Wildfires are known for their destructive capacity towards ecosystems and devastating impacts to human life and property. Regions in the western United States are particularly prone to such events, including large crown fires. However, there is limited research on the potential for smoke from these wildfires to carry and redistribute nutrients in the form of aerosols. Here I show that higher levels of airborne nutrients, specifically iron, phosphorus, and potassium, can be associated both with the timing of active wildfires and a known smoke tracer, elemental carbon. Data from the Interagency Monitoring of Protected Visual Environments (IMPROVE) show a strong correlation with fire presence and local peaks in levels of potassium, with less strong associations with phosphorus and iron. Across a multi-year period, there is also a correlation between the number of acres burned in a particular year and the average concentration of iron and potassium in that region. Using data from the NASA DC-8 airborne laboratory corroborates this information, with calculated potassium concentrations matching those on the ground. These findings indicate the wildfire plumes have a potential to be significant sources of nutrients in the short term. This is especially relevant for areas impacted by wildfires, as these nutrients are key for plant growth and development. These elements may also be carried into the ocean and affect the aquatic biosphere. All of the information gathered will help improve our understanding of the complex networks that make up Earth’s biogeochemical cycles.

Newly developed, rational syntheses of the phosphaethynolate anion have taken it from an academic curiosity to a useful synthetic reagent. Since the new state-of-the-art synthetic method was published by Grützmacher et al. in 2014, many studies have emerged investigating the phosphaethynolate anion’s possibilities as a synthetic building block, cycloaddition reagent, and P atom transfer agent. I have performed very preliminary work (cut short by COVID-19) on using this anion to generate phosphide thin film materials. Metal phosphide thin films have many applications in advanced electronics and thus are a valuable manufacturing target. The solution-processed, electrochemical method proposed would have marked advantages over current methods to generate phosphide thin films such as MOCVD and MBE. If realized, this unique method of producing metal phosphide thin films would be entirely unprecedented.

Embodiments of additive manufacturing that utilize hydrogels as building materials have recently received much attention for their ability to construct biological systems in vitro. These embodiments, often referred to as 3D bioprinters, build up layers of cell-laden hydrogel by rasterizing two dimensional patterns of material deposited in a layer-by-layer fashion; the most common mechanisms of pattern deposition include extrusion, where a shear-thinning gel is forced through a thin nozzle, and light-induced polymerization, where a laser polymerizes material from a vat of liquid hydrogel precursor solution. These methods of material deposition work well for hydrogels which are designed and optimized for their respective deposition method, however, many unique and useful designer hydrogels cannot be printed using conventional 3D bioprinters. Herein, we describe a novel method for layer-by-layer fabrication of hydrogel structures using open microfluidic patterning. For each layer of a printed hydrogel structure, a hydrophilic track or “rail” is manually placed parallel to and several hundred micrometers above the previously patterned layer. Hydrogel precursor solution is then introduced between the underlying layer and the rail, and flows along the rail via capillary action. The cross-sectional geometry of the rail constrains fluid flow to the space directly under the rail, meaning that the pattern of each layer of hydrogel is defined by the pattern of the rail corresponding to that layer. We show that this methodology can be used to fabricate relatively large (1 cm^3) structures of agarose gel, as well as cell laden structures of collagen and a novel peptide-based synthetic hydrogel. Finally, we show that the patterning rail can constrain fluid flow via differential surface chemistry, opening up the possibility for an automated 3D printer and advancing the commercial viability of the method.

Native mass spectrometry (MS) experiments provide direct mass measurements of intact proteins and protein complexes. Protein samples for native MS are prepared in solutions that mimic physiological conditions, which maintain a protein’s native folded state before entering the gas phase of the mass spectrometer. Ammonium acetate solution is typically used due to its volatility and relevant ionic strength. However, protein purification protocols typically require inorganic salts and detergents to maintain protein stability. Native MS experiments can be hindered or made uninterpretable by those salts and detergents. Furthermore, the presence of protein modifications or multiple proteins can make native mass spectra difficult to interpret. Anion exchange chromatography (AEX) is well suited for the requirements of native MS, as it can simultaneously desalt, remove non-ionic detergents, and separate proteins or proteoforms directly into an ammonium acetate solution. This project seeks to develop a comprehensive method for desalting, removing non-ionic detergents, and separating proteins through an ammonium acetate-based anion exchange chromatography method. Preliminary experiments in egg whites, a complex matrix with a high sodium concentration, showed separation and four distinct proteins using an AEX pH gradient from pH 10 100 mM ammonium acetate to pH 4 100 mM ammonium acetate. Native MS analysis showed low interference from sodium or other contaminants and the various modified forms of those proteins were identified. Refinement of this preparation technique can result in the improvement and efficiency of native MS analysis of proteins.

