Mountain Scholar :: Browsing by Author "Henry, Charles, committee member"

Browsing by Author "Henry, Charles, committee member"

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A metabolomics approach for examining synbiotic protection against infectious enteric pathogens
(Colorado State University. Libraries, 2019) Nealon, Nora Jean, author; Ryan, Elizabeth P., advisor; Dean, Gregg, committee member; Henry, Charles, committee member; Tobet, Stuart, committee member
Infectious gastrointestinal diseases contribute to billions of global cases of human illness annually. Salmonella enterica serovar Typhimurium and human rotavirus represent two human health challenges, where escalating multidrug resistance and poor vaccine efficacy warrant the development of alternative treatments. Health-promoting probiotic microorganisms are becoming increasingly studied for their production of bioactive small molecules that confer protective effects against enteric pathogens. Among probiotics, Lactobacilli, Bifidobacteria and E. coli Nissle form synbiotics with rice bran, the prebiotic-rich outer coating of brown rice, to enhance animal protection against S. Typhimurium infection and human rotavirus diarrhea compared to probiotics or rice bran alone. Despite these beneficial interactions of probiotics and rice bran, a knowledge gap exists in our understanding of the synbiotic small molecules driving these protective effects, especially across probiotic species differences in small molecule production. To test our overarching hypothesis that probiotic species would metabolize rice bran into distinct suites of small molecules that suppressed pathogen function, we first applied the cell-free supernatant from L. paracasei, L. fermentum, and L. rhamnosus cultured with rice bran to S. Typhimurium and observed magnitude-dependent growth suppression across synbiotics. Both L. paracasei and L. fermentum supernatants exhibited enhanced growth suppression compared to their probiotic-only treatments and contained differentially abundant antimicrobial lipids, amino acids, and nucleotides that have not been previously characterized for antimicrobial functions. The cell-free supernatant of the L. paracasei and L. fermentum synbiotics were fractionated and applied to S. Typhimurium to identify the small molecules driving their enhanced Salmonella growth suppression. Metabolite profiles were also compared across synbiotics. Each synbiotic produced several bioactive fractions that suppressed Salmonella growth. While both L. fermentum and L. paracasei bioactive fractions contained abundant lipids, L. fermentum fractions were selectively-enriched in the energy metabolite fumarate and L. paracasei fractions were uniquely-enriched with amino acids (imidazole lactate, ornithine) suggesting that Lactobacillus spp. probiotics could differentially metabolize rice bran to drive Salmonella growth suppression with different suites of small molecules. To examine probiotic metabolism of rice bran in mammalian systems, we compared the intestinal and blood metabolomes of healthy adult mice and gnotobiotic, neonatal pigs that were fed combinations of probiotics and rice bran to the metabolomes of animals consuming rice bran or probiotics alone. In mice, a notable difference following 15 weeks consumption of B. longum fermented was that the arginine metabolite N-delta-acetylornithine was significantly increased in B. longum fermented rice bran compared to rice bran alone and was elevated in both the colon tissue and blood of mice consuming fermented rice bran compared to rice bran alone. In gnotobiotic neonatal pigs, three weeks of prophylactic supplementation with E. coli Nissle and L. rhamnosus GG and rice bran were more effective at reducing human rotavirus diarrhea compared to pigs given these probiotics or rice bran alone. Approximately 300 colon and blood metabolites that were differentially-abundant between synbiotic-consuming pigs versus pigs consuming probiotics alone were identified, over 50% of which were lipids and amino acids. Similar modulations lipid and amino acid metabolites (sphingolipids, diacylglycerols, arginine metabolites) were identified in the colon tissue and blood of mice and pigs consuming the synbiotic treatments. Consequently, the association of these metabolite profiles with human rotavirus diarrhea protection, when combined with their presence in two mammalian models, provides strong rationale for these infectious enteric disease protective roles harbored by these metabolites. The results of these studies provide a role for synbiotics in the prevention of infectious gastrointestinal diseases. For the first time, high-throughput metabolomics analyses were applied to identify differential bioactive metabolite production by Lactobacillus spp. + rice bran synbiotics that suppressed S. Typhimurium growth, as well as to compare bioactive metabolites produced by B. longum, L. rhamnosus GG, and E. coli Nissle in mice and pigs that were protective against human rotavirus diarrhea. The contributions of amino acids and lipids to the enhanced capacities of these synbiotics compared to probiotics or rice bran alone can be studied further for their mechanisms of action on pathogens. Ultimately, these bioactive synbiotic metabolites can guide the optimization and development of broad-spectrum antimicrobials and other prophylactic agents that protect against infectious enteric diseases across the human and animal lifespan.
