Browsing by Author "Snow, Christopher, committee member"
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Item Open Access Development of computational tools to model molecular interactions for medicinal chemistry(Colorado State University. Libraries, 2017) Ford, Melissa Coates, author; Ho, P. Shing, advisor; Cohen, Robert, committee member; Snow, Christopher, committee member; McCullagh, Martin, committee memberMedicinal chemistry has evolved over the past 40 years to rely heavily on the computationally aided design of new drugs. The work in this dissertation focuses on developing computational tools for the application of medicinal chemistry. For computational techniques to be dependable, important interactions must be properly modeled and the techniques must be rigorously tested. In this work, I first introduce an important interaction for drug design, the halogen bond (X-bond). We consider how decades of work has come closer to properly modeling the X-bond, yet there remain many unexplored areas. Two areas are addressed in this dissertation: the structure-energy relationship of 1) a Br…S- X-bond in a DNA junction and 2) Br…O and I…O X-bonds in T4 Lysozyme (T4-L). Using these systems, we can better understand the X-bond and further test computational tools. One such tool, a molecular mechanics/dynamics package, TINKER, does not model X-bonds. Thus, I then incorporate a force field for a broad range of X-bonding molecules into TINKER, creating X-TINKER. X-TINKER reproduces the energies and geometries of the X-bond in the DNA and T4-L systems. Last, I will discuss testing a different software developed by Schrödinger, FEP+. We find FEP+ can effectively predict protein stability; however, it still has areas that need improvement. Together, the findings of this dissertation emphasize the importance of understanding molecular interactions, improving algorithms, and testing current programs to find remaining failures. By continuing to use this cycle, we hope to see the impact of computational tools in medicinal chemistry.Item Open Access Development of fluidic devices to facilitate more accessible monitoring of human health(Colorado State University. Libraries, 2024) Cherwin, Amanda E., author; Henry, Charles S., advisor; Tobet, Stuart A., advisor; Snow, Christopher, committee member; Abdo, Zaid, committee memberIn December of 2023, the World Health Organization (WHO) Director-General Tedros Adhanom Ghebreyesus outlined the 'Five P's' of global health priorities: Promoting health, Providing health, Protecting health, Powering health, and Performing for health. Despite the mantra of 'prevention is better than cure,' many countries still prioritize treating the sick over proactive health promotion, leading to inadequate prevention of non-communicable diseases (NCDs). Access to healthcare services poses a significant barrier to early recognition and treatment of health issues, particularly in low-income communities. To address these challenges, harnessing the power of science and technology becomes imperative. Powering health involves leveraging scientific research and collaboration to understand disease mechanisms better. Physiologically relevant models, such as microfluidic systems, offer insights into disease progression. Microfluidics, especially when combined with 2D and 3D culture systems, enhances functionality by mimicking physiological conditions. These devices provide cost-effective solutions for diagnostic challenges, bridging the gap between in vitro and in vivo studies. Protecting health requires a deeper understanding of organ systems. Chapter 2 examines a microfluidic model of the gut, an organ that plays a critical role in maintaining overall health. Two devices are discussed, an organotypic device for maintaining ex vivo gut tissue explants, and an electrochemical sensor module for monitoring relevant molecules such as oxygen or hydrogen peroxide within the tissue media. Dysbiosis in the gut microbiome has been linked to various pathologies, emphasizing the need for accurate models for studying gut barrier integrity. Ex vivo models using microfluidic devices offer promising avenues for studying disease mechanisms. The devices described in Chapter 2 serve as an effective model of the intestinal barrier that can be closely monitored in real-time. Providing health involves making effective healthcare solutions universally accessible. Point-of-care (POC) diagnostics, facilitated by microfluidics, enable rapid and cost-effective disease detection. Capillary-driven flow microfluidic devices enhance accessibility by eliminating the need for bulky external pumps, making POC testing feasible even in resource-limited settings. Combining the concepts of Powering and Providing health leads to the