Cells use a variety of mechanisms to regulate gene expression. One way cells control gene expression is by forming structures such as DNA loops to position genes in 3D space near regulators. My lab is developing tools to synthetically control 3D genome structure and assess the relationship between positioning and expression. Our strategy is to engineer DNA loops by fusing DNA binding domains to targeting domains that dimerize and bring the two sites together. To ensure the binding domains bind to each other to form a loop, we are developing an allosteric sensor of DNA binding where the dimerization motifs are only active when bound to DNA. Without this switch, the looping interaction would be outcompeted by free binding domains that are not attached to DNA. We used LOCKR, a bioactive protein switch comprised of a protein cage that switches to an ON-state in the presence of a key protein. These proteins are tethered to DNA using dCas9 as a programmable binding domain. I have tested different system parameters, including the length of the linker between dCas9 and the key protein, for increased activation of green fluorescent protein, a reporter gene that is expressed when the switch is activated. My data show that the switch is effective with a variety of linker lengths. I am also exploring other parameters for optimization, such as changing the relative orientation of the dCas9 complexes. Exploring these parameters is important because the switch might be sensitive to small changes in structure or orientation, and we want to identify the optimal arrangement of dCas9 and switch proteins for effective switch function. After this system is optimized, we will be able to direct our efforts toward looping DNA, which will allow us to address broad questions about the relationship between gene position and expression.

Quantum confined nanomaterials have become an important field of study with many applications from color displays to low-energy alternative lighting sources. Discovered in the early 1980s, these semiconducting nanocrystals continue to draw attention; their unique properties differ from their bulk counterpart’s due to a quantum confinement effect rising from their small nanometer-scale size. Indium phosphide (InP), a group III-V semiconductor, is a promising nontoxic, environmentally innocuous material. My research is currently focused on investigating the implications of destabilizing kinetic InP intermediates. I explore how creating small nuclei with the same zinc-blende structure as quantum dots (QDs) can potentially lead to unique shelling applications to increase photoluminescence (PL) and color purity. I have developed a synthesis employing a destabilizing additive to yield the small nuclei, which I then shell with zinc chalcogenides to produce luminescent core-shell QDs. I characterize all material via UV-vis and emission spectroscopy as well as powder X-ray diffraction. My goal is to optimize the shelling procedure to produce QDs suitable for use in the market, particularly as blue emitters with a narrow emission range and high PL quantum yield. In optimizing this process, I can contribute meaningfully to the nanocrystal field and the display industry by presenting a unique strategy for making small materials through careful control over crystal phases.

The effectiveness of different catalysts during biofuel production was investigated to determine if a heterogeneous catalyst, eggshells, could perform as well as a traditional homogenous catalyst, Sodium Hydroxide. Eggshells add up to a significant amount of waste each year. If they were repurposed, that would not only get rid of that waste factor but also serve as an alternative to fossil fuels. The biodiesel was synthesized from sunflower oil. The amount of each catalyst was fixed at 2.25 grams and the yield of each fuel was documented. Fifty milliliters of the fuels produced were individually tested to measure their efficiency. The fuels were tested in a pop-can calorimeter. The pre-calculated combustion of ethanol in said calorimeter was used as a baseline for calculating relative efficiency. From there, we were able to determine the richness of the fuels produced by the two catalysts. The biodiesel catalyzed by Sodium Hydroxide produced 38.17 kilojoules per gram and the one catalyzed by Waste Egg shells produced 34.81 kilojoules per gram.