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A selection of nitric oxide-releasing materials incorporating S-nitrosothiols
(Colorado State University. Libraries, 2017) Lutzke, Alec, author; Reynolds, Melissa, advisor; Henry, Charles, committee member; Kennan, Alan, committee member; Kipper, Matthew, committee member
Nitric oxide (NO) is a diatomic radical that occurs as a crucial component of mammalian biochemistry. As a signaling molecule, NO participates in the regulation of vascular tone and maintains the natural antithrombotic function of the healthy endothelium. Furthermore, NO is produced by phagocytes as part of the immune response, and exhibits both antimicrobial and wound-healing effects. In combination, these beneficial properties have led to the use of exogenous NO as a multifunctional therapeutic agent. However, the comparatively short half-life of NO under physiological conditions often renders systemic administration infeasible. This limitation is addressed by the use of NO-releasing polymeric materials, which permit the localized delivery of NO directly at the intended site of action. Such polymers have been utilized in the development of antithrombotic or antibacterial materials for biointerfacial applications, including tissue engineering and the fabrication of medical devices. NO release from polymers has most frequently been achieved through the incorporation of functional groups that are susceptible to NO-forming chemical decomposition in response to appropriate environmental stimuli. While numerous synthetic sources of NO are known, the S-nitrosothiol (RSNO) functional group occurs naturally in the form of S-nitrosocysteine residues in both proteins and small molecule species such as S-nitrosoglutathione. RSNOs are synthesized directly from thiol precursors, and their NO-forming decay has generally been established to produce the corresponding disulfide as a relatively benign organic byproduct. For these reasons, RSNOs have been conscripted as practical NO donors within a physiological environment. This dissertation describes the synthesis and characterization of RSNO-based NO-releasing polymers derived from the polysaccharides chitin and chitosan, as well as the development of amino acid ester-based NO-releasing biodegradable poly(organophosphazenes) (POPs). The broad use of chitin and chitosan in the development of materials for tissue engineering and wound treatment results in a significant overlap with the therapeutic properties of NO. NO-releasing derivatives of chitin and chitosan were prepared through partial substitution of the carbohydrate hydroxyl groups with the symmetrical dithiols 1,2-ethanedithiol, 1,3-propanedithiol, and 1,6-hexanedithiol, followed by S-nitrosation. Similarly, thiol-bearing polyphosphazenes were synthesized and used to produce NO-releasing variants. Polyphosphazenes are a unique polymer class possessing an inorganic backbone composed of alternating phosphorus and nitrogen atoms, and hydrolytically-sensitive POP derivatives with organic substituents have been prepared with distinctive physical and chemical properties. Although POPs have been evaluated as biomaterials, their potential as NO release platforms has not been previous explored. This work describes the development of NO-releasing biodegradable POPs derived from both the ethyl ester of L-cysteine and the 3-mercapto-3-methylbutyl ester of glycine. The NO release properties of all polymers were evaluated at physiological temperature and pH, and the results suggested potential suitability in future biomaterials applications.
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Analytical methods to enhance detection of anthropogenic radionuclides in environmental matrices
(Colorado State University. Libraries, 2016) Rosenberg, Brett L., author; Brandl, Alexander, advisor; Borch, Thomas, committee member; Henry, Charles, committee member; Pinder, John, committee member; Steinhauser, Georg, committee member
The efficacy of methods that are used to detect radionuclides is dependent on the properties of the radionuclides and the matrices being analyzed. Gamma spectroscopy is an excellent tool for detecting very low quantities of a short-lived gamma-emitting radionuclide. However, as the probability of gamma ray emission decreases and the half-life increases, greater quantities of a radionuclide are required for detection by gamma spectroscopy. Since most transuranic actinides are usually not present in such quantities or concentrations in the environment, mass spectrometry is the preferred tool. For tritium, 90Sr, and other lower-Z elements that emit no easily detectable gamma rays, liquid scintillation counting is commonly used to measure the beta particles they emit. However, this methodology requires radiochemical extraction procedures to ensure a maximized ratio between signal and background. Nondestructive gamma spectroscopy was used to evaluate radiocesium content in soil and vegetation samples collected from the Fukushima prefecture exclusion zone in 2013 and 2014. Liquid scintillation counting was used for quantifying 3H in samples collected in 2013 and 90Sr in samples collected in 2013 and 2014. The radiocesium and 90Sr activities were found to have decreased from 2013 to 2014. Although 3H activities could be quantified in most samples, a sample from Chimeiji had a specific activity that statistically exceeded background (1.2 ± 1.6 Bq mL-1); further investigation is required to ascertain if 3H is present within that sample. Reports generated by TEPCO were also evaluated; radiocesium ratios and 131I/132Te ratios calculated from the reports reveal the importance of considering counting statistics and spectroscopic interference when drawing conclusions about the presence of anthropogenic radionuclides in environmental samples. Gamma spectroscopy was then applied to explore radiochemical separation techniques that can enhance detection of anthropogenic radionuclides, especially gamma-emitting actinides like 239Np shortly after a nuclear event. Ion specific extraction chromatography was found to be effective at minimizing spectroscopic interference from fission products, and addition of stable iodide carrier and a precipitating agent facilitated decreasing radioiodine activity within environmental samples. Extraction chromatography was found to reduce 131I interference by at least one order of magnitude, making it preferred for reducing 131I activity within an environmental sample. Extraction chromatography also avoids the potential of precipitating any analyte. The separation and measurement techniques utilized herein have effectively enhanced the ability to detect low-activity anthropogenic radionuclides; supplemental measurements gathered from the exclusion zone confirm the observed trends and prove the necessity of minimizing interference.
Open Access
Biophysical, structural, and functional studies of histone binding proteins
(Colorado State University. Libraries, 2010) Sudhoff, Keely B., author; Luger, Karolin, advisor; Chen, Chaoping, committee member; Henry, Charles, committee member; Woody, Robert, committee member; Hansen, Jeffrey C., committee member
Eukaryotic genomes are extensively compacted with an equal amount of histone proteins to form chromatin. A high level of control over chromatin structure is required to regulate critical cellular processes such as DNA replication, repair, and transcription. To achieve this feat, cells have developed a variety of means to locally or globally modulate chromatin structure. This can involve covalent modification of histones, the incorporation of histone variants, remodeling by ATP-dependent remodeling enzymes, histone chaperone-mediated assembly/disassembly, or any combination of the above activities. To understand how chromatin structure is affected by histones, it is essential to characterize the interactions between histones and their associated proteins. In Saccharomyces cerevisiae, the multi-subunit SWR1 complex mediates histone variant H2A.Z incorporation. Swc2 (Swr1 complex 2) is a key member of the SWR1 complex and is essential for binding and transfer of H2A.Z. Chz1 (Chaperone for H2A.Z/H2B) can deliver H2A.Z/H2B heterodimers to the SWR1 complex in vitro. Swc2 1-179 (a domain of Swc2 that retains histone binding and the apparent preference for variant dimers) and Chz1 are intrinsically disordered, but become more ordered upon interaction with histones. Quantitative measurements done under physiological in vitro conditions demonstrate that Chz1 and Swc2 1-179 are not histone variant-specific. They bind to histones with an affinity lower than that of previously described histone chaperones, and lack the ability to act on nucleosomes or other histone-DNA complexes. Small-angle X-ray scattering demonstrates that the intrinsic disorder of the proteins allows them to adopt a multitude of structural states, perhaps facilitating many different interactions and functions. We show that Swc2 1-179, despite its overall acidic charge, can bind double stranded DNA, in particular, 3-way and 4-way junction DNA. These junctions are thought to mimic the central intermediates found in DNA damage repair. This characteristic is unique to Swc2 1-179. Consistent with this unexpected activity, yeast phenotypic assays have revealed a role for SWC2 in DNA damage repair, as indicated by sensitivity to DNA damaging agent methane methylsulfonate. Importantly, our data has exposed a novel role for Swc2 in DNA damage repair. In an independent study, we investigated the histone chaperone Vps75, a Nap1 homolog. Rtt109 is a histone acetyltransferase that requires a histone chaperone for the acetylation of histone H3 at lysine 56 (H3K56). Rtt109 forms a complex with the chaperone Vps75 in vivo and is implicated in DNA replication and repair. We show that deletion of VPS75 results in dramatic and diverse mutant phenotypes, in contrast to the lack of effects observed for the deletion of NAP1. The flexible C-terminal domain of Vps75 is important for the in vivo functions of Vps75 and modulates Rtt109 activity in vitro. Our data highlight the functional specificity of Vps75 in Rtt109 activation.
