for the potential use of HBO for tissue engineering applications. This study will further the applicability of HBO as a tool for enhanced tissue engineering methods.

Advancements in the design of robotic prostheses have provided promising new prototypes as well as highlighted the need for continued development in myographic sensors essential for fine control of prosthetic limbs and other assistive devices. Force myography (FMG) sensor matrices have the potential to improve upon the granularity of motion control present in current non-invasive robotic prostheses. FMG utilizes pressure sensors to detect flexion and extension of muscle fibers, requires no complex signal processing, and has the potential for improved accuracy and robustness over other myographic interfaces, which have known issues of data clarity. In this study, FMG was investigated through sensor matrix design and data collection. A matrix of force sensors was constructed for use in human-computer interaction, using the open-source prototyping platform Arduino for data collection and mechanism control. The sensors were constructed from pressure-sensing material (Velostat) layered between conductive foil, arrayed in a grid, and housed in an adjustable sleeve. Data collection was focused on identifying force patterns which correspond to the eight classes of motion critical to a robust transradial prosthesis. Preliminary prototyping and data analysis indicate that simple, widely-available materials provide sufficient pressure sensitivity for prosthetic applications when compared to more common, prohibitively expensive sensors. The matrix returns pressure data regardless of radial position on a user’s arm, such that pattern recognition algorithms will likely be sufficient to translate user intent into action. This approach will provide a framework for further development of low-cost prosthetic control alternatives, of particular interest for developing countries with limited funds for healthcare.

Alkanes are the major constituents of natural gas and petroleum. The development of catalysts for the efficient and cost-effective catalytic conversion of alkanes to functionalized organics is highly desirable. Previously, Nishiyama and coworkers found that the Ir^III complex (^dmPhebox)Ir(OAc)₂(OH₂) (Phebox = 3,5-dimethylphenyl-2,6-bis(oxazolinyl)) could activate the C-H bonds of n-octane at high temperatures under nitrogen. Goldberg and coworkers found upon increasing the temperature, β-H elimination could be promoted, giving isomers of n-octene. Reactions of similar Ir complexes with alcohols, ketones and other organic substrates will be presented.

Stimuli-responsive hydrogels represent a promising class of inks for use in 3D-bioprinting. These materials respond to external stimuli such as heat, light, pH, and pressure, and researchers have utilized these different stimuli responses to develop inks for direct-write 3D printing. The objective of our work is to understand how the composition of poly(alkyl glycidyl ethers)--more specifically, allyl-, ethyl-, isopropyl, n-propyl-, and methyl-glycidyl ether homopolymers--affects their thermal response in aqueous solutions. UV-Vis Spectroscopy was used to determine the lower critical solution temperature (LCST) of each homopolymer, varying the molecular weight, concentration and polymer composition to demonstrate poly(alkyl glycidyl ethers) versatility as a platform for synthesis of stimuli-responsive hydrogels. We determined that the identity of the alkyl group significantly affected the temperature-response of the respective hydrogel. These studies will guide our work toward developing temperature and shear-responsive inks for 3D-bioprinting. In particular, these materials can be used to develop printed hydrogel lattices for whole-cell catalysis to produce medicinal compounds.

Each year bee specialists report detection of larger numbers of bee colonies suffering from colony collapse disorder. One of the suggested causes is the increasing use of neonicotinoid pesticides in farming agriculture. To study the effects these pesticides have on the environment, it is necessary to establish a method to quantify the amount of imidacloprid in an environmental sample. Previously, we found that off-line C-18 cartridges were suitable for enriching imidacloprid from complex matrices and for preparing samples for analysis in GC-MS (gas chromatography-mass spectrometry). Our previous work primarily focused on achieving a reliable calibration curve by enhancing signal-to-noise in the GC-MS. To further evaluate our findings, a spiked environmental sample was subjected to C-18 extraction and analyzed. This method resulted in a limit of detection as low as 160 micromolar, which is similar to the 120 to 320 micromolar range for limits of detection achieved by other research groups using more complex extractions. This is important to the future study of imidacloprid because it allows us to progress towards the analysis of environmentally relevant concentrations of imidacloprid in soil, plant and honey samples.