Wastewater treatment has been a major issue in undeveloped and developing countries due to the lack of access to water filtration systems to treat wastewater. Without clean water resources, it has affected agriculture, water storage, and daily activities. According to Global Affairs Canada, eighty percent of illnesses in developing countries are linked to a lack of proper wastewater treatment. In this project, an eco-friendly and lower cost of chemical, ferrate, has been generated and analyzed to combat global wastewater treatment.To generate more stable and user friendly ferrate. The precursors of ferrate are synthesized in solutions. These solutions are freeze dried to form stable precursors in solid form. These precursors can be mixed to give an easy route to make ferrate on demand. More importantly, they can be added directly to wastewater to generate ferrate for disinfection. In the second part of this project we have come up with an analytical technique to quantify the concentration of ferrate in solutions using UV instrument.The ferrate solution can be easily transferred to developing countries to use in wastewater treatment without generating harmful chemicals to humans or the environment. In future research, the research will focus on the mass production of ferrate as well as creating a more stable form.

In recent decades, there has been massive growth in consumer demand for products containing live bacterial cultures, or "probiotics", driving a $75B market. Yet, even as market share has expanded, the relative effectiveness of different probiotic products is still not fully understood. Such products require further scientific substantiation before manufacturers can claim health benefits. Few studies have been conducted on how wide-ranging and adverse conditions in the gastro-intestinal tract can influence ingested "pro-biotic" culture function and viability. This research attempts to close this knowledge gap, providing a formal method of characterizing bacterial function under various gut conditions through the identification of biomarkers that are indicative of healthy “probiotic” cultures. L. Bulgaricus, L. Acidophilus and S. thermophilus cultures were evaluated after exposure to conditions simulating major components of the gastro-intestinal tract, their protein expression analyzed and correlated with growth. Simulated colonic conditions maximized bacterial growth, while simulated gastric conditions minimized it. The validity of the experimental model was thus reinforced, as it accurately reflected previous in vivo analysis of bacterial growth in different components of the GI tract. By linking growth and protein expression, the gene, oppa1, was identified as a possible biomarker of cell growth. This gene, activated in conditions that conferred sub-standard growth relative to a positive control, seems to present a key to understanding bacterial population health. This research presents a step forward in the evaluation of the quality of various “probiotic” products by understanding the influence of the human digestive system on live cultures.

Separation of proteins using metal ligand complexes is a well-established practice in the field of bioengineering and biochemistry.  electrospray ionization mass spectrometry (ESI-MS) can be used to identify bio and organic molecules. Previous studies have detected chelated metal ions using ESI-MS, this project focuses on the identification of a metal ligand complex comprised of a tridentate chelating agent Iminodiacetic acid (IDA) and a bidentate ligand, Histidine. By coordinating copper with IDA a binary complex is formed, this allows for the detection of copper by proxy of the IDA. By selecting a unique fragment related to IDA we can target in tandem mass spectrometry (MS/MS) for greater sensitivity; the complex can be selected for analysis out of solution. We will use this novel approach to build a parent-ion scanning technique to monitor metal-ligand complexes extracted from environmental matrices. first the ideal solution parameters are determined to maximize the complex formation and detection of the Cu-IDA complex. So far, a high ratio of copper to IDA coupled with a basic buffer have yielded the best data. Creating a ternary complex comprised of copper IDA and an imidazole ring containing compound, histidine. Selectively tuning to the peaks associated with the copper IDA complex, the ternary complexes can be selected for in depth analysis of its structure and bonding properties. Future work could focus on identification of metal ligand complexes from soil samples with other compounds containing imidazole rings such as the neonicotinoid imidacloprid which has been indicated in bee colony collapse. By chelating solid with IDA complex formed from pentacoordinate copper ions could be detected despite the low relative concentration.