Open Access
Computer vision algorithm to extract color data of pixels in microfluidic paper based analytical devices
(Colorado State University. Libraries, 2021) Deotale, Saurabh, author; Beveridge, James Ross, advisor; Blanchard, Nathaniel, committee member; Henry, Charles, committee member
Microfludic paper-based devices are fast becoming an inexpensive and faster option than traditional methods for substance detection and chemical measurements. These devices are designed to be used in the field for quicker result. One hurdle towards that goal is a manual step of data extraction from the images of these devices for further analysis and results. This involves identifying and extracting color data from specific regions of interest. The color data is the color values in BGR and HSV color channels of the pixels lying in these regions of interest. The manual demands labor and time that can avoided by automating this process using computer vision techniques. The goal of this thesis is to aid chemists by automating the data extraction process. This thesis presents a layered algorithm which uses simple techniques like region growing and thresholding in conjunction with leveraging the knowledge of the device design to extract the required data. This data is then labeled and compiled in CSV file for further analysis.
Open Access
Development of a nitric oxide measurement method in tissue media
(Colorado State University. Libraries, 2012) Bishop, Cherelle M., author; Reynolds, Melissa, advisor; Henry, Charles, committee member; Tobet, Stuart, committee member
Nitric oxide (NO) is involved in many biological pathways such as vasodilatation and cellular migration. The biological roles of NO have been most heavily investigated using cell and tissue culture models. The limitations with current analytical measurement methods used most commonly with these studies, however, are that they often do not record in real-time or measure NO directly. This makes it difficult to understand the concentration dependent response activity of NO. To overcome these limitations, a measurement method has been developed that enables the real-time measurement of NO in buffered tissue media (pH 7.4, buffered with CO2 gas, 37 °C). The design of our system included multi-volume custom sample cells with a pH probe and multiple gas supply inputs, a flow regulated CO2 gas system and a chemiluminescence detector. Results demonstrated the expected first-order NO release kinetics using a model NO donor (MAHMA/NO) in phosphate buffered saline (PBS) over a specified volume range. The following half-lives were found: 63±2 s (2 mL), 65±2 s (6 mL), 63±4 s (8 mL) and 67±9 s (10 mL). Using this method at these buffer volumes, an experiment was conducted using 11 mM MAHMA/NO stock used to demonstrate that NO release was linearly proportional with respect to buffer volume with a linear fit of R2 =0.9936. The linearity of NO release allowed NO release measurements of 4.4 x 10-7 M MAHMA/NO concentration in 10 mL PBS achieving NO recovery of 117±2 and MAHMA/NO decomposition half-lives 66±2. The analysis of a 10-7 M MAHMA/NO was not measurable previously using other chemiluminescence methods. Subsequent results in tissue media buffered with 5% CO2 at a controlled rate of 20 mL/min showed statistically similar kinetic rates 68±5 s (2 mL) to that of the PBS, demonstrating the ability to measure NO in real time under tissue conditions. The simultaneous pH measurements confirmed that the pH was constant at 7.4 during the NO release portion of the experiment, an important aspect to maintain accurate kinetics. Using this method for NO release measurement in tissue media, another NO donor, DETA/NO, was used to look at steady-state release for 1.5 h. The total NO release was 0.12±0.02 (nmol) and the NO release rate was 22±3 (fmol/s). This is the first analytical measurement method that enables detection of NO release from NO donors in buffered tissue media method mimicking in vitro condition.
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Development of an artificial temporomandibular joint disc replacement
(Colorado State University. Libraries, 2023) Kuiper, Jason Paul, author; Puttlitz, Christian M., advisor; Prawel, David, committee member; McGilvray, Kirk, committee member; Henry, Charles, committee member
The temporomandibular joint (TMJ) is a complex bilateral ginglymoarthroidal joint containing a fibrocartilaginous disc and is essential for chewing, speaking, and swallowing. Due to the high loading frequency, small imbalances in joint homeostasis can overcome the natural capacity for adaptation and lead to a cascade of degenerative changes. For progressive TMJ disorders, resection of the TMJ disc is the leading treatment, but disc resection inherently increases stress and friction on the articular cartilage surfaces, leading to a progression to total joint replacement in 11.7% of patients. The current methods of treatment for disorders of the TMJ musculoskeletal complex are predominantly palliative and do not reliably address disorders of arthrogenous origin. Unfortunately, no synthetic TMJ disc replacements currently exist due to profound implant failures in earlier attempts. Introduction of a robust artificial TMJ disc replacement after resection will prevent further joint degradation and improve patient outcomes. Rigorous preclinical evaluation of artificial TMJ disc replacement strategies must be conducted to support future translation to humans. Therefore, the following aims are proposed: (1) Characterize the biomechanical behavior of the ovine temporomandibular joint soft tissues, (2) identify and evaluate a material candidate for a temporomandibular joint disc replacement, (3) develop in silico and in vitro methods for evaluating design candidates for artificial TMJ disc replacement, and (4) implement a temporomandibular joint disc replacement strategy in an ovine model.