development of innovative diagnostic devices. Capillary-driven flow microfluidics enables the development of portable devices for diagnosing conditions from viscous sample matrices like blood and saliva. These devices offer less invasive and more accessible alternatives to traditional diagnostic methods, potentially revolutionizing healthcare delivery. Chapter 3 describes a capillary flow device used to quantify levels of two salivary biomarkers (Galectin-3 and S100A7) correlated to Heart Failure (HF) outcomes. This rapid, noninvasive, accessible POC test can drastically improve the quality of life for HF patients, particularly in rural and resource-limited areas. Using an electrochemical detection method, we demonstrate successful multiplexed detection of both biomarkers in spiked buffer solutions. Chapter 4 focuses on microfluidic devices probing rheological properties of whole blood related to Sickle Cell Disease (SCD) and clotting using capillary flow. For the SCD device, our goal was to develop a low-cost Point-of-Care (POC) multiplexed device for rapid and accurate identification of SCD phenotypes using three key reagents tied to altered sickle cell blood rheology: calcium chloride, sodium metabisulfite, and adenosine diphosphate. We developed an integrated device where whole blood reacts with reagent pads, enabling rapid assessment of a patient's SCD phenotype to inform appropriate treatment. We also introduced the Paper-based Clotting Analysis Test (PCAT) for efficient, low-cost analysis of primary hemostasis. Current methods for monitoring hemostasis are expensive and slow. Our capillary flow device uses whole blood moving at high flow rates for sustained durations to induce thrombus formation. This dissertation bridges the gap between effective health monitoring and accessibility through fluidic devices using either pump-driven or capillary-driven flow. Chapters detail the development of microfluidic systems for monitoring intestinal barrier function, detecting biomarkers in saliva for Heart Failure prognosis, and processing blood samples for Sickle Cell Disease phenotyping and clotting analysis. Ultimately, these devices hold the potential to transform healthcare management, particularly in underserved communities.Item 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 memberThe 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.Item Open Access Exploration of protein engineering methods towards biomaterials, therapeutic protein scaffolds, and localization detection within mammalian cells(Colorado State University. Libraries, 2019) Bjerke, Jennifer N., author; Kennan, Alan, advisor; Snow, Christopher, committee member; Williams, Robert M., committee member; Nyborg, Jennifer, committee memberProteins are large biomolecules entangled with complex chemistry that uniquely control the processes, function and efficiency of everyday life. These macromolecules are the final product of the central dogma, and as in any synthesis, the proper reactants must combine in order to produce the correct products. As technologies develop to understand their production and resulting structure, sequence and activities, they have increasingly become a bulk chemical platform in which nearly any desired application can be engineered- ranging from materials to therapeutics. The first half of this dissertation will discuss the potential to break down the central dogma in order to create new biological materials through incorporation of novel building blocks, known as amino acids. The second half will focus solely on the design and analysis of engineered proteins to create scaffolds for biological therapeutics that have the capability to bind almost any disease relevant target. The second chapter of this dissertation describes the synthesis of novel non-canonical amino acids that add new chemistries to any protein of interest. Using amber codon suppression, and careful engineering of tRNA synthetase and cognant tRNA's, these new amino acids can be site selectively incorporated. To start, derivatives of phenylalanine are selected since this machinery has been successfully implemented for many new amino acids. The synthesis of bithiophene explores the utilization of phase transfer catalysis and transition metal cross- coupling to deliver a racemic mixture of the non-canonical amino acid with a bithiophene moiety. The third chapter of this dissertation will discuss the engineering potential of nanobodies, a monomeric protein responsible for all binding interactions, isolated from the variable heavy chain of camelid antibodies. We had previously reported a cationic resurfacing strategy that endowed mammalian cell penetration in three nanobody scaffolds. We have since explored and refined this strategy, along with endowing