Browsing by Author "Chung, Jean, committee member"
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Item Open Access Adsorptive separations of phytocannabinoids and pesticides in the liquid phase(Colorado State University. Libraries, 2022) Cuchiaro, Jamie H., author; Reynolds, Melissa, advisor; Farmer, Delphine, committee member; Chung, Jean, committee member; Reardon, Ken, committee memberTo view the abstract, please see the full text of the document.Item Open Access Bioengineering of cloneable inorganic nanoparticles(Colorado State University. Libraries, 2023) Hendricks, Alexander Ryo, author; Ackerson, Christopher J., advisor; Chung, Jean, committee member; McNally, Andy, committee member; Cohen, Bob, committee memberWhen a defined protein/peptide (or combinations thereof) control and define the synthesis of an inorganic nanoparticle, the result is a cloneable NanoParticle (cNP). This is because the protein sequence/structure/function is encoded in DNA, and therefore the physicochemical properties of the nanoparticle are also encoded in DNA. Thus the cloneable nanoparticle paradigm can be considered as an extension of the central dogma of molecular biology (e.g. DNA -> mRNA -> Protein -> cNP); modifications to the DNA encoding a cNP can modify the resulting properties of the cNP. The DNA encoding a cNP can be recombinantly transferred into any organism. Ideally, this enables recombinant production of cNPs with the same defined physiochemical properties. Such cNPs are of primary interest for applications in biological imaging as clonable contrast agents. The advancement of cNPs for broader and more rigorous applications in imaging (and elsewhere) requires further development through multidisciplinary approaches. Described in This Thesis is a bioengineering approach to improve the cNP platform through development of the enzymes responsible for nanoparticle formation.In the first chapter, background and significance is given to provide rationale behind the cNP platform. Among all the modalities of biological imaging, there is no 'one-size-fits-all' solution. Biological fluorescence microscopy (FM) and electron microscopy (EM) are the preferred methods of choice when imaging at cellular levels. Although the relatively recent advent of fluorescent proteins and super-resolution microscopy have ushered major scientific breakthroughs, FM is resolution-limited: only the cellular components which are labeled by fluorophores can be resolved – everything else in a cell (~99% of components) is imaged with low resolution owing to the diffraction limit of light. Biological EM comparatively can image widefield cells at atomic-level resolution yet lacks an analogous toolset to fluorescent proteins. cNPs are proposed as a multimodal, uniform, and precise means of clonable contrast for biological EM (and other modalities) analogous to fluorescent proteins. In the second chapter, a tellurium reductase is isolated and characterized from screened environmental bacterial cultures collected throughout the Colorado Mineral Belt. A strain of Rhodococcus erythropolis PR4 was found to be highly resistant to a broad range of metal(loid) species at toxic concentrations – notably 4.5 mM TeO32− determined by broth microdilution. Through screening of cell lysate in the presence of metal(loid) substrates, a mycothione reductase was characterized as a Te-specialized enzyme which reduces Te preferentially over Se. This is a surprising finding on the basis of reduction potentials for the two substrates. The standard reduction of potential for the reaction TeO32− + 3 H2O + 4e− ← → Te + 6 OH− is −0.57 V vs Hydrogen. The corresponding reduction of SeO32− is −0.366 V. Thus, SeO32− is the preferred substrate for reduction in the absence of a mechanism for substrate selectivity. We hypothesize that the R. erythropolis mycothione reductase may form the basis of a cloneable tellurium nanoparticle (cTeNP). In the third chapter, metal(loid) reductase substrate specificity is developed through directed evolution of a glutathione reductase-like metalloid reductase (GRLMR). The native substrate of GRLMR is selenodiglutathione (GS-Se-SG), where zerovalent selenium nanoparticles are formed in the presence of NADPH. Error prone polymerase chain reaction was used to create a library of ~100,000 GRLMR variants. The library was expressed in Escherichia coli with 50 mM SeO32− to select a GRLMR variant with 2 mutations. One mutation (a D to E) appears to be silent, whereas the other (L to H) resides within 5Å of the active