Since the 20th century, the concentration of atmospheric carbon dioxide has increased dramatically, inducing several environmental issues. Our research seeks to reduce this excess carbon dioxide in our atmosphere by using a chemical method to capture and utilize carbon dioxide. In our research, we have developed a bimetallic complex coordinated by a multidentate carbene-phosphine ligand. We hypothesize that the carbon dioxide molecule will be cooperatively reduced by the two metal centers. Using nuclear magnetic resonance and crystallography techniques we have been able to analyze our complex's molecular structure. So far, we have concluded that this complex successfully reduces a molecule with a shape and stability similar to that of carbon dioxide. Moving forward in our research, this bimetallic complex should behave similarly with the bonds of carbon dioxide and thus represents a potential method of reducing the amounts of carbon dioxide in our atmosphere. 

Cells can be thought of as aqueous solutions of proteins enclosed within a lipid membrane. A surprising and exciting discovery in the past decade is that several proteins inside of cells aggregate to form separate liquid droplets. This discovery has potential medical applications because the types of proteins that undergo aggregation are associated with prion diseases. In the lab, researchers use a specific type of alcohol (1,6-hexanediol) to disrupt droplets in protein solutions. However, it is unknown whether hexanediol disrupts lipid membranes at the same concentration that it is used to disrupt liquid droplets. If so, then hexanediol cannot be used in cell systems. By probing the effects of 1,6 hexanediol on model lipid membrane physical properties, this study will determine the suitability of 1,6 hexanediol for in vivo studies of liquid droplets.

Ion Mobility Mass Spectrometry (IMMS) has become an important tool for separating small molecules such as peptides, glycans, and petroleum molecules. Ion Mobility is most often performed in He, N₂ or air. There is a great interest in using alternative drift gases to increase selectivity of ion mobility separations, specifically for complex mixtures like petroleum. In this study we measured arrival time distributions (ATD) of a set of double bond equivalents (DBE) in four different gases (He, N_2, CO₂, Ar) which span a range of masses and polarizabilities. Quinoline, isoquinoline, and their DBE were chosen because they are aromatic compounds that are easily protonated due to the presence of nitrogen and are similar to molecules found in petroleum. The measured ATDs were plotted as a function of reciprocal drift voltage; the resulting slope is inversely proportional to mobility. Using the mobility and the Mason-Schamp equation, we determined the collision cross section of each molecule with each drift gas. We also investigated the relationship between the dipole moment of an ion and its collision cross section. In general, collision cross sections increase with drift gas size and polarizability. Additionally some of the DBE were better separated with larger, more polarizable drift gases (N₂ and CO₂). We have shown that IMMS can be used with different gases to improve separation between ions of similar size and shape. This method can be used in future investigations of petroleum mixtures to potentially identify a wider range of molecules.

The addition of cholesterol to pure phospholipid bilayers changes the structural properties of the composite membrane. A well-known example of this change is the condensing effect: the average thickness of the bilayer increases, and the average area per molecule decreases, upon addition of cholesterol. We study the spatial extent and variation of these structural changes. We use all-atom Molecular Dynamics computer simulations of mixed cholesterol/dipalmitoylphosphatidylcholine (DPPC) bilayers to quantify lipid structure in a spatially resolved manner. We use order parameters such as area per lipid, tail ordering, and head group tilt angle to measure the ordering potency of a single cholesterol molecule. One challenge when working on a molecular scale is to define a unique area for each lipid in the bilayer, which we have achieved using the Voronoi tesselations of the leaflets. We show that significant changes in phospholipid structure can be observed within the first two solvation shells of the sterol molecule. Our findings provide a simple and direct way to test the controversial Umbrella Model, describing phospholipid-cholesterol interactions and the structure of the bilayer, to improve and expand the theory.