Exhibiting high photoluminescence quantum yield, tunable surface functionalization and well-defined emission linewidths, colloidal semiconductor nanostructures are promising materials for a wide range of applications from lignocellulosic depolymerization to low power nonlinear optics. In past decades, synthesis of zero-dimensional (quantum dots) and one-dimensional (nanorods) systems has attracted much interest. In contrast, the potential of two-dimensional (nanoplatelets) and three-dimensional (nanotetrapods) structures remains to be tapped. As a consequence of their anisotropic morphology, the tunable emission frequency and linewidth of nanoplatelets and nanotetrapods renders them excellent candidates for single-photon emitters in devices such as hybrid photonic integrated circuits. Integrated photonics are devices that utilize light-matter coupling with embedded light-emitting media to achieve state-of-art engineering processes like ultralow threshold lasing and quantum many-body simulations. However, it remains a challenge to find suitable photoluminescent agents with outstanding desirable features. The subject of this research presents a solution to this problem—nanotetrapods and nanoplatelets used for coupling to the nanocavities in the integrated circuits. Owing to their tunable surface chemistry, nanotetrapods and nanoplatelets have the potential to be optimized for efficient processing with photonic cavities, with clear pathways for deterministic positioning. Moreover, the structures’ adjustable sizes and dimensions allow for maximization of single-photon behavior, including high quantum yield and narrow emission linewidth. Herein, I report the seeded-synthesis of several II-VI and III-V nanotetrapods and two approaches to growing nanoplatelets via a solution-phase decomposition procedure and the colloidal atomic layer deposition pathway. To understand both the spectral and structural properties of the nanostructures, I characterize the products by UV-vis and fluorescence spectroscopy as well as transmission electron microscopy. By exploring different routes to synthesizing the highly absorbing and emissive anisotropic colloidal nanostructures and investigating strategies for modifying the materials’ surface chemistry, it will be possible to achieve specialized spectral functionalities with these nanostructures.

Though renewed efforts in tuberculosis (TB) research have facilitated massive strides in treating Mycobacterium tuberculosis (Mtb), TB remains a global health problem with an estimated 10 million infections and 1.5 million deaths in 2018. The ability of the pathogen to sequester itself inside a granuloma, a mass of immune cells whose precise mechanism of regulation is unknown, prevents the simple study of Mtb pathogenesis and subsequent treatment discovery. Current in vivo models have been established to study TB infection using animal models or tissues, limiting biological relevance of human disease while current in vitro models lack components of the complex lung microenvironment during infection. We present the creation of a novel microscale infection model, which uses open and suspended microfluidic principles to enable spatial and temporal manipulation of cultures in suspended hydrogel plugs. Utilizing the ‘stacking’ feature of the device, we demonstrate the ability of a model granuloma consisting of M.bovis BCG (Mycobacterium bovis bacille Calmette-Guérin) and monocyte-derived macrophages to interact with a model vasculature layer consisting of endothelial cells. Analysis of soluble factors for proinflammatory cytokines and characterization of infection-dependent angiogenesis in the vasculature layer are used to verify crosstalk between cultures. In the future, we envision this model expanding to contain multiple immune cell types and to incorporate additional aspects of the lung anatomy to approach a more accurate pathophysiological model as a tool for other researchers’ studies.

There are an estimated 300 million people worldwide who are affected by asthma, a respiratory condition in which a person has inflammation and swelling in the airways. Asthma patients also experience increased vasodilation in their lungs, i.e. the widening of blood vessels, which causes increased blood flow and results in increased inflammation. The goal of this project is to create a device that makes free standing hydrogel rings, modelling the structure of blood vessels, offering a simple approach to better understand asthma and potential treatments. The device used to form the rings is 3D printed and can be printed in a range of sizes. The rings are composed of collagen I that has been seeded with smooth muscle cells. Once the hydrogel rings are cast, they can be transferred to a 96 well plate and be free standing of any rigid structures. The ability to be free standing allows us to measure the ring diameter and wall thickness, as well as measure any change in size when a vasodilator is added. Future steps to be taken with this project include optimizing the size for biological relevance, introduce endothelial cells to create multiple layers of cells that are involved in signaling for vasodilation, and increase the responsiveness that the rings have to constriction factors as well as dilators.