site. Compared to the GRLMR parent enzyme, the evolved enzyme became less capable of reducing reduced glutathione (GSSG) and GS-Se-SG in favor of SeO32−. The evolved enzyme also gained an ability to reduce SeO42−. We have described this enzyme as a selenium reductase (SeR). This is the first known instance of the substrate specificity profile of a metal(loid) reductase changing as a result of directed evolution. In the fourth chapter, the cNP concept is discussed in greater detail. The cNP synthesis paradigm is loosely defined as a system of ligands, reductants, and inorganic cations – where ligands are peptides, reductants are enzyme-cofactor pairs, and inorganic cations are dietary or supplemented metal(loid) ions. This modular platform is adaptable to a wide variety of metal(loid)/enzyme/peptide systems. The story of the creation of a cloneable Se nanoparticle (cSeNP) is also retraced. Briefly, a bacterial endophyte Pseudomonas moraviensis subsp. Stanleyae was found to be capable of efficient selenium reduction under aerobic conditions. Continued characterization led to the discovery of GRLMR which unraveled the cellular mechanism for reducing SeO32−. The enzyme can endow host cells with selenium resistance through nanoparticle formation when cloned. GRLMR was further modified through the fusion of a selenium nanoparticle-binding peptide which improved overall kinetic rates, nanoparticle retention, and nanoparticle uniformity. In the fifth chapter, preliminary work is described which may enable further development of the next generation of cNPs through reduced enzyme mass/mericity and 'multicolored' nanoparticles. Work is described which investigates the plasticity of GRLMR towards reducing other metals such as bismuth. Fluorescence assisted cell sorting (FACS) was used to determine if the relative quantity of intracellular metal(loid) nanoparticles can be differentiated, which is hypothesized to correlate to relative metal(loid) reductase activity. Whereas selenium content could be discerned between active and inactive GRLMR-expressing bacteria, relative bismuth content has yet to be analogously discerned. On the other hand, work was done towards rationally designing a monomeric GRLMR; there are ongoing efforts to use machine learning to graft the active site of GRLMR into a different monomeric template. Finally, a Muchor racemosus cytochrome b5 reductase (Cb5R) was identified in the literature which may serve as an ideal candidate to develop more minimalistic cSeNPs. Initial work has revealed that the enzyme is particularly resistant to soluble expression, which may hinder its ability to function as a clonable contrast agent. However, ongoing work is being done to 'supercharge' the enzyme to enable more facile expression.Item Open Access Development of paper-based devices for point-of-need, bioanalytical applications(Colorado State University. Libraries, 2020) Noviana, Eka, author; Henry, Charles S., advisor; Reynolds, Melissa M., committee member; Chung, Jean, committee member; Geiss, Brian J., committee memberThe growing demand for reliable analytical tools to perform testing at the point-of-need has necessitated the development of novel sensors that are low cost (USD 1-10), portable, sensitive, selective, easy to use, and rapid (i.e. provide results within minutes or a few hours). Miniaturization of the sensors into microfluidic platforms has become a promising approach to achieve these sensors. However, traditional microfluidics often require relatively expensive and complicated pumping mechanisms that increase the cost and limit the portability of the sensors. From a material perspective, cellulosic paper is an attractive substrate for constructing point-of-need sensors due to its affordability, vast availability, self-pumping ability via capillary action, and easy fabrication using various printing and patterning techniques. My dissertation research has been focused on developing paper-based devices to address several key gaps that exist between the current technologies and the desired properties of point-of-need sensors. Chapter 2 describes the development of a steady flow paper device that enabled a function similar to conventional flow injection analysis (FIA) without external pumps. Two-layer paper devices increased the attainable flow rate and reduced the analysis time to only a minute, compared with 10-20 min analysis time reported in previous paper-based FIA. Disposable Pt microwire electrodes were used as a detector in the electrochemical paper-based device (ePAD) and