the capability to bind new targets. In the end we found that, when fused to sfGFP, a polyarginine version (PolyR-NB) increased internalization over the original cationic GFP nanobody (CatNB), but the original CatNB scaffold was more adaptable to extensive mutagenesis. Therefore, the PolyR nanobody scaffold can be utilized to aid the internalization of therapeutic cargo proteins that would otherwise not be able to cross the lipid bilayer, and the CatNB scaffold can become a therapeutic itself, modified to bind any intracellular target of disease relevant proteins. To further prove the later point, the complimentary determining regions (CDRs) of a separate nanobody, the BC2 nanobody, were grafted upon the CatNB framework, keeping all resurfacing and structure intact. Remarkably, the CatNB-BC2 CDR nanobody was able to maintain binding to its wild type partner. The final chapter will showcase the efforts towards development of a facile luminescent assay that detects protein delivery to the cytosol of cells. Adapted from the NanoBit assay developed by Promega to identify protein-protein interactions, our assay utilizes the split nanoluciferase technology to produce a luminescent signal once an exogenous protein has recombined with its other half in the cytosol. This system, in theory, could be applied to identify endosomal release of virtually any therapeutically relevant protein.Item Open Access Investigating the role of the cellular RNA decay pathway during flavivirus replication(Colorado State University. Libraries, 2022) Denis, Denis, author; Geiss, Brian, advisor; Willusz, Jeffrey, committee member; Ebel, Gregory, committee member; Snow, Christopher, committee memberThe Cellular RNA decay pathway is an important regulatory system that affects both the quality and quantity of mRNA within the cells. Previous studies have shown that RNA viruses develop evasion mechanism to the cellular RNA decay pathway due to its antiviral nature. In eukaryotic cells, 5'-3' exonucleolytic decay mediated by XRN1 is known to be the major RNA decay pathway that interacts with RNA viruses. To provide RNA substrates for XRN1 degradation, cellular mRNA and viral RNA need to be processed by decapping enzymes such as DXO and DCP2 or RNA triphosphatases such as DUSP11 and NUDT2. Currently, there are few studies that have examined the roles of these proteins during RNA virus replication. In this study, we performed a loss-of-function experiment utilizing siRNA-mediated knockdown to reduce various RNA decay proteins and examine their effects on flavivirus replication. Collectively, our data suggested that knocking down DUSP11 and NUDT2 did not significantly affect the replication of infectious flaviviruses, whereas depletion of DCP2, showed significantly diminished West Nile virus and Zika virus replication but not on yellow fever virus. The results of this project indicate that DCP2 acts as a proviral factor for several but not all flaviviruses during infection and provide new insight into how flaviviruses may generate RNAs for XRN1-mediated degradation and subsequent XRN1 inhibition.Item Open Access Large margin methods for partner specific prediction of interfaces in protein complexes(Colorado State University. Libraries, 2014) Minhas, Fayyaz ul Amir Afsar, author; Ben-Hur, Asa, advisor; Draper, Bruce, committee member; Anderson, Charles, committee member; Snow, Christopher, committee memberThe study of protein interfaces and binding sites is a very important domain of research in bioinformatics. Information about the interfaces between proteins can be used not only in understanding protein function but can also be directly employed in drug design and protein engineering. However, the experimental determination of protein interfaces is cumbersome, expensive and not possible in some cases with today's technology. As a consequence, the computational prediction of protein interfaces from sequence and structure has emerged as a very active research area. A number of machine learning based techniques have been proposed for the solution to this problem. However, the prediction accuracy of most such schemes is very low. In this dissertation we present large-margin classification approaches that have been designed to directly model different aspects of protein complex formation as well as the characteristics of available data. Most existing machine learning techniques for this task are partner-independent in nature, i.e., they ignore the fact that the binding propensity of a protein to bind to another protein is dependent upon characteristics of residues in both proteins. We have developed a pairwise support vector machine classifier called PAIRpred to predict protein interfaces in a partner-specific fashion. Due to its more detailed