Probiotics are living organisms that when ingested have been linked with health benefits to the gut and improvement of conditions such as irritable bowel syndrome (IBS). The gut contains a plethora of microorganism populations that make up the microbiota. To understand how these populations communicate with each other, and the tissues surrounding them, it is imperative to identify and characterize the method of communication. Extracellular vesicles are one such possible method. Extracellular vesicles (EVs) are lipid-bilayer delineated sacks of material secreted from cells. It has been established that EVs are used as a waste disposal system. However, new research revealed that EVs can be used by the cell for methods of communication. Furthermore, EVs are now being linked to cell-to-cell and cell-to-organism communication. If EVs have been linked to communication, then characterizing them is one step closer to understanding how probiotic bacteria function. Previous studies have mainly characterized EVs by their size and divided them into 3 main groups: exosomes (40-150 nm), microvesicles (100-1000 nm), and apoptic bodies (>2000 nm). However, an analysis of proteins found in these EVs has not been performed yet. Here we compare EV proteins to proteins in the cell, to determine which protein fractions are secreted by cells through vesiculation for signaling purposes. To separate the cellular fraction from the EVs fraction, cell suspensions were centrifuged. First, the cells were pelleted and collected at 300x g. The leftover supernatant was spun at 16,000x g to pellet the EVs. Then, proteins from cells and EVs were solubilized and digested with trypsin. The tryptic peptides will be analyzed using liquid chromatography-mass spectrometry. A comparison of proteins in cells and EVs, and their relative concentrations can help us learn more about how probiotic EVs function in the gut.

Scaffold proteins, which assemble enzymes and their substrates into multiprotein complexes, are critical for many cellular functions. By bringing enzymes and their substrates into close proximity, scaffold proteins are thought to enhance the rates of enzymatic reactions. For instance, Axin is a scaffold protein in the Wnt pathway that binds the transcription factor, ß-catenin and the kinase, GSK3ß. This tethering method is predicted to enhance the rate of ß-catenin phosphorylation. An outstanding question is whether a scaffold protein that binds to both GSK3ß and ß-catenin is sufficient to observe these effects or whether Axin has other structural properties that are not currently understood. Although there is no detailed structural information about Axin, it is predicted to be disordered. To address this gap, we have engineered a synthetic scaffold protein that can bind both GSK3ß and ß-catenin and contains a linker that is also presumed to be disordered. Previous work in the lab has reconstituted the reaction between Axin, ß-catenin and GSK3ß in vitro and found that Axin enhances the rate of ß-catenin phosphorylation. Using enzyme kinetics, I have compared the rate of ß-catenin phosphorylation of the engineered scaffold to that of Axin. Identifying the quantitative kinetic comparisons between the engineered scaffold protein with the natural scaffold protein could tell us more about what aspects of a tethering system make it effective at enhancing the rates of reactions. We are expecting to see similar tethering effects between the two scaffold proteins, as their linkers are both disordered and equivalent in length. 

Probiotics are found in numerous fermented foods such as yogurt, sauerkraut, and kombucha. Research shows that eating live probiotics are beneficial for the gut because it helps supplement the plethora of native bacteria. Now, these microorganisms are gaining popularity throughout the world—people are ingesting them in the form of food, drink, and even pills. Most probiotics are anaerobic. In fact, excess oxygen can damage organelles, create ionic imbalance and eventually even kill the cells. However, in the process of manufacturing these fermented foods, the probiotics in them often get exposed to oxygen, for example, due to leaks in packaging. Even the FDA does not have a rule about standard manufacturing processes regarding anaerobic conditions and yogurt. This could mean that the probiotics people eat are highly compromised: damaged or dead. I hypothesize that probiotics exposed to aerobic environments for extended periods of time will express more DNA repair enzymes such as DNA pol. 1 and 2, p53 or photolyase. This is because oxygen will cause probiotic cells to change function, and therefore, they will start to express enzymes, such as these DNA repair enzymes, to protect them from oxidative damage. In order to test this, I left probiotics out to oxidize for various amounts of time—0 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, and 1 hour. After oxidizing, I cultured the cells for 16 hours. I then pelleted the cells by centrifugation at 300 x g and washed and lysed them. Then, I tryptically digested cell proteins and analyzed them by LCMS (Liquid Chromatography Mass Spectrometry) for identification. In the future, these proteins can provide insights to how these oxidized probiotics impact the gut. Are they really the miracle microorganisms that benefit the gut, or could their compromised condition end up being harmful?