the proposed sensor has been used to detect the activity of β-galactosidase (a bacterial indicator for coliform detection and a common detection label in enzyme-linked immunosorbent assay). Similar enzyme kinetics to those reported in the literature was obtained using the proposed sensor, showing a great promise for semi-automation in bioanalysis. Implementing a similar flow ePAD, the goal has now expanded toward improving the detection sensitivity as well as reducing the cost of the sensors. In Chapter 3, low-cost (~1 USD) and reusable thermoplastic electrodes (TPEs) were fabricated by mixing carbon and a plastic binder and pressing the material into an acrylic mold. These TPEs showed an improved electrochemical activity over conventional carbon paste electrodes typically used in ePADs. In addition, electrode arrays can also be fabricated using the technique to improve detection sensitivity via a generation-collection experiment, where the first electrode in the array oxidizes the analyte, the second reduces it, and the process is repeated across the entire array to provide an enhanced cumulative signal. Nanomolar detection limits were achieved using TPEs in both single detector and detector arrays configurations. A 5× improved sensitivity was obtained by employing electrode arrays over the single detector. In Chapter 4, the dissertation shifts focus to a more specific application, detecting nucleic acid, an important biological analyte that has been largely targeted to diagnose various diseases including genetic disorders, cancer, neurodegenerative, and infectious diseases. This chapter describes the integration of nuclease protection assay (NPA), a highly specific hybridization-based technique, with a reader-free colorimetric detection via lateral flow assay (LFA). In NPA, the hybridization of an antisense probe to the target sequence is followed by single-strand nuclease digestion. The protected double-stranded target-probe hybrids are then captured on the LFA device, followed by the addition of a colorimetric enzyme-substrate pair for signal visualization. The proposed paper-based NPA can detect sub-femtomole (~108 copies) of target DNA with high specificity. While the paper-based NPA can serve a good screening tool for several types of chronic infection in which large copies of pathogen DNA is present in the samples, the high detection limit hinders the application of this method for early disease diagnosis and detecting pathogens in environmental samples. In Chapter 5, polymerase chain reaction (PCR), a nucleic acid amplification technique, was coupled to the colorimetric LFA to improve the detection limit and enable the detection of antimicrobial-resistant (AMR) genes and bacteria in environmental samples. Six orders of magnitude lower detection limit (i.e. 102 plasmid DNA copies) was achieved by the PCR-LFA. The proposed method can be applied for rapid detection (less than 3 h) of AMR bacteria in environmental samples. Several works presented in this dissertation provided different approaches to achieve viable paper-based sensors for point-of-need applications. Progress has been made in improving both analytical figures of merit (i.e. sensitivity and detection limit) and practical specifications of the paper sensors (i.e. reduced sensor cost, semi-automation via an external pump-free flow-based system, instrument-free colorimetric readout, and improved assay time).Item Open Access Modulating translation dynamics with tunable optogenetic protein recruitment(Colorado State University. Libraries, 2024) Fixen, Gretchen M., author; Stasevich, Timothy, advisor; Nishimura, Erin, committee member; Chung, Jean, committee memberGenes encoded in our DNA are fundamental to human health and well-being. Their imperative role requires tight regulation throughout their journey to becoming functional proteins. These regulations, when disrupted, have been linked to many neurodegenerative disorders and cancers, stressing the importance of deconvolving their components. Translation is one of the final steps in this journey that has been extensively explored, resulting in a recent technique developed known as nascent chain tracking (NCT) coupled with MS2 stem loop tagging. Using this technique, we are able to track translation dynamics in real-time and in live cells. Despite this, there are still limitations in spatially and temporally tracking the recruitment of translation effectors to translation sites and accurately measuring these dynamics. With the incorporation of optogenetic