model of the problem, PAIRpred offers state of the art accuracy in predicting both binding sites at the protein level as well as inter-protein residue contacts at the complex level. PAIRpred uses sequence and structure conservation, local structural similarity and surface geometry, residue solvent exposure and template based features derived from the unbound structures of proteins forming a protein complex. We have investigated the impact of explicitly modeling the inter-dependencies between residues that are imposed by the overall structure of a protein during the formation of a protein complex through transductive and semi-supervised learning models. We also present a novel multiple instance learning scheme called MI-1 that explicitly models imprecision in sequence-level annotations of binding sites in proteins that bind calmodulin to achieve state of the art prediction accuracy for this task.Item Embargo Mapping the metabolic protein interactome that supports energy conservation at the limits of life(Colorado State University. Libraries, 2024) Williams, Seré Anne, author; Santangelo, Thomas, advisor; Hansen, Jeffrey C., committee member; Pilon, Marinus, committee member; Anderson, G. Brooke, committee member; Snow, Christopher, committee memberDistinct metabolic strategies yield energetic gains from a wide variety of substrates, yet only three overarching methods of energy conservation have been defined: substrate level phosphorylation, the generation of a charged membrane, and electron bifurcation. The dominant theme of known energy conservation mechanisms suggests that energy is conserved through the selective movement and management of electrons, thus essentially all life relies on redox (reduction and oxidation) reactions. Small molecule redox cofactors (such as NAD(P)+) and proteinaceous electron carriers (such as ferredoxins) are employed as electron carriers throughout the biosphere. Proteinaceous electron carriers offer the potential for selective protein-protein interactions to bridge reductive flow from catabolic reactions to the membrane, providing a "proteinaceous electron highway" for efficient electron shuttling. Specific redox protein partnerships have been shown to adapt to changing physiological conditions, suggesting that proteinaceous electron flux is tunable and provides a level of selectivity not possible with small molecule electron transport. While electron flux through a tunable and regulated system of protein interactions can offer exceptional energy conservation strategies, large gaps remain in our knowledge of how electron flux is regulated in vivo. Identification of bona fide in vivo protein assemblies – and how such assemblies dictate the totality of electron flow and thus cellular metabolism – is an important milestone to understand the regulation imposed on metabolism, energy-production, and energy conservation. Resolving the dynamic nature of nanoscale interactions in living systems is arguably the current frontier of molecular biology, and combinatorial methods – which layer multiple in vitro and in vivo techniques with large data analysis – have come to the forefront. This dissertation addresses energy conservation strategies of in vivo protein associations in a model, genetically accessible, hyperthermophilic archaeon (Thermococcus kodakarensis) by mapping the metabolic protein interactome using affinity purification mass spectrometry (AP-MS) and generating engineered strains where fusion proteins selectively redirect electron flux in vivo. Twenty-five proteins involved in distinct metabolic functions were tagged to reveal that each tagged-protein interacts with ~ thirty proteins on average. These interactions connected disparate functions suggesting catabolic and anabolic activities may occur in concert -- in temporal and spatial proximity in vivo. The AP-MS method also refined our understanding of previously determined stable complexes suggesting that protein complexes in vivo likely adapt to redox conditions. Engineered strains linking a proteinaceous electron donor to a proposed electron acceptor were viable and impacted electron flux in vivo. Fusion strains linking a ferredoxin to the hydrogen-generating respiratory system increased hydrogen gas output ~8% on average with one strain showing a ~45% increase over wild type. Fusion strains impacting lipid saturation were shown to inhibit saturation, and future studies aim to determine if electrons can be redirected from the vast reductant sink of lipids to the generation of hydrogen gas, a valuable biofuel.Item Open Access N,N-Diaryl Dihydrophenazine photoredox catalysis for organocatalyzed atom transfer radical polymerization(Colorado State University. Libraries, 2019) Ryan, Matthew