Open Access
Development of an asymmetric NHC-catalyzed cascade reaction and studies towards the asymmetric aminomethylation of enals
(Colorado State University. Libraries, 2015) Ozboya, Kerem, author; Rovis, Tomislav, advisor; Henry, Charles, committee member; McNally, Andrew, committee member; Kennan, Alan, committee member; Inamine, Julia, committee member
A cascade reaction is developed to form complex cyclopentanones using an asymmetric Michael/Benzoin sequence. This reaction employs simple aliphatic aldehydes and ketoesters in conjunction with a chiral amine catalyst and a chiral NHC catalyst. Further investigation reveals a surprising interplay between these two catalysts. This relationship is manifested in a pseudo-dynamic kinetic resolution, which is responsible for the high diastereoselectivity observed. Subsequent work details the discovery of the aminomethylation of enals using NHC catalysis. This reaction utilizes an iminium source as well as cinnamaldehyde derivatives to form gamma-amino butyrate derivatives. Rendering this reaction asymmetric has proven a challenge, despite extensive effort to resolve these issues. In the course of these studies, an unexpected NHC-catalyzed Morita-Baylis-Hillman reaction was observed. Optimal conditions for this reaction were established, proving access to useful amino-enals. In an effort to design suitable catalysts for the asymmetric aminomethylation reaction, a strategy for the late-stage manipulation of NHC catalysts was developed. Key to this strategy is the `protection' of the triazolium salt by reduction to the triazoline. An aryl C-Br bond is then exploited for cross-coupling reactions, building a small library of new catalysts. The triazolium salt is then recovered by oxidation with a trityl salt.
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Development of an ultrasensitive ELISA for the detection of Mycobacterium tuberculosis antigens: an impossible challenge or a promising feat?
(Colorado State University. Libraries, 2022) Early, Kala, author; Dobos, Karen, advisor; Mehaffy, Carolina, advisor; Schenkel, Alan, committee member; Henry, Charles, committee member
Tuberculosis (TB) has been classically characterized as a two-state disease with active and latent phases. Latent TB infection (LTBI) is diagnosed by either the tuberculin skin test (TST) or the Interferon Gamma Release Assay (IGRA) test. However, both diagnostic tests are unable to differentially diagnose active TB and LTBI and perform poorly in immunocompromised patients. The TST is further complicated by cross-reactivity with BCG vaccination. Therefore, further diagnostic discovery for LTBI is needed for differential diagnosis and to identify those at risk of progression to active TB for subsequent treatment. Extracellular vesicles (EVs) are nanovesicles released by eukaryotic cells. EVs from TB patients contain Mycobacterium tuberculosis (Mtb) proteins, and these protein biomarkers show promise for TB and LTBI diagnostics. Our lab previously identified 31 Mtb peptides in trypsin-treated serum EVs isolated from patients with LTBI using multiple reaction monitoring-mass spectrometry (MRM-MS) methods. MRM-MS is a highly sensitive technology but is not feasible for widespread use as a diagnostic. The goal of this study was to develop an ultrasensitive ELISA against Mtb proteins for potential use as a point-of-care diagnostic. A sandwich ELISA was initially developed against Mtb proteins DnaK, Mpt32, and GroES. Reagent development for the sandwich ELISA included polyclonal antibody production using a rabbit model, murine monoclonal antibody purification and biotinylation from an existing collection of hybridoma cell lines for each antigen, and detection using a streptavidin-HRP system with a chemiluminescent substrate for signal expansion. We observed that the sandwich ELISA was complicated by non-specific binding of the DnaK and GroES antigens to the BSA block. We hypothesized that the chaperone function of these two proteins influenced them to bind to BSA. This non-specific interaction was further characterized using SPR technology and demonstrated a concentration dependent binding of DnaK to BSA. A direct-biotinylated ELISA was subsequently developed and optimized. Limit of detection (LOD) and limit of quantification (LOQ) of the direct-biotinylated ELISA was determined for each antigen: 1) GroES had an LOD of 1.959 ng/mL and an LOQ of 6.531 ng/mL, 2) Mpt32 had an LOD of 1.884 ng/mL and an LOQ of 6.278 ng/mL, and 3) DnaK had an LOD of 6.310 ng/mL and an LOQ of 21.032 ng/mL. This direct-biotinylated ELISA platform demonstrated high sensitivity with low background for all three antigens. Thus, we successfully developed and optimized an ultrasensitive ELISA for the detection of Mtb antigens.