High-resolution structures of membrane proteins are necessary for guiding the rational design of novel therapeutics. Membrane proteins make up 30% of our proteome and roughly 60% of all drug targets, yet they only represent ~4% of the structures in the Protein Data Bank. This is because membrane proteins have proven to be extremely difficult to crystallize for traditional X-ray crystallographic studies. Recent advances in single-particle electron cryo-microscopy (CryoEM) technology are beginning to revolutionize the field of membrane protein structural biology by providing near-atomic resolution structural information without the need for protein crystallization. Despite these advances, CryoEM is limited in its ability to characterize small membrane proteins (less than ~200 kDa in size) and dynamic protein-protein interactions. Several important classes of membrane proteins are “too small for CryoEM”, such as the G-Protein Coupled Receptors and the Major Facilitator Superfamily transporters. Moreover, our ability to capture membrane proteins in complex with cytosolic signaling proteins is commonly limited by their inherently short-lived low-affinity interactions. Our research group aims to overcome these hurdles to characterize the regulatory complex formed between the ~100 kDA small membrane water channel, Aquaporin-0 (AQP0) and the calcium signaling protein, Calmodulin (CaM). We intend to enhance the small size of the AQP0 water channel by applying Nanodisc technology, which offers the added benefit of a near native lipid bilayer environment to study the membrane protein. While the system requires further optimization, a preliminary Nanodisc reconstitution has an enhanced mass of ~250 kDa, which is sufficient for high-resolution CryoEM analysis. We are also exploring the use of engineered avidity as a novel approach to enhance the native multi-valent interaction between CaM and AQP0 tetramers. We believe these strategies represent general approaches that may be applied to thousands of other small membrane proteins and transient protein complexes to facilitate their structural characterization. "

 Two in three American adults are considered overweight or obese and research done in the last decade has indicated possible treatment options through the introduction of probiotics to an individual's diet. While there is research indicating the efficacy of probiotic use in the management of obesity, there is little available regarding the specifics of how that process works. Previous studies have shown that Elongation Factor-Thermounstable (EF-Tu) mediates the binding of Lactobacillus to mucins and epithelial cells in the intestine, but not the stomach. Our goal was to evaluate if (and how) EF-Tu production would change as Lactobacillus moved from the low-pH environment in the stomach to the more neutral intestine. We hypothesized that the rate of external EF-Tu expression would increase at more neutral pHs to facilitate gut colonization. To quantify levels of EF-Tu in different parts of the cell, we measured bacterial growth and EF-Tu levels in cultures of pH 3, 6, and 7.4 representing the averages at each stage in the GI tract. This was done by culturing bacteria in an incubator at each pH for six hours and separating supernatant and cell fractions by centrifugation. Membrane and cytosol were separated using freeze-thaw lysis in liquid nitrogen. SDS-PAGE was then used to isolate EF-Tu, followed by in-gel tryptic digestion. LCMS (a method of molecule separation and identification) was used to detect and quantify EF-Tu from each fraction. Initial results from growth showed the highest turbidity (cloudiness) from the pH 3 culture. Turbidity began before inoculation, indicating turbidity at this pH is not a sufficient measure of cell growth. Understanding how Lactobacilli produce and deploy EF-Tu in different pH regimes may help to optimize the use of probiotics in controlling obesity and improving digestive health.

In many parts of the world, the lack of fresh water is the single largest factor limiting sustainable development. Water scarcity contributes to poverty and poor public health outcomes, and it can exacerbate ethnic and international conflicts in arid regions. This study aims to determine whether forward osmosis (FO) is a viable method for desalination of seawater. FO is an emerging technology for desalination, in which osmotic pressure is used as a driving force for separation. By using a concentrated solution of high osmotic pressure, called the draw solution, water is induced to flow from saline water across the membrane, rejecting the salt. To obtain potable water, the diluted draw solution is recovered and then recycled. Two chemicals – ammonium bicarbonate (NH₄HCO₃) and magnesium chloride (MgCl₂) – were compared in order to determine which one results in a more effective FO process, in terms of flow and chemical composition of the produced water. The results showed that the FO process is viable in terms of water flow and salt rejection. The effectiveness of the FO process does not depend on the type of draw solution, but instead depends on concentration, volume ratio of feed and draw solutions, and type of mixing. More experiments are required in order to develop the optimal mathematical relationship between the concentration of draw solution, and volume ratio of feed solution to draw solution. Potable water that complies with EPA standards, was obtained only from recovery of MgCl₂ diluted draw solution. Methods of NH₄HCO₃ removal described in the FO scientific literature did not result in production of potable water. Further analysis is required to identify possible techniques for NH₄HCO₃ removal from draw solution.