blue-light-sensitive proteins, we can generate inducible biomolecular condensates that recruit green fluorescent protein (GFP)-tagged proteins and our reporter mRNAs. Using this controlled test-tube-like environment, we can discover the direct effects ribosomal quality control proteins have on translation dynamics. A main quality control pathway involves ZNF598, GIGYF2, and 4EHP proteins that mediate translation control during ribosome stalling. We discovered that both GIGYF2 and 4EHP can be recruited to these clusters and co-localize with our active translation sites in live cells. Further exploration found that 4EHP alone cannot fully cause translation inhibition with our system. Despite this, we do see translation initiation occurring over time due to complex formation with HIF-2∝. However, GIGYF2 has distinct effects on these kinetics that are variable. This tool, when optimized, will be able to describe different proteins' effects on translation kinetics in an isolated environment in live cells.Item Open Access Physicochemical modification of gliadin by black tea polyphenols: insight towards a nutraceutical therapy for celiac disease(Colorado State University. Libraries, 2022) Mathews, Paul, author; Van Buiten, Charlene, advisor; Gentile, Chris, committee member; Chung, Jean, committee memberCeliac disease is an autoimmune disorder that affects approximately 1% of the global population. The pathogenesis of celiac disease is complex, involving the innate and adaptive immune responses. Exposure to gluten amongst genetically susceptible individuals initiates and propagates the disease process, with autoimmunity against endogenous tissue-transglutaminase enzymes manifesting intra- and extra-intestinal symptoms. Currently, the only mitigation strategy for celiac disease is an adherence to a gluten-free diet, which can be difficult to maintain. Recent advances in synthetic and natural products chemistry may offer therapeutic alternatives to the total abstinence from gluten containing products. The overarching objective of our research is to develop a nutraceutical approach to treating celiac disease using dietary polyphenols from tea. Within this thesis, we used a multi-spectroscopic approach to show that black tea polyphenols, which are rich in theaflavins and other flavanols, interact with gluten proteins in vitro to form colloidal complexes that result in structural change to the protein. These changes have the potential to reduce the immunogenicity of gluten via interference with digestion, sequestration, and conformational changes which may reduce recognition of the protein by immune cells. The interactions investigated here offer promise as a nutraceutical, plant-based therapy to acute gluten exposure in susceptible individuals.Item Open Access Ring-conversion and functionalization of nitrogen-containing heterocycles(Colorado State University. Libraries, 2024) Josephitis, Celena M., author; McNally, Andrew, advisor; Bandar, Jeff, committee member; Chung, Jean, committee member; Reisfeld, Bradley, committee memberPyridines and related azines are ubiquitous in pharmaceuticals and agrochemicals development. Chemist rely on the development of new synthetic methods to modify these heterocycles. Described herein are the development of methods to functionalize azines and convert pyridines and diazines into new heterocycles. Novel hydrogenation and molecular editing strategies were designed and leveraged to accomplish this goal. Chapter one introduces the importance of pyridines and related heterocycles in pharmaceuticals as well as methods to access and functionalize these molecules. Both classical and contemporary methods for functionalization and hydrogenation of pyridines are discussed to provide context for this work. Chapter two describes a novel method to selectively reduce pyridines to dihydropyridines, tetrahydropyridines, and piperidines. This method offers a complementary alternative to current hydrogenation or reduction methods, in which the degree of saturation cannot be controlled, and applies to complex azine starting materials. Chapter three explains the importance of structure-activity relationship (SAR) studies and its implications on the drug-discovery process. It also describes classical and contemporary strategies that apply to SAR diversification including de novo heterocycle synthesis and molecular editing strategies. Finally, chapter four presents a novel method for SAR diversification of pyrimidine containing molecules using a deconstruction/reconstruction