Open Access
Development of electrochemical assays and biosensors for detection of Zika virus
(Colorado State University. Libraries, 2019) Filer, Jessica, author; Geiss, Brian, advisor; Chen, Tom, advisor; Henry, Charles, committee member; Wilusz, Jeff, committee member; Ebel, Greg, committee member
Zika virus (ZIKV) emerged as a significant public health concern after the 2015-2016 outbreak in South and Central America. Severe neurological complications and birth defects in adults and children respectively underscore the need for quick and accurate diagnosis so that proper medical observation and intervention can be done. Electrochemical assays and biosensors are attractive as alternative diagnostic tools due to their sensitivity and ease of miniaturization. This dissertation describes three novel electrochemical assays and biosensors to detect ZIKV specific nucleic acid, antibodies, and virus particles. A nuclease protection ELISA (NP-ELISA) was developed for nucleic acid detection by enzymatic readout. The assay was validated using synthetic complementary oligos for absorbance, chemiluminescence, and electrochemical enzymatic readout. Two horseradish peroxidase substrates, 3,3',5,5'-Tetramethylbenzidine (TMB) and hydroquinone, were characterized electrochemically and compared for electrochemical assay use. Electrochemical TMB readout demonstrated better sensitivity compared to all tested detection modalities with a limit of detection of 3.72×103 molecules mL-1, which compares well to the amount of ZIKV RNA in clinical samples and to other approved assays like the CDC's Trioplex assay. For serological analysis, a capacitive microwire biosensor was developed and validated using immunized mouse sera to detect a ZIKV antibody response. Measurements were taken through a wide serial dilution range of 1:1018 to 1:103 and two dilutions (1:1012 and 1:106) were used for analysis for optimal sensitivity. A statistically significant immune response was detected four days after immunization at a 1:1012 dilution and was specific for ZIKV when compared with Chikungunya virus (CHIKV). These results indicate that serological analysis can be performed four days earlier with the wire sensor compared to ELISAs using ultra-dilute samples. The sensor also was used to differentiate between IgG and IgM antibodies and compared well with ELISA results. Lastly, an impedance array sensor was designed and validated for detection of ZIKV particles. The array allows for simultaneous handling of many electrodes, which increases throughput compared to other biosensor designs. The sensor demonstrated good sensitivity with an LOD of 22.4 focus forming units (FFU) which compares well to other reported sensors. In addition, it was optimized for specificity and tested using Sindbis virus (SINV) as a negative control. These novel platforms comprise new advancements in biosensor technology by simplifying existing assays, increasing sensitivity, and providing a new platform for handheld measurements.
Open Access
Developments in automated electrochemical biosensors to improve point of care diagnostics
(Colorado State University. Libraries, 2022) Schenkel, Melissa, author; Kennan, Alan, advisor; Henry, Charles, committee member; Snow, Christopher, committee member; Ross, Eric, committee member
The onset of the COVID-19 pandemic brought public attention to the pre-existing need for developments in diagnostics, especially at the point of care. While traditional techniques, like PCR, can be highly sensitive and specific, they are also time consuming, expensive, and require trained personnel in a laboratory setting and expensive equipment. The need for point of care diagnostic options was made evident in early 2020 when laboratories could not keep up with the high demand for COVID-19 testing. Lateral flow assays (LFAs) like home pregnancy tests offer a platform that is inexpensive, easy to use, and can produce results rapidly at the point of care. Unfortunately, LFAs usually exhibit poor sensitivity and limits of detection compared to traditional techniques. Electrochemical biosensors can provide a diagnostic platform that is quick, cost effective, accurate, highly sensitive, and quantitative. While electrochemical biosensors incorporated in lateral flow devices have improved sensitivity, they typically require complex fabrication techniques, and the nitrocellulose platform can limit electrochemical performance. The Henry group has recently reported a new class of capillary-driven fluidic devices using alternating layers of patterned polyethylene terephthalate (PET) films and double-sided adhesives (DSA) that can control flow for sequential delivery of reagents. This work presents recent developments in automated electrochemical biosensors to improve point of care diagnostics. The incorporation of electrochemical biosensors with the aforementioned novel fluidic devices provides a diagnostic platform that has the potential to achieve the sensitivity and selectivity rivaling that of traditional techniques while maintaining the ease of use of an LFA. Chapter 2 of this dissertation first presents an electrochemical immunosensor for detection of SARS-CoV-2 N-protein. This sensor was then adapted and optimized for compatibility in a fluidic device. This included optimizing ease of functionalization with manufacturing-friendly techniques, exploring different buffers for assay steps, and optimizing assay components Ultimately, these studies led to automated, concentration-dependent detection of SARS-CoV-2 N-protein upon a single sample addition step. Chapter 3 of this dissertation presents a novel device design that improved flow rates, decreased device malfunctions, and incorporated commercial electrodes. This device was developed for measurements of C-reactive protein, a common biomarker of inflammation. Utilizing gold electrodes has the potential for more sensitive detection compared to carbon electrodes and aptamers as biological recognition elements provides many advantages as well. While work on this project is still underway, the results presented herein demonstrate the ability of this novel diagnostic device to be adapted for various analytes. Future work includes continued assay and device optimization, with the intent for multiplexed detection of multiple analytes. Overall, the work presented here provides a novel platform for point of care diagnostics and demonstrates its application to two different analytes.
Open Access
Electrochemically prepared metal antimonide nanostructures for lithium ion and sodium ion battery anodes
(Colorado State University. Libraries, 2016) Jackson, Everett D., author; Prieto, Amy, advisor; Rappe, Anthony, committee member; Dandy, David, committee member; Bailey, Travis, committee member; Henry, Charles, committee member
The use of energy fundamentally enables and globally supports post-industrial economies and is critical to all aspects of modern society. In recent years, it has become apparent that we will require superior energy technologies to support our society, including improved methods of generating, storing, and utilizing energy resources. Battery technology occupies a critical part of this new energy economy, and the development of electrochemical energy storage devices will be a critical factor for the successful implementation of renewable energy generation and efficiency strategies at the grid, transportation, and consumer levels. Current batteries suffer from limitations in energy density, power density, longevity, and overall cost. In addition, the inherent tradeoffs required in battery design make it impossible to create a single battery that is perfect for all applications. To overcome these issues, the development of low-cost and high-throughput methods, new strategies for materials design, and a comprehensive understanding of electrochemical mechanisms for battery performance is necessary. Herein, an in-depth study on the electrochemistry of a model anode system for rechargeable batteries based on metal antimony alloys produced through an electroplating approach is detailed. The first chapter of this dissertation provides a brief introduction of lithium ion and sodium ion battery technology. In the second chapter, a detailed review of the literature on antimony and metal antimonide alloys for battery anodes is provided. The third chapter details a study on copper antimonide thin films with varying stoichiometry produced through a facile electrodeposition process. In the fourth chapter, stoichiometric Cu2Sb thin films are studied as potential anodes for sodium ion batteries. The fifth chapter details the development of a process for electroplating zinc-antimony alloy thin films onto zinc and their electrochemical properties in sodium ion cells. The sixth and seventh chapters report the synthesis and characterization of copper-antimony alloy nanowire arrays produced through an alumina-templated process. These nanowire arrays are first used in an electrolyte-additive study to show the importance of surface stabilization for high surface area electrodes in chapter five. In chapter six, the rate performance is characterized under different thermal conditions for different compositions of copper-antimony alloy nanowire arrays as an assessment of the kinetic limitations of this electrode. The final chapter briefly describes some preliminary experiments that have been performed on characterizing the electrochemistry of metal salts in a deep eutectic solvent as a potential method for co-deposition of new metal antimonides.
