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.

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.

Traumatic spinal cord and brain injuries may lead to a devastating loss of neurological function. After the traumatic primary insult, a secondary injury phase ensues, which greatly increases the extent of the injury. Inflammation is a dominant component of secondary injury, which includes the immune response in which free radicals and proinflammatory cytokines are released that induce the death of surrounding neurons. Dexamethasone, a commonly used corticosteroid, has been shown to produce a neuroprotective effect by inhibiting inflammation and reducing cytokine release. However, the clinical application of large systemic doses of steroids is limited by side effects, such as sepsis and pneumonia. For this reason, a localized microneedle delivery system may be favored to allow for a therapeutic dose at the injury site, with reduced side effects. The goal of this work is to design a local, controlled drug release system, in which the drug could be given for a sustained duration at a therapeutic dose at the site of injury. Using a mixture of polyvinylpyrrolidone (PVP) and poly (ethylene glycol) diacrylate (PEG-DA), a biodegradable microneedle array can be formed that can encapsulate dexamethasone that then slowly release into the tissue over 72 hours. With a 3D printed system, various sizes can be quickly developed and produced to create customizable arrays for various target locations. Using a 2-photon polymerization laser, Nanoscribe, arrays can be formed on a scale of <300 μm as well as larger arrays using traditional printers. The needles are developed using a three-stage molding method, casting customized 3D printed arrays in a silicon elastomer, PDMS, and polymerizing our monomer-drug solution to the desired shape. Current results indicate sustained release over the target window and are currently branching into trans-membrane in-vitro studies.

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.

A wide range of diseases and disabilities are associated with mitochondrial dysfunction including diabetes, cancer, Alzheimer’s disease, cardiovascular disease, and Parkinson’s disease. Mitochondrial dysfunction triggers activation of a novel mitochondrial retrograde response pathway in Caenorhabditis elegans termed the PMK-3 Retrograde Response, which upon activation leads to a reduction in mitochondrial stress and extension of lifespan. A mitogen-activated protein kinase (MAPK) cascade comprised of DLK-1, SEK-3, and PMK-3 forms the signaling core of the PMK-3 Retrograde Response. In this presentation, I will outline my attempts to develop photo-regulatable PMK-3 and DLK-1 kinases which are tools that can aid in the identification of other proteins involved in this pathway. These photo-regulatable kinases were made by engineering a recombinant form of the fluorescent protein Dronpa into two discrete sites of DLK-1 and PMK-3 to confer photosensitivity of the kinases. Photoregulation of the kinase occurs through dimerization of Dronpa in violet light (inactive kinase) and dissociation in cyan light (active kinase). After determining the identities of phosphorylated proteins purified from nematodes exposed to cyan and violet light using mass spectroscopy, a comparative analysis between the two datasets will suggest which proteins are involved in the PMK-3 Retrograde Response. Further investigation into these proteins could elucidate the role of these proteins in mitigating mitochondrial stress.

Mutations in Complex I (NADH:Ubiquinone Oxidoreductase) of the mitochondrial electron transport chain have been reported in up to 30% of pediatric mitochondrial diseases (MDs) and affect 1 in 5000 live births. One of such pathologies, Leigh Syndrome, is often fatal in the first three years of life and has no known cure. Knock-out (KO) of Ndufs4 in mice recapitulates several aspects of the disease, including lethargy, encephalopathy and retarded growth. Acarbose delays the onset of neurological symptoms and prolongs the lifespan of both Ndufs4−/− mice and heterogeneous wild type mice. Acarbose is a type 2 diabetes drug that inhibits alpha glucosidases, resulting in delayed absorption of complex carbohydrates and increased activity of the intestinal flora, including increased levels of short-chain fatty acids (SCFAs) and other fermentation products. No formal study has been conducted to determine whether increased SCFA concentration mediates the effects of acarbose on longevity and MD suppression. Our goal is to elucidate the mechanistic basis of acarbose. We hypothesize that acarbose delays MD progression in Nduf4-/- mice by increasing circulating levels of SCFAs. Thus, SCFAs supplementation should recapitulate the effects of acarbose. We will feed chow containing tributyrin to control and mutant mice from weaning until humane endpoint. We will measure body weight, observe the incidence of forelimb clasping behavior (a widely tested neurological symptom), and record percent survival daily. We expect to observe a delay in clasping behavior and prolonged survival in acarbose-treated KO mice compared to that of untreated KO mice, and that these effects are recapitulated more pronouncedly with higher tributyrin doses. SCFAs are promising therapeutics because they are natural metabolites that are inexpensive and can be manufactured into easy consumable pills. A successful outcome of this study will help progress therapies for patients with MDs and for anti-aging purposes.

Mitochondrial diseases are frequently characterized by mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like symptoms. Leading causes of mitochondrial diseases are mutations affecting the assembly of complex-I, which is located in the electron transport chain and is partially responsible for energy production during cellular respiration. Such mutations reduce the ability of complex-I to couple electron transfer to proton pumping, ATP production, and efficient energy production. The ND2 (mitochondrially encoded NADH dehydrogenase 2) gene encodes the production of the ND2 protein, a subunit of complex-I. In Drosophila melanogaster, the ND2^del1 mutation is associated with signs of progressive neurodegeneration and paralysis, similar to the effects of mutations in human ND2. These behaviors are known as ‘bang sensitivity’, characterized by sudden paralysis after Drosophila are vigorously shaken in a test-tube (bang assay). The objective of our research was to understand how nuclear genetic variation present in a population can exacerbate or ameliorate the effects of the ND2 gene mutation. To model genetic variation in a large population, we used the Drosophila Genetic Reference Panel (DGRP). These consist of 200 fully sequenced, inbred lines derived from a genetically diverse wild population. We crossed males from 20 DGRP lines to ND2 females, collected their progeny at 21 days of age, and subjected the progeny to a bang assay. Our results demonstrated significant variation across differing genotypes (Kruskal Wallis: χ2 = 266.85, df = 21, P < 2.2e-16). These results point to significant epistatic interactions between nuclear and mitochondrial alleles for a mitochondrially encoded mutation associated with neurodegeneration. Previous studies have demonstrated links between neurodegenerative disease susceptibility, variable NAD+ concentrations, and complex-I dysfunction. Our unpublished research has identified correlations between the Drosophila metabolome and age-related disease phenotypes. As such, future research will test the hypothesis that variation among genotypes is associated with variable NAD and other metabolite profiles.