Neonicotinoid pesticides have been identified as a possible contributor to colony collapse disorder (CCD), decimating honeybee populations. Development of methods to extract neonicotinoids from environmental matrices are important in exploring the correlation between pesticide exposure and CCD. Imidacloprid was chosen as representative neonicotinoid given it is the most commonly used agricultural pesticide in its class. Two extraction methods, QuEChERS and SPE C18, were evaluated for their effectiveness in quantifying imidacloprid concentrations in honey and water. The effectiveness of each extraction method was evaluated using gas chromatography-mass spectrometry (GC-MS). QuEChERS kits use a buffered extraction technique to absorb excess water and stabilize the pH of the solution and cleanup with weak, anionic and cationic exchange resins to remove complex sugars/macro organic molecules. The SPE C18 extraction method used a non-polar solid phase to isolate Imidacloprid from other undesirable solutes in the solution. Preliminary experiments have indicated the SPE C18 extraction method appeared to have better performance with water based solutions. However, honey solutions were ineffective due to more organic interference. Additionally, QuEChERS extraction appears to be more efficient for isolating analyte, but did not concentrate as efficiently compared to SPE C18 extraction. We hypothesize that QuEChERS and SPE C18 extraction methods will be more effective in tandem, as QuEChERS will remove organic interference, while SPE C18 extraction will concentrate the samples. The impact of pH adjustment of samples on peak area and shape was also investigated in both polar and non-polar GC-MS columns. The results obtained will help identify best practices for extraction and detection of imidacloprid in honey samples.

Probiotics are living microorganisms that, when administered in adequate amounts, confer health benefits on the host. Many studies indicate that there are several ways in which probiotics, which include lactic acid bacteria (LAB), have health benefits. These bacteria survive in a wide range of temperatures and pHs, from storage to digestion. In this study, I investigated the effect of varying pH and temperature on LAB growth in a model stomach with MRS broth, one of the most common growth mediums used by microbiologists to grow LAB. I isolated LAB from Yogourmet® yogurt starter, which contains L. Bulgarius, S. Thermophilus, and L. Acidophilus, and grew the bacteria in MRS broth at temperatures of 4°C, 22°C, and 37°C to imitate conditions under which various probiotic products are stored. I also grew LAB at pHs of 2, 4, 6.2, and 8 to replicate variations of pH that come with digestion. I measured bacterial growth at optical density (OD) 600 after 24 hours for all trials. I found that optimal LAB growth at the standard pH of the MRS broth (6.2 ± 0.2) and at the standard temperature of the human stomach (37°C) was expected and confirmed through several trials. When conditions deviated from standard, there was always less LAB growth. I concluded that variations in pH and temperature from standard stomach conditions result in smaller amounts of probiotic growth. This finding is important because a smaller amount of bacterial growth in the stomach will result in reduced health benefits on the host.

The need for energy efficient chemical reactions is growing in response to the world’s energy crisis. Reactions must not only be high yielding but also require low energy input. Many redox processes of interest involve the transfer of electrons from the surfaces of electrodes and need an electrical input as driving force. The ability to promote redox reactions photochemically would greatly reduce the energy input needed. As of yet, interfacing electrochemical reactions with colloidal photosensitizers in order to promote them photochemically has not been done. This project experiments with CdSe quantum dots as the colloidal photosensitizer by absorbing visible light from an LED light source and transferring an electron in order to reduce ferrocenium to ferrocene as a model reaction. The goal of this project is display the ability of CdSe in the productive electron transfer promoted by visible light to ferrocenium. Knowledge of this model system can be applied to more complex reactions in the future and the work done here will help understand how to drive industrial chemical reactions via sunlight. 

Photovoltaic devices based on polycrystalline films of hybrid organic-inorganic lead perovskite semiconductors have demonstrated impressive power conversion efficiencies of over 22% while still retaining the advantage of low-cost solution processability. However, a fundamental understanding of many of the photophysical and charge transport properties of these materials must be further developed. In this project, we synthesize a series of n-type doped single crystals of organic-inorganic methylammonium lead bromide (CH₃NH₃Pb_1-xBi_xBr₃) and all-inorganic cesium lead bromide (CsPb_1-xBi_xBr₃) by varying the amounts of Bi3+ cation and use optical spectroscopy and x-ray diffraction to characterize their optical and structural properties. In collaboration with the Chu group in the UW Department of Physics, we use these crystals to compare the charge transport properties of organic-inorganic to all-inorganic perovskite as a function of temperature and doping concentration, in order to understand the mechanisms that determine the charge carrier mobility in these materials. Insight gained from studies on these crystals can be applied to improve device fabrication procedures, ultimately improving perovskite device performance and stability and thereby hastening the introduction of efficient perovskite-based materials into the emerging solar energy market.