Open Access
Enhanced surface functionality via plasma modification and plasma deposition techniques to create more biologically relevant materials
(Colorado State University. Libraries, 2013) Shearer, Jeffrey C., author; Fisher, Ellen R., advisor; Henry, Charles, committee member; Szamel, Grzegorz, committee member; Bailey, Travis, committee member; Buchanan, Kristen, committee member
Functionalizing nanoparticles and other unusually shaped substrates to create more biologically relevant materials has become central to a wide range of research programs. One of the primary challenges in this field is creating highly functionalized surfaces without modifying the underlying bulk material. Traditional wet chemistry techniques utilize thin film depositions to functionalize nanomaterials with oxygen and nitrogen containing functional groups, such as -OH and -NHx. These functional groups can serve to create surfaces that are amenable to cell adhesion or can act as reactive groups for further attachment of larger structures, such as macromolecules or antiviral agents. Additional layers, such as SiO2, are often added between the nanomaterial and the functionalized coating to act as a barrier films, adhesion layers, and to increase overall hydrophilicity. However, some wet chemistry techniques can damage the bulk material during processing. This dissertation examines the use of plasma processing as an alternative method for producing these highly functionalized surfaces on nanoparticles and polymeric scaffolds through the use of plasma modification and plasma enhanced chemical vapor deposition techniques. Specifically, this dissertation will focus on (1) plasma deposition of SiO2 barrier films on nanoparticle substrates; (2) surface functionalization of amine and alcohol groups through (a) plasma co-polymerization and (b) plasma modification; and (3) the design and construction of plasma hardware to facilitate plasma processing of nanoparticles and polymeric scaffolds. The body of work presented herein first examines the fabrication of composite nanoparticles by plasma processing. SiOxCy and hexylamine films were coated onto TiO2 nanoparticles to demonstrate enhanced water dispersion properties. Continuous wave and pulsed allyl alcohol plasmas were used to produce highly functionalized Fe2O3 supported nanoparticles. Specifically, film composition was correlated to gas-phase excited state species and the pulsing duty cycle to better understand the mechanisms of allyl alcohol deposition in our plasma systems. While these studies specifically examined supported nanoparticle substrates, some applications might require the complete functionalization of the entire nanoparticle surface. To overcome this challenge, a rotating drum plasma reactor was designed as a method for functionalizing the surface of individual Fe2O3 nanoparticles. Specifically, data show how the rotating motion of the reactor is beneficial for increasing the alcohol surface functionality of the nanoparticles when treated with pulsed allyl alcohol plasmas. Plasma copolymerization was used to deposit films rich in both oxygen and nitrogen containing functional groups using allyl alcohol and allyl amine plasma systems. Functional group retention and surface wettability was maximized under pulsed plasma conditions, and films produced under pulsed plasma conditions did not exhibit hydrophobic recovery or experience loss of nitrogen as the films aged. Plasma surface modification with N2/H2O and NH3/H2O, and plasma deposition with allyl alcohol and allyl amine, were used to increase the wettability of poly(caprolactone) scaffolds while simultaneously implanting functional groups onto the scaffold surface and into the scaffold core. While plasma deposition methods did not modify the internal core of the scaffold as much as modification methods, it afforded the ability to have higher water absorption rates after a three week aging period. Additionally, cell viability studies were conducted with N2/H2O plasma treated scaffolds and showed enhanced cell growth on plasma treated scaffolds over non plasma-treated scaffolds.
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Evaluation of nitric oxide releasing polymers for wound healing applications
(Colorado State University. Libraries, 2015) Wold, Kathryn A., author; Reynolds, Melissa, advisor; Henry, Charles, committee member; Kipper, Matt, committee member; Popat, Ketul, committee member; Williams, John, committee member
Chronic, non-healing wounds afflict millions of Americans and represent a costly burden to the healthcare industry. In addition, the overuse and misuse of antibiotics has triggered the widespread emergence of drug-resistant bacteria, making the treatment of infected wounds more challenging. As a result, improved methods for wound care incorporating antibiotic-alternative bactericidal agents are in high demand. Recent wound care advances have focused on the development of dressings incorporating physical structures and biological components which mimic those encountered in a natural wound environment. Nitric oxide (NO), an endogenously produced molecule upregulated to promote cellular function and bactericidal activity during wound healing, has been harnessed in material systems and studied for wound healing potential. This work describes the characterization, bactericidal activity, cell functionality and processing of two NO-releasing polymer systems, one water-soluble and another water-insoluble. The results of this work demonstrate the capability of these polymeric NO-releasing materials to promote high log reductions of planktonic bacteria. Additionally, polymer dosages that promote cell survival and induce cytotoxicity in eukaryotic cells have been determined and nano-scale polymer fibers that maintain NO release properties have been processed. These results represent qualities beneficial towards the development of enhanced materials for the treatment of chronic infected wounds.