David, author; Miyake, Garret, advisor; Chen, Eugene, committee member; Kota, Arun, committee member; Snow, Christopher, committee memberThe synthesis, application, and mechanistic investigation of the 5,10-diaryldihydrophenazine catalyst family as applied to organocatalyzed atom transfer radical polymerization is presented in this dissertation. The N,N-Diaryl Dihydrophenazine catalyst family, which will be referred to in this dissertation as the phenazines, are an appealing class of molecules due to their strongly reducing excited states, accessed through modular syntheses enabling a wide range of photophysical and electrochemical properties. This class of molecules represented the first example of organic catalysts capable of operating a controlled, visible light driven, organocatalyzed atom transfer radical polymerization for the precision syntheses of (meth)acrylic polymers. Phenazine catalysts were shown to polymerize (meth)acrylic monomers to polymers of very low dispersities (< 1.10) in a process with quantitative initiator efficiency; both features crucial to produce precision polymeric materials poised for myriad applications. Supported by computational efforts, mechanistic understanding and structure-property-catalyst activity relationships were identified and harnessed to design optimal polymerization conditions, which have laid the groundwork for new research efforts into highly reducing, visible light absorbing, organic photocatalysts.Item Open Access Nitrogen utilization in heterotrophic Chlamydomonas reinhardtii(Colorado State University. Libraries, 2017) Sweeley, Justin Brye, author; Reardon, Kenneth F., advisor; Peebles, Christie, committee member; Snow, Christopher, committee member; Peers, Graham, committee memberThe aim of this dissertation research is to bring better understanding to the process of nitrogen adaptation in heterotrophic Chlamydomonas reinhardtii. Microalgae are a diverse group of aquatic photosynthetic organisms that account for almost 50% of the photosynthetic productivity on Earth. There is immense interest in using the unique ability of microalgae to convert sunlight to triacylglycerides (TAGs) for industrial purposes. However, to date there has been little success in implementing these systems at scale and price parity with non-biological methods. Microalgae can modify their metabolism to adapt to the surrounding environment. Under certain circumstances, including nutrient stress, microalgae divert carbon flow away from biomass production and into TAG accumulation. The most common nutrient stress used to trigger TAG accumulation is nitrogen stress, most often induced by transferring a cell from a nitrogen replete medium to a deficient one. The goals of this research were to understand this process, develop methods to manipulate the stress response, and ultimately, to find a way to decouple lipid production from nutrient depletion entirely. Chapter 1 introduces the concepts and research referenced throughout the dissertation including: a background of the C. reinhardtii species, the cultivation techniques that have been applied to cultivation, the physiology behind nitrogen stress, the mechanism that algae use to incorporate nitrogen into the cell, and finally an introduction to the global nitrogen regulator, PII. Chapters 2 through 4 present research into the nitrogen stress pathway and its modification. Chapter 2 discusses a simple method of cultivation used to bring about new insights into the nitrogen stress response, as well as a proposed technique for increasing cellular lipid production. Through differential nitrogen feeding, significantly different effects on cell growth were observed, demonstrating that the response to nitrogen availability is a continuous effect as opposed to an all or nothing "stress response". Chapter 3 describes experiments in which C. reinhardtii was genetically modified to increase understanding of the nitrogen stress response. A nitrogen regulatory protein, PII, was downregulated via amiRNA. Cultures of a mutant strain with lower levels of PII exhibited slow adaptation to fresh nutrient-replete medium but achieved a higher final cell number, final mass concentration, and total neutral lipid content. Similar results were obtained in cultures shifted to nitrogen-free medium. Chapter 4 employs proteomics to identify differences in the specific protein expression pattern between a functional PII strain and a knock-down mutant. Chapter 5 demonstrates a unique approach to producing an engineered nutrient-limited environment in a continuous stirred tank bioreactor. Chapters 7 and 8 summarize the research findings and offer possible direction for future research. Through this research work, new information was obtained on the effects of PII on the cellular response to