Bacteria are widely used in metabolic engineering because they can synthesize chemical products from cheap, renewable carbon sources. A major current problem, however, is that toxic intermediates can build up when bacteria express heterologous biosynthetic pathways. In principle, we can control metabolic gene expression levels so that downstream enzymes quickly consume toxic intermediates. To achieve this goal, we use a programmable gene regulatory tool called the CRISPR-Cas system. To optimize gene activation activity of CRISPR-Cas in bacteria, we sought to identify modular transcriptional activation domains that could be recruited to specific genes to activate transcription. This strategy has been previously used successfully in eukaryotic cells, but with only limited success in bacteria. We used CRISPR-Cas to recruit a number of potential synthetic transcriptional activators to specific loci in E. coli, and identified candidates that can activate a fluorescent reporter gene. We also used protein-engineering approaches to optimize the gene activation activity of our CRISPR-Cas system. The transcription factor screening and protein engineering we did gave an 80-100-fold increase in activation compared to basal transcription levels. To make this new tool useful for practical metabolic engineering, we plan to use this system to dynamically respond to metabolic stresses. This tool could potentially be used to optimize biosynthesis of useful polymer precursors such as lactate and p-aminostyrene. Production of these types of cheap, biosynthetic precursors will help us move towards a greener industrial future.

The obligate human pathogen, Mycobacterium tuberculosis (Mtb), is the primary causative agent of the infectious disease tuberculosis (TB) and ranks as one of the world’s deadliest pathogens; infecting about 1.5 billion people worldwide. Mtb evades the host immune response via intracellular persistence in alveolar macrophages, where it faces chemical stresses such as a drop in pH, various hydrolytic enzymes, increased toxic ion concentration, and highly reactive oxygen (ROI) and nitrogen intermediates (RNI). Modification of mycobacterial proteins by the prokaryotic ubiquitin-like protein (Pup) and their subsequent proteasomal degradation plays a critical role in Mtb survival in this harsh environment. Thus enzymes associated with protein pupylation may be valuable targets in anti-tubercular therapy. This knowledge is exploited in a rational approach to drug design wherein known structural information about the sole Pup ligase, PafA, is used to develop specifically targeted inhibitors. These inhibitors take the form of stabilized short α-helical peptides, which mimic the hydrophobic and electrostatic interactions in Pup essential for binding to PafA. Production of these compounds was achieved using both automated and manual solid-phase peptide synthesis, as well as 'Click' chemistry to modify cyclized variants. Quantitation of inhibitor performance was accomplished by densitometric analysis of SDS-PAGE results from in vitro pupylation assays with purified proteins. Here I report my work on the successful design, synthesis, and testing of helical peptides which display significant activity as PafA inhibitors.

The incorporation of trivalent ytterbium ion (Yb³⁺) impurities, or dopants, into nanoscale materials is of significant interest due to the unique optical properties of the Yb³⁺ ion. The electron configuration of Yb³⁺, 4f¹³, is associated with just one f-f electronic transition (²F_5/2↔²F_7/2) that is narrow and lattice-independent with an energy of ~1.27 eV. This makes luminescent ytterbium doped colloidal semiconductor nanocrystals, which are solution processable, ideal for use in silicon-based solar energy applications like luminescent solar concentrators. Doping colloidal nanocrystals with ytterbium ions, however, is not necessarily sufficient for creating a material with luminescent ytterbium. It is necessary for ytterbium to be sensitized by energy transfer from a visible light absorber in order to be emissive. Previous synthetic work indicates that the most promising route for sensitizing ytterbium is the direct doping of ytterbium ions into visibly absorbing host nanocrystals such as chalcogenide semiconductors. Incorporating ytterbium ions into chalcogenide semiconductor hosts, however, poses a significant synthetic challenge. Additionally, it is essential to optimize synthetic reaction conditions such as growth temperature, growth time, coordinating solvent used, precursor concentration in coordinating solvent, and ytterbium dopant concentration in order to produce a material with luminescent ytterbium. These synthetic variations are informed by physical characterization of the morphology, absorbance, and photoluminescence of previously synthesized ytterbium doped colloidal nanocrystal samples by transmission electron microscopy, absorbance spectroscopy, and photoluminescence spectroscopy, respectively. It is expected that current efforts to optimize these synthetic parameters and interface ytterbium doped colloidal nanocrystals with compatible ionic materials in a process known as ‘shelling’ will allow the reproducible creation of a novel nanoscale material with bright ytterbium luminescence at room temperature. Upcoming fundamental studies of these ytterbium doped colloidal nanocrystals will probe the dynamics of energy transfer by time-resolved photoluminescence spectroscopy and provide insight into the kinetics of ytterbium sensitization.