Open Access
Evaluation of sodium bismuthate chromatographic systems for the separation of americium from curium
(Colorado State University. Libraries, 2023) Labb, Samantha A., author; Sudowe, Ralf, advisor; Brandl, Alexander, committee member; Johnson, Thomas, committee member; Henry, Charles, committee member
The development of a successful and efficient americium (Am) and curium (Cm) separation method is necessary for stockpile stewardship science and for the simplification and improvement of currently proposed reprocessing schemes towards the closure of the nuclear fuel cycle. However, the similar chemical properties of these radionuclides (e.g., similar ionic radii, ionic bonding, and predominant trivalent oxidation states in acidic media) makes this difficult to achieve. Differences in redox chemistry can be exploited based on the fact that Am can be oxidized to higher oxidation states in acidic media while Cm cannot. Recently, the ability of solid sodium bismuthate to oxidize Am and its ion exchange properties were demonstrated in solvent extraction and chromatographic systems, but were limited by oxidation stability, kinetics, and flow rates. This dissertation focuses on evaluating and characterizing new solid-liquid chromatographic systems that combine both the oxidation and ion exchange mechanisms into one material for a continuous separation process. In addition, the solution behavior of NaBiO3 in nitric acid and the effect on acidity and dissolution kinetics is determined.
Open Access
Expanding and evaluating sense codon reassignment for genetic code expansion
(Colorado State University. Libraries, 2017) Biddle, C. William, author; Fisk, John D., advisor; Ackerson, Chris, committee member; Henry, Charles, committee member; Stasevich, Tim, committee member
Genetic code expansion is a field of synthetic biology that aims to incorporate non-canonical amino acids (ncAAs) into proteins as though they were one of the 20 "natural" amino acids. The amino acids which naturally make up proteins are chemical limited, and ncAAs can carry new chemical functionality into proteins. Proteins are of interest because they are simple to produce with good consistency and have immense potential due to the diversity of structure and function. Incorporating ncAA into proteins expands the scope of function of proteins even further. Two methods have been widely used for genetic code expansion, global amino acid replacement and amber stop codon suppression. Global amino acid replacement exchanges one of the natural amino acids for a ncAA, producing an altered 20 amino acid genetic code. Amber stop codon suppression incorporates ncAA in response to the UAG stop codon making a 21 amino acid genetic code, but is limited in incorporation efficiency and producing proteins with multiple instances of a ncAA is challenging. We wanted to use a third genetic code expansion system called sense codon reassignment which has not been widely employed at all but should enable multisite incorporation of ncAAs. When the work presented in this dissertation was started, a single report of sense codon reassignment existed in the literature. We set out to improve and expand sense codon reassignment for the incorporation of multiple copies of ncAAs into proteins. We quickly discovered disparities in what was known regarding the variables that could be used to manipulate genetic code expansion, and the focus of our work shifted to systems for improving sense codon reassignment using quantitative measurements. The first chapter of this dissertation is an introduction to genetic code expansion and the processes of translation and gene expression that are likely involved or could be involved in genetic code expansion. The three following chapters will build upon the fundamentals described in Chapter 1. The second chapter is a complete story about how a screen to quantify sense codon reassignment was developed. The fluorescence based screen was used in a high throughput fashion to screen a directed evolution library of variants for increased sense codon reassignment efficiency at the Lys AAG sense codon. While evaluating various sense codons for potential reassignment efficiency, the AUG anticodon was found to be incapable of discriminating between the CAU and CAC codons. This was anomalous relative to the other anticodons we tested. Chapter 3 describes how unintended modifications to an engineered tRNA were identified and then how the fluorescence based screen was used to engineer the tRNA further for increased sense codon reassignment efficiency and to avoid the unintentional modification. Most applications of genetic code expansion rely on modifications to tRNAs but few reports actually consider them, The final chapter of this dissertation is a manuscript in preparation describing the reassignment of a rare sense codon to incorporate ncAAs. The chapter focuses on how improvements made in a system specific for an amino acid can be transferred to systems specific for other ncAAs. Over 150 different ncAAs have been incorporated into proteins using genetic code expansion technologies, but the extent to which the various systems are combinable has barely been evaluated. This dissertation is a story about developing sense codon reassignment to functional levels and quantifying the effects of different variables along the way.
Open Access
Interrogating reactions of gold nanoclusters: insights into catalysis and the Brust-Schiffrin synthesis
(Colorado State University. Libraries, 2017) Dreier, Timothy Andrew, author; Ackerson, Christopher J., advisor; Kennan, Alan J., committee member; Henry, Charles, committee member; Peebles, Christie, committee member
Over the past several decades, interest in the synthesis and behavior of atomically precise gold nanoclusters has gained substantial momentum. Herein, both catalytic behavior and synthetic mechanisms are explored using techniques more typically applied to organic chemistry. In the case of catalysis, Au25(SR)18 has emerged as a well-studied model system. In an effort to investigate their potential as intact, homogeneous, unsupported catalysts, we have discovered that Au25(SR)18 clusters are not stable in oxidizing conditions reported for catalytic styrene oxidation. Further investigation suggests that the active catalytic species is an Au(I) species resulting from oxidative decomposition of the starting gold cluster. Equally important to chemical behavior is an understanding of the reaction dynamics during the synthesis of atomically precise clusters. Because the Brust-Schiffrin method is the standard procedure by which gold nanoclusters are synthesized, the role of oxygen in it has been investigated for both organic and aqueous systems. In either case, it is clear obtaining the desired product depends on a radically mediated etching step. These results give new insight into how the Brust-Schiffrin method might be modified to further synthesis of uniquely interesting nanocluster systems.