nitrogen limitation. By increasing our understanding of this basic mechanism, we have proposed several processing conditions that may be implemented to increase microalgal productivity. Furthermore, the homology between microalgae and terrestrial plants suggests the possibility that the results discussed within could give genetic engineers new targets for creating crops with decreased nitrogen demands and increased nitrogen-stress tolerance traits.Item Open Access Novel applications of advanced integral-equation theories to various polymeric systems(Colorado State University. Libraries, 2021) Wang, Yan, author; Wang, Qiang, advisor; Snow, Christopher, committee member; Bailey, Travis, committee member; Grzegorz, Szamel, committee memberTo view the abstract, please see the full text of the document.Item Open Access Novel in silico-designed SMYD3 inhibitors eliminate unrestrained proliferation of breast carcinoma cells(Colorado State University. Libraries, 2021) Alshiraihi, Ilham Mohammed, author; Brown, Mark, advisor; Kato, Takamitsu, advisor; Reynolds, Melissa, committee member; Snow, Christopher, committee memberSMYD3 is a lysine methyltransferase that regulates the expression of over 80 genes and is required for the uncontrolled proliferation of most breast, colorectal, and hepatocellular carcinomas. Elimination of SMYD3 restores normal expression patterns of these genes and halts aberrant cell proliferation. In this study, we used in silico screening to identify potential small molecule inhibitors of SMYD3 and tested the ability of these inhibitors to reduce its methyltransferase activity in vitro. Using breast cancer cell lines that overexpress SMYD3 and normal breast epithelial cell lines, we have confirmed the ability of one of these inhibitors, Inhibitor-4, to reduce cell proliferation, arrest the cell cycle, and induce apoptosis in breast cancer cells without affecting normal cell behavior. Our results provide a proof of concept for the in silico design of small molecule enzyme inhibitors and for the use of such an inhibitor to target SMYD3 for the treatment of cancer.Item Open Access Protein engineering strategy for the stabilization of HIV-1 α-helical peptides(Colorado State University. Libraries, 2019) Tennyson, Rachel Lee, author; Kennan, Alan, advisor; Ackerson, Christopher, committee member; Snow, Christopher, committee member; Gustafson, Daniel, committee memberMany disease-relevant protein-protein interactions (PPIs) contain an alpha helix and helical binding cleft at their interface. Disruption of these interactions with helical peptide mimics is a validated therapeutic strategy. However, short peptides typically do not fold into stable helices, which significantly lowers their in vivo stability. Researches have reported methods for helical peptide stabilization but, these approaches rely on laborious, and often expensive, chemical synthesis and purification. The research I have preformed aims to stabilize disease-relevant helices through protein engineering. In contrast to chemically constrained helical peptides, a protein can be expressed in a cellular system on a much larger scale. Recently, we reported a new strategy termed "helix-grafted display" that overcomes the traditional hurdles of helical mimics and applied it to the challenge of suppressing HIV entry. Our helix grafted proteins, potently inhibits formation of the extracellular PPI involving C-peptide helix, and HIV gp41 N-peptide trimer, as tested in HIV CD4+ cells. Further optimization of the helical sequence by yeast display yielded new proteins that suppress HIV-1 entry and express substantially better in E. coli. Furthermore, fusion proteins designed to improve the serum stability of these helix grafted proteins have been made that potently suppress HIV-1 entry. Collectively, I report a potential cocktail of evolved HIV-1 entry inhibitors that are functional against an Enfuvirtide-resistant strain and are designed for serum stabilities that rival current monoclonal antibody drugs.Item Open Access Protoporphyrinogen oxidase: origins, functions, and importance as an herbicide target site(Colorado State University. Libraries, 2021) Barker, Abigail, author; Dayan, Franck, advisor; Snow, Christopher, committee member; Pilon, Marinus, committee member; Gaines, Todd, committee memberProtoporphyrinogen IX oxidase (PPO)-inhibiting herbicides are effective tools to control a broad spectrum of weeds, including those that have evolved resistance to glyphosate. Their utility is being threatened by the appearance of biotypes that are resistant to PPO inhibitors. While the chloroplastic PPO1 isoform is thought to be the primary target of PPO herbicides, evolved resistance mechanisms elucidated to date are associated with changes to the