The purpose of this research project is to demonstrate the feasibility of incorporating a new photocatalytic Diels-Alder reaction into the curriculum of undergraduate organic chemistry laboratories at University of Washington. Although the traditional Diels-Alder reaction is thermally allowed and does not require UV light catalysis, it fails to succeed when both the dienophile and diene are electron rich due to electrostatic repulsion between the reagents. Recently, an alternative photocatalytic Diels-Alder reaction was reported in the chemical literature involving a ruthenium bipyrazine catalyst, Ru(bpz)₃X, where X denotes the counter anion. This new photocatalytic method is advantageous because it allows the Diel-Alder reaction to work when both the diene and the dienophile are electron rich. Consequently, we believe that the new photocatalytic Diels-Alder reaction would be an attractive addition to our undergraduate laboratory curriculum by increasing the versatility of Diels Alder reagents to accommodate both electron rich substrates. Because the ruthenium catalyst is not commercially available, it must be synthesized from chloropyrazine. Two equivalents of chloropyrazine were coupled together under heated conditions and a Pd(Ph₃)₄ catalyst to yield a solution mixture containing bipyrazine, which was purified via silica flash chromatography. Subsequently, bipyrazine was reacted with RuCl₃xH₂O in ethylene glycol at 170°C to afford a solution mixture containing Ru(bpz)₃Cl₂. This chloride salt was reacted with NaBArF to precipitate the desired catalyst, Ru(bpz)₃X, where X represents the anion BArF-. Finally, the catalyst was purified through alumina flash chromatography. We intend to test the efficacy of this new photocatalytic method using the electron rich substrates trans anethole (dienophile) and isoprene (diene) in the upcoming months. We expect that the two photocatalysts we have synthesized will catalyze the cycloaddition reaction between isoprene and trans anethole with endo stereoselectivity.

The goal of this project is to analyze and compare the carbonyl (C=O) stretch of ethylene carbonate before and after deuteration. This molecule is commonly used as a primary component of electrolytes in batteries, and exhibits a strong C=O stretching mode. The C=O mode is a sensitive reporter to an electric field through a process known as the Stark effect. A shift in peak position is associated with a quadratic Stark effect, while a widening of the lineshape is representative of a linear Stark effect. Many times, a combination of the linear and quadratic Stark effect manifest in analysis of the lineshape. Unfortunately, the absorption peak of C=O stretching is split into a doublet due to the presence of a fermi resonant mode, causing a significant misrepresentation of the C=O lineshape. This would result in a poor estimate of an electric field associated with a linear and/or quadratic Stark effect. Substituting the hydrogen atoms on ethylene carbonate for deuterium results in a reduction of the previously mentioned fermi resonant effect, enabling a better interpretation of C=O lineshape for the analysis of Stark effects. Recently, we have observed a shift in the C=O peak position of carbonate-based electrolytes at the interface silicon nanoparticle-based electrodes. It is our hypothesis that this change in vibrational peak position is due to the previously mentioned Stark effect, a measurable electric field at the nanoparticle interface. This is significant because one could correlate the magnitude of this hypothetical interfacial electric field with the performance of a battery containing Si nanoparticles. To better estimate the linear and quadratic Stark effect contributing to the magnitude of this interfacial electric field in future studies, we have characterized the lineshape of deuterated vs. non-deuterated ethylene carbonate, as well as attempted to synthesize the material in both forms using inorganic catalysts.