approach.Item Embargo Site-selective pyridine functionalization via nucleophilic additions to activated pyridiniums(Colorado State University. Libraries, 2024) Nguyen, Hillary M. H., author; McNally, Andrew, advisor; Bandar, Jeff, committee member; Chung, Jean, committee member; Shoemaker, Mark, committee memberPyridines and diazines are important heterocycles commonly found in pharmaceuticals, agrochemicals, ligands, and various other organic molecules. Pyridines existing in these molecules usually have multiple bonds connected to them that contribute to their reactivity and characteristics. Therefore, there are ongoing efforts l to find new methods to functionalize these heterocycles. Our lab has contributed to this field by developing methods to functionalize pyridines directly from the C–H bond through phosphonium salts or Zincke imines. Chapter One gives an overview of the current methods for pyridine functionalization and their limitations. Chapter Two describes the synthesis of N-Tf Zincke imines and their use for regioselective 3-position pyridine functionalization. Bipyridines and pyridine-piperidine coupled products are accessed through this method. Chapter Three discusses using N-Tf Zincke imines to form 15N pyridines and coupled with deuteration forms higher mass isotopologues. Chapter Four describes the formation of N-alkyl pyridinium salts from N-Tf Zincke imines. This chapter focuses on optimizing the ring-opening of 2-ester pyridines and ring-closing them with amino esters to access pipecolic esters for macrocyclization. Chapter Five highlights direct nucleophile additions to the 4-position of N-Tf pyridinium salts for pyridine functionalization. 4-aminated pyridines are formed with both aliphatic amines and anilines from the C–H bond. The regioselectivity of this amination is controlled by the basicity of the reaction. In addition, 4-NH2 pyridines are achieved through this method by adding benzophenone imine, an ammonia surrogate. This reaction extends to adding in heteroatom nucleophiles including alcohols, thioesters, amides, and sulfonamides.Item Open Access Towards elucidating photochemical reaction pathways in nickel catalyzed cross coupling and organocatalyzed Birch reduction(Colorado State University. Libraries, 2021) Kudisch, Max, author; Miyake, Garret, advisor; Finke, Richard, committee member; Chung, Jean, committee member; Reisfeld, Brad, committee memberCarbon-nitrogen (C─N) bond forming reactions to couple aryl halides with amines are essential for the discovery and production of medicinal compounds. The state-of-the-art method uses a precious metal palladium catalyst at high temperatures which poses sustainability concerns. Recently, a method was reported in which an iridium photocatalyst (PC) works in tandem with a nickel catalyst under blue light irradiation to achieve C─N bond formation at room temperature. Herein, it was discovered that the iridium PC could be omitted if 365 nm light is used, constituting a precious metal-free approach. This discovery suggests that a nickel-centered excited state can mediate C─N bond formation, raising the possibility of an energy transfer type pathway in dual catalytic systems. The nickel complexes formed were identified for the first time and mechanistic evidence was found that is consistent with energy transfer with both [Ru(bpy)3]2+ (bpy = 2,2'-bipyridine) and a phenoxazine PC. A series of [NiBr2(amine)n] complexes were isolated, characterized, and detected in C─N coupling reaction mixtures. A theoretical framework for predicting energy transfer rate constant ratios based on Förster theory and UV-visible spectroscopy was developed. The phenoxazine PC was both predicted and found to exhibit faster energy transfer and enhanced reaction performance when compared with [Ru(bpy)3]2+. In addition, a light-driven, organocatalyzed system for Birch reduction was developed. Historically, Birch reduction to reduce an arene to a 1,4-cyclohexadiene has been limited by the required use of alkali metals which are pyrophoric and can be explosive. Under violet light, a benzo[ghi]perylene imide PC was found to reduce challenging arenes such as benzene, constituting the first visible light driven approach capable of this reactivity. Mechanistic studies were performed that are consistent with a catalytic cycle involving addition of OH─ to the PC to form an adduct, [PC─OH]─. Photolysis of the adduct forms OH• and the PC radical anion which subsequently undergoes photoionization, ejecting a solvated electron that reduces the substrate.