Growing sustainable crops on Mars is an important aspect of developing a colony on the red planet. The goal of this research is to modify Martian regolith simulant to support plant growth. Results will be presented for the readjustment of the pH of Mars soil (pH 8) simulant to match that of typical fertile earth soil (pH 6) using nitric and phosphoric acid. The acids used were chosen based on their viability for transport to Mars and their ability to add crucial nutrients for plant growth in the unfertile soil. During the project, both acids effectively lowered the regolith pH, but in the hours and days following the pH increased significantly, which has motivated testing the buffer capacity of Mars soil simulant. The data collected was used to prepare three samples of Mars soil simulant; the first was modified with phosphoric acid, the second with nitric acid, and the third was also modified with nitric acid and had a buffer of dihydrogen phosphate added. The growth of kale was measured in the three modified soils, each mixed with equal parts potting soil. Kale growth was compared to trials performed without the acidification or buffering of Mars simulant soil.  Our research presents progress towards growing food in Mars regolith to sustain colonization efforts on the planet. This work can also be applied to the potential need to grow food in adverse conditions on Earth as the human population increases and the impacts of climate change advance.

Harnessing the power of enzymes to carry out synthetically relevant reactions is rapidly emerging as a powerful tool for sustainable chemistry. Iron-dependent enzymes have been of particular interest to the field because of their ability to facilitate complex reactions with broad substrate scope. Recently, we found that Fe(II) 2-oxoglutarate dependent hydroxylases (Fe(II)/2OGs) exhibit non-native activity towards either epoxidation or allylic hydroxylation. Oxyfunctionalizations are important in chemical synthesis as they allow for diversification to a range of more complex molecular scaffolds from simple olefinic precursors. Because of this, reactions that produce hydroxides or epoxides are of high interest, especially in an asymmetric fashion. However, current methods are not broadly applicable, as they are limited in substrate scope, selectivity, and tolerance towards other functional groups. Enzymes could rival traditional methods, offering greater selectivity and substrate scope while using inexpensive and Earth-abundant reagents. Here, we explore the ability for Fe(II)/2OGs to catalyze non-native asymmetric epoxidations or allylic hydroxylations. First, we will determine whether the reaction taking place is an epoxidation or allylic hydroxylation. Second, we will identify any additional Fe(II)/2OGs capable of oxyfunctionalization beyond our initial hit. Third, we will use directed enzyme evolution to improve oxyfunctionalization activity and enantioselectivity of a selected enzyme candidate. Fourth, we hope to expand substrate scope of an evolved Fe(II)/2OG to develop a general “epoxidase” or hydroxylase capable of reacting with a variety of functionalized olefinic small molecules in an asymmetric fashion. Generally, our lab seeks to use Fe(II)/2OGs mutagenesis libraries and a developed liquid chromatography-mass spectrometry screening platform to exploit the existing wide catalytic diversity of Fe/2OGs into a useful array of synthetically relevant transformations.

With the aim of better understanding developmental disorders, researchers have expended great energy to elucidate the mechanisms that regulate the development of the peripheral nervous system (PNS). Larval Drosophila melanogaster presents a highly useful model organism for studying PNS development due to the larva’s rapid development and neurological parallels to mammals. Thus, to investigate the development of pain-sensing neurons in the PNS, we studied nociceptive class IV dendrite arborization (C4da) neurons in larval D. melanogaster. Randomly inducing mutations with the mutagen ethyl methanesulfonate ultimately identified a miRNA, miR-14, that when absent perturbs C4da neuron development. Confocal microscopy revealed that miR-14 knockout results in greater detachment of neurons from the extracellular matrix (ECM) than those in wildtype specimens. Furthermore, imaging showed that miR-14 knockout leads to far greater ensheathment of c4da dendrites in the epithelial cell-cell junctions than observed in wildtype specimens. These observations suggest that miR-14 plays a critical role in directing proper C4da neuron development. Additionally, relative to wildtype specimens, miR-14 mutants demonstrated significantly increased responsiveness to numerous stimuli including noxious touch, noxious chemicals, and blue light but not noxious heat. These findings indicate that miR-14 influences pain sensitivity in a modality-specific manner. Finally, analysis of miR-14 mutant transcriptomes by RNA-seq demonstrated decreased expression of innexin and integrin—proteins associated with epithelial cell-cell junction formation and ECM adhesion, respectively. These results suggest that miR-14 regulates proteins that enable proper attachment of C4da neurons to the ECM and prevent abnormal C4da ensheathment in epithelial cell-cell junctions. Taken together, these data support the hypothesis that miR-14 regulates sensitivity to pain by controlling C4da neuron-epithelial interactions.