Currently, only photo- or thermochromic filaments exist. We are the first group to synthesize a professional grade mechanochromic filament for extrusion 3D printing. The mechanochromic unit used in this poly(ε-caprolactone) (PCL) polymer-based filament is spiropyran. By shining UV light, or stretching the filament, the spiropyran activates and undergoes a reversible isomerization from the yellow-colored closed spiropyran form to its purple-colored open merocyanine counterpart. This filament could have a huge range of uses. For example, it could be used in safety and sports equipment where high impact, potentially concussive forces would result in activation of the polymer, and notify the wearer to seek medical attention. It could also be used as a coating that allows earthquake assessment of steel beams, as well as a diagnostic tool in orthotics and wearable prosthetics to determine if they need to be exchanged. We made this filament by sending our precipitated polymer to Functionalized Incorporated, who integrated it into a blend with more PCL to create over 25 kilograms of filament. We are now testing the filament on our extrusion 3D printers. Future work will focus on blending the spiropyran-PCL polymer with a softer, more elastomeric filament such as Ninjaflex to achieve lower strain activation that is also reversible, allowing for repeated use of a single printed object. Once we have our first samples, we will analyze the mechanical properties of the blends, their changes in strain of activation, and the potential for multiple cycles of activation. We intend to commercialize this filament, and make the material available to other research groups to study the material mechanics and mechanochemical properties. This is the only mechanochromic filament in existence, and its potential uses within these customizable print materials are numerous and important.

One of the research goals of the Stoll lab is to characterize how the behavior of tryptophan radicals is influenced by their nano-environment. Tryptophan is an aromatic amino acid that facilitates electron transport during reduction or oxidation reactions within a protein. The ability for a protein to fine-tune tryptophan’s reduction potential by modulating its nano-environment is a crucial aspect of the function of enzymes such as cryptochromes, which are involved in regulation of circadian rhythm, and photolyases, which are involved in DNA repair. In order to study the relationship between protein environment and tryptophan oxidation, we have designed and synthesized model peptides to engineer specific environments. The peptides used in this study fold into small cages surrounding a tryptophan residue. Mutations to the peptide change the local environment around tryptophan from charged to hydrophobic with varying levels of solvent exposure. The reduction potential of the tryptophan in these peptides was then determined using differential pulse voltammetry experiments. Each peptide was tested over a pH range from 1-7.5 in order to determine how the pH dependence of tryptophan's reduction potential responds to changes in its nano-environment. We have identified some initial trends such as the tendency for more cationic environments to increase the potential at which tryptophan oxidation occurs. Further analysis will allow us to develop a model which will be able to directly predict tryptophan oxidation behavior as a function of the pKas of surrounding residues and a weighting metric based on solvent exposure. We hope to extend this model to be able to describe the tryptophan redox behavior in full proteins.

MicroRNAs (miRNA) are non-coding RNA (ncRNA) which post-transcriptionally regulate gene expression. In cancer, many miRNAs are overexpressed leading to the down-regulation of tumor suppressing genes. While the general processing steps of miRNA are known, the specific reason certain miRNAs are overexpressed in cancer remains unclear. Our particular miRNA of interest is miR-21, which inhibits genes encoding tumor suppressing proteins, and upregulation is associated with cancer. Specifically, we are interested in understanding how the pre-miRNA-21 processing step is regulated to generate functionally mature miRNA, and what proteins are involved in this process. Many RNA binding proteins are involved in miRNA biogenesis and influence transcription levels. This project explores the possible role of the RNA binding protein NOVA. NOVA is a neuron-specific RNA-binding protein exclusively expressed in the brain and neuronal tissues except for patients with Paraneoplastic opsoclonus-myoclonus ataxia, where it is found site of malignancy. NOVA is composed of 3 K-homology RNA binding domains selective for the sequence 5’ -YCAY-3’ (Y = pyrimidine). This KH domain recognition sequence is present in the hairpin loop in pre-miR-21. Additionally, in-vitro DICER processing of pre-miR21 is very slow relative to other miRNAs, however, if NOVA interacts with the pre-miR-21, this could stimulate its processing. We are trying to determine a possible mechanism of NOVA stimulated miR-21 expression through binding a conserved YCAY sequence. MiR-21 and NOVA have been isolated and purified, and their binding affinities are being evaluated using electro mobility shift assays. If binding is indicated, gene knockdowns will be preformed in cells to assess how NOVA affects miR-21 levels. It is difficult to design microRNA specific inhibitors, however, finding inhibitors for proteins is possible. If NOVA is found to lead to miR-21 up-regulation, a possible cancer treatment could be designed by targeting NOVA to lower levels of miR-21. 