mitochondrial PPO2 isoform, suggesting that the importance of PPO2 has been underestimated. Our investigation of the evolutionary and structural biology of plant PPOs provides some insight into the potential reasons why PPO2 is the preferred target for evolution of resistance. The most common target-site mutation imparting resistance involved the deletion of a key glycine codon. The genetic environment that facilitates this deletion is apparently only present in the gene encoding PPO2 in a few species. Additionally, both species with this mutation (Amaranthus tuberculatus and Amaranthus palmeri) have dual targeting of PPO2 to both the chloroplast and the mitochondria, which might be a prerequisite to impart herbicide resistance. The most recent target-site mutations have substituted a key arginine residue involved in stabilizing the substrate in the catalytic domain of PPO2. This arginine is highly conserved across all plant PPOs, suggesting that its substitution could be equally likely on PPO1 and PPO2, yet it has only occurred on PPO2, underscoring the importance of this isoform for the evolution of herbicide resistance. As glyphosate resistance becomes widespread, weed control turns to older mechanisms of action with less resistance. Protoporphyrinogen oxidase (PPO) inhibitors are a versatile class of herbicides that have been used since the 1960's, with active ingredients that work in pre-emergent and post-emergent applications. Differential efficacy of PPO inhibitors applied pre-emergent, early post-emergent and late post-emergent has been observed in multiple species and settings. Understanding the cause of higher efficacy in younger plants could preserve these important weed control tools. To understand the differing efficacies elements that affect the mechanism of action of PPO inhibitors were analyzed over the course of plant growth including target site transcript levels and protein levels, herbicide uptake, antioxidant capacity, and indicators of flux through the pathway. Data show levels of PPO do not explain differential efficacy. Increases of glutamate, the pathway precursor, do increase damage due to PPO inhibitor treatment, but increased levels are not observed in younger plants. Differential efficacy is likely due to a combination of increase in antioxidant capacity and a decrease in herbicide uptake. Other possible factors such as metabolism will need to be measured in future work. Protoporphyrinogen oxidase (PPO) is a critical enzyme across life as the last common step in the synthesis of many metalloporphyrins. The reaction mechanism of PPO was assessed in silico and the unstructured loop near the binding pocket was investigated. The substrate, intermediates, and product were docked in the catalytic domain of PPO using a modified Autodock method, introducing flexibility in the macrocycles. Sixteen PPO protein sequences across phyla were aligned and analyzed with Phyre2 and ProteinPredict to study the unstructured loop from residue 204–210 in the H. sapiens structure. Docking of the substrate, intermediates, and product all resulted in negative binding energies, though the substrate had a lower energy than the others by 40%. The α-H of C10 was found to be 1.4 angstroms closer to FAD than the β-H, explaining previous reports of the reaction occurring on the meso face of the substrate. A lack of homology in sequence or length in the unstructured loop indicates a lack of function for the protein reaction. This docking study supports a reaction mechanism proposed previously whereby all hydride abstractions occur on the C10 of the tetrapyrrole followed by tautomeric rearrangement to prepare the intermediate for the next reaction. Weed control is essential in modern agriculture, though it becomes more difficult with increasing resistance levels to current herbicides and a slow process to register a new mechanisms of action because of safety concerns and current methods. Agrematch provides a new method to identify possible herbicide candidates using an artificial intelligence algorithm that takes into effect biological parameters with the goal of reducing R&D time on new herbicides. Herein we describe the discovery of 4-chloro-2-pentenamides as novel inhibitors of protoporphyrinogen oxidase, a known herbicide target site, by the Agrematch AI. The herbicidal activity is confirmed in greenhouse assays, with the highest performing AGR001 showing good activity pre-emergent at 150 g/ha and post emergent as low as 50 g/ha on the troublesome weed palmer amaranth (Amaranthus palmeri). A lack of activity is shown on PPO resistant palmer amaranth carrying the ΔG210 deletion mutation. The mechanism of action is confirmed by the herbicide dependent accumulation of protoporphyrin IX, subsequent light