Open Access
Laboratory evaluation of a microfluidic electrochemical sensor for aerosol oxidative load
(Colorado State University. Libraries, 2012) Shapiro, Jeffrey, author; Volckens, John, advisor; Henry, Charles, committee member; Peel, Jennifer, committee member
Human exposure to particulate matter (PM) air pollution is associated with both human morbidity and mortality. The mechanisms by which PM impacts human health are yet unresolved, but evidence suggests that PM intake leads to cellular oxidative stress through the generation of reactive oxygen species (ROS). Therefore, reliable tools are needed for estimating the oxidant generating capacity, or oxidative load, of PM. The most widely reported method for assessing PM oxidative load is the dithiothreitol (DTT) assay. The traditional DTT assay utilizes filter-based PM collection in conjunction with laboratory analysis. However, the traditional DTT assay suffers from poor time resolution, loss of reactive species during sampling, and high limit of detection. Recently, a new DTT assay was developed by coupling a Particle Into Liquid Sampler with microfluidic-electrochemical detection. This 'on-line' system allows continuous monitoring of PM reactivity (~three minute measurement resolution) from substantially reduced sample masses (nanograms). This study reports on a laboratory evaluation of the on-line DTT approach. A standard urban dust sample was aerosolized in a laboratory test chamber at three atmospherically-relevant concentrations allowing comparison of the on-line and traditional DTT methods. The on-line system gave a stronger correlation between DTT consumption rate and PM mass (R2 = 0.93) than the traditional method (R2 = 0.29). The on-line system also reported ~1.4 times greater relative reactivity for a given PM sample compared to the traditional method (p = 0.022) indicating improved efficiency for the capture and detection of redox-active species. These results suggest that on-line methods for PM sampling and reactivity analysis may improve our ability to study impacts of PM exposure on human health.
Open Access
Obesity accelerates mammary carcinogenesis in a rat model of polygenic obesity susceptibility
(Colorado State University. Libraries, 2015) Matthews, Shawna Beth, author; Thompson, Henry J., advisor; Henry, Charles, committee member; Hickey, Matthew, committee member; Melby, Christopher, committee member
Given the ongoing obesity epidemic, in which more women in the US are overweight or obese than are lean, the impact of obesity on the development of breast cancer is an important public health concern. Obese women with breast cancer generally have larger tumors and poorer prognosis than lean women with breast cancer. In an effort to deconstruct the biological mechanisms that link obesity and breast cancer, we have developed a novel rat model with high relevance to the polygenic development of obesity and breast cancer in humans. These rats have differing susceptibility to obesity when fed a diet of similar macronutrient composition as that consumed by the average American woman. Diet susceptible (DS) rats rapidly accumulate excess body fat and display metabolic perturbations, including resistance to insulin and leptin, which normally provide "stop eating" anorexigenic cues. In contrast, diet resistant (DR) rats remain lean despite being fed the same diet. Findings from experiments conducted in our novel rat model have provided several critical pieces of information. When DR and DS rats were treated with a chemical carcinogen, DS rats displayed markedly accelerated mammary cancer formation compared to DR rats, including higher cancer incidence, multiplicity, and tumor burden, in conjunction with reduced cancer latency. The larger tumor mass in DS rats was found to be attributable to higher growth rates in DS vs. DR tumors, due to a combination of accelerated cell cycle progression and reduced apoptotic efficiency. Importantly, DS rats tended to develop more tumors that were negative for sex hormone receptor expression, a subtype of breast cancer with high rates of breast cancer mortality. This observation was corroborated by an endocrine ablation experiment, i.e., bilateral ovariectomy. Removal of the ovaries puts a strong selection pressure on expansion of cell populations that can grow in the absence of high circulating levels of sex hormones. In addition to removal of the primary source of circulating sex hormones, several experiments failed to provide evidence in support of peripheral production of estrogen by adipose tissue. In spite of the lack of estrogen at the host systemic and local (mammary gland) level, ovariectomized DS rats displayed elevated cancer multiplicity and sum tumor weight compared to ovariectomized DR rats, indicating that obesity in DS rats promotes the growth of cancer cells in an estrogen-independent manner. Clinically, chronic inflammation in adipose tissue as a consequence of obesity has been shown to create a permissive environment for the development of breast cancer. While DS rats display evidence of heightened fat storage in the form of adipocyte hypertrophy, there was no evidence of inflammation accompanying this hypertrophy in the rat mammary gland in the current studies. Thus, peripheral production of estrogen by fat tissue and chronic inflammation in fat tissue—two of the mainstream mechanisms proposed to link excess fat and breast cancer—do not appear to be obligatory biological processes for the effect of obesity on the increased cancer response in DS rats in our model. These findings suggest that our novel rat model represents a preclinical tool that facilitates investigation of mechanisms beyond those currently considered to link obesity to carcinogenesis of the breast. Breast cancer is a highly heterogeneous disease, and the integrated rat model reported herein is a tool that complements monogenic models of obesity and breast cancer in an effort to deconstruct the complex problem of breast cancer in clinical subpopulations whose disease is not explained via traditional mechanisms.
Open Access
Photoelectrochemical microscopy studies of transition metal dichalcogenides nanoflakes: addressing open questions of structure-function relationships
(Colorado State University. Libraries, 2022) Van Erdewyk, Michael, author; Sambur, Justin, advisor; Krummel, Amber, committee member; Henry, Charles, committee member; Stasevich, Tim, committee member
Transition metal dichalcogenides (TMDs) are exciting materials for applications in solar energy conversion. However, to advance technologies that leverage these materials, a strong understanding of fundamental photoelectrochemistry and related processes is necessary. Photoelectrochemical microscopy methods are well poised in this aspect. Methods like scanning photoelectrochemical microscopy allow for the excitation of small, localized region of a material with a focused laser and the subsequent measurement of the photocurrent. The measured photocurrent can be related to the position of the laser and the physical attributes of the material surface at the location, and variations in the photocurrent across the surface can be tracked. In this way, the technique offers insight into how different surface motifs affect the photoelectrochemical behavior of the material. This method can be combined with other spectroscopies, such as photoluminescence or Raman, to can further understanding about the studied material. The following work details the use of photoelectrochemical microscopy methods to answer questions relating to both the structure and underlying properties of mechanically exfoliated TMD nanoflakes.