    Optical microscopy is a cornerstone of biological research. It reveals to scientists phenomena that explain many of life’s secrets. Despite its capabilities, traditional optical microscopy techniques are limited to a spatial resolution of ~250 nm due to the diffraction limit of light. Consequently, features smaller than ~250 nm cannot be distinguished. However, the development of super-resolution microscopy techniques such as the Stochastic Optical Reconstruction Microscopy (STORM) now enables resolution up to ~60 nm. An essential criteria of STORM requires that the fluorophores turn on and off (photoswitch) stochastically. Up to date, the fluorophores used in these techniques are commercially available fluorophores with photophysical properties optimized for traditional optical microscopy methods. Since the photoswitching properties of these commercial dyes for use in STORM is not well understood, my project thus involves tuning the electronic properties of the cyanine scaffold by adding various functional groups to the dye in order to improve the photoswitching properties. I am currently working on synthesizing cyanine fluorophores in the red region that have electron-withdrawing groups on the indole molecule and a carboxylate linker attached for bioconjugation. Organic synthetic chemistry techniques will be used in the creation of these dyes. The structure of the dyes will then be characterized by proton NMR and mass spectrometry, while the photophysical properties will be characterized by Ultraviolet-Visual spectroscopy. The dyes will eventually be applied to fixed sample staining and STORM. If successful, these new dyes will improve multi-color STORM and researchers will be able to view cells in greater detail.

Expansion Microscopy (ExM) is a revolutionary fluorescence microscopy technique that enables super-resolution imaging of fixed biological specimens on ordinary microscopes. By chemically incorporating the specimens into a hydrogel polymer matrix, enzymatically digesting the proteins that hold the structures together, and physically expanding the hydrogel complex, this technique yields expansion of fourfold along each x/y/z axis and a spatial resolution of ~70nm. The original ExM approach utilized special tri-functional antibodies containing a DNA-dye-linker complex to attach the fluorescent dyes to the expandable polymer. Previously, our group has developed an improved and simplified ExM protocol, which is compatible with conventional antibodies and fluorescent proteins, by using only commonly available reagents, yielding increased fluorophore retention for cultured cells and tissue specimens. In this project, we extend this new approach of ExM to enable ultrahigh resolution imaging of Drosophila melanogaster tissues which had previously been incompatible with expansion. To do so, we adapted our existing ExM protocol to Drosophila embryos, larval brains, and larval and adult body walls by utilizing an enzyme system that can efficiently digest the tough outer cuticle of the organism. We assessed the uniformity of expansion achieved by our new procedure using correlative pre-expansion and post-expansion imaging and found the distortions to be minimal (<2%). With our new ExM approach, we were able to resolve features of presynaptic active zone structure and study the interactions between the epidermis and dendrites of somatosensory neurons in Drosophila. This work should open up this powerful super-resolution microscopy method to a large community of researchers who utilize Drosophila as an important model system in biology.

Despite their inability to express proteins, long intervening noncoding RNAs (lincRNAs) have demonstrated significant biological function; they regulate both cancer and physiological epidermal development and target chromatin-remodeling genes. Their repertoire of activity is constantly growing with the discovery of each new lincRNA. The lincRNA OIP5-AS1 (Cyrano) is a 4.5kb transcript required for proper embryogenesis in zebrafish, and unlike most lincRNAs, possesses multiple stretches of conservation found in at least 52 vertebrates. While the mechanism for how Cyrano regulates embryogenesis is unknown, it is believed that Cyrano functions primarily through a 26-nucleotide region that is perfectly conserved in every tetrapod; perturbation of this region resulted in a lack of proper development of zebrafish embryos. The secondary structure of the 26-nucleotide conserved region was revealed to contain an RNA binding domain, 5’–CCAU-3’, for RBMX, an RNA binding protein that is highly conserved in mammalian species. I hypothesize that Cyrano interacts with RBMX to aid in the process of embryogenesis, not only in zebrafish, but in other species as well. By using in vitro gel shift assays and in cellulo pull-down experiments, I aim to determine if the lincRNA Cyrano binds to the RBMX protein. Understanding this protein-lincRNA interaction will give more insight to the process of embryogenesis, and how lincRNAs function overall.