dependent loss of membrane integrity, and direct inhibition of protoporphyrinogen oxidase in an in vitro assay. Modeling of the docking of these inhibitors in the active site of protoporphyrinogen oxidase confirms the target.Item Open Access Relationships between hydrogen bonds and halogen bonds in biomolecular engineering(Colorado State University. Libraries, 2019) Hartje, Rhianon Kay Rowe, author; Ho, P. Shing, advisor; Reynolds, Melissa, committee member; Snow, Christopher, committee member; Stasevich, Tim, committee member; Woody, Robert, committee memberIn this dissertation, we will explore the interconnectedness between halogen bonds (X-bonds) and hydrogen bonds in rational biomolecular engineering efforts. As X-bonds are not readily designed into biomolecules, we aim to show how they can be advantageous for molecular design. We will begin by considering how X-bonds compare to H-bonds and show how the two can work in harmony to provide enhanced stabilizing potential. In two unique protein engineering efforts we will show 1) how the X-bond can be just as specifying in terms of molecular assembly as compared to the H-bond, and 2) how it can coordinate with the H-bond to increase protein stability. One study shows the specifying potential the X-bond possesses in terms of coiled-coil assembly. While the study points to a direct application of a sensing probe, the scope of the work will aid others using coiled-coils for materials purpose, designing protein interfaces or potential ligand binding sites. In the other protein engineering study, we will survey how a protein with an intrinsically disordered region responds to hydrogen enhanced halogen bond engineering. We show how we can drastically increase the thermal stability of the protein through minimal change to its primary sequence. This study lends itself to exploring bigger structure-function questions and how the stabilizing capacity of halogen bonds fits into this. Through this work we aspire to show how useful X-bonds can be for biological engineering efforts by exhibiting their specifying and stabilizing characteristics in these settings.Item Open Access The flavivirus NS3 helicase Motif V controls unwinding function and alters viral pathogenesis in mosquitoes(Colorado State University. Libraries, 2020) Du Pont, Kelly Elizabeth, author; McCullagh, Martin, advisor; Geiss, Brian J., advisor; Szamel, Grzegorz, committee member; Snow, Christopher, committee member; Krummel, Amber, committee member; Ho, Shing, committee memberOver half of the world's population is at risk of flavivirus (e.g. dengue virus, West Nile virus, Japanese Encephalitis virus, and Zika virus) infection making it a global health concern. These specific mosquito-borne flaviviruses are responsible for causing a variety of symptoms and outcomes including flu-like fevers, encephalitis, hemorrhagic fevers, microcephaly, Guillain-Barré syndrome, and death. Unfortunately, vaccines and anti-viral therapeutics are not always effective in protecting against and treating viral infections. Sometimes these therapies cause more severe symptoms through an antibody dependent enhancement. Therefore, there is a pressing need for the development of effective anti-viral therapies against each flavivirus. For the advancement of these interventional strategies, a fundamental understanding of how flaviviruses replicate within hosts, including the mosquito vector, is required. This dissertation investigates how flaviviruses regulate viral replication, pathogenesis and mosquito transmission through the nonstructural protein 3 (NS3) helicase structure and function. A combination of virology, biochemistry, and computational simulations will be utilized to address how NS3 plays a role in viral infection, viral replication, and viral protein structure. An essential aspect of flaviviral genome replication is the unwinding of the double-stranded RNA intermediate via the C-terminal helicase domain of NS3. NS3 helicase translocates along and unwinds the double-stranded nucleic acids in an ATP-dependent manner. However, the mechanism of energy transduction between the ATP- and RNA-binding pockets is not well understood. Previous simulations in the group led us to hypothesize that Motif V is a critical component of the transduction mechanism. Here, we tested Motif V mutations in both sub-genomic replicon and recombinant protein systems to examine viral genome replication, helicase unwinding activity, ATP hydrolysis activity, and RNA binding affinity activity. NS3 helicase mutants, T407A and S411A, indicated reduced viral genome replication and increased turnover rates of helicase unwinding activity by a factor of 1.7 and 3.5 respectively. Additionally, we simulated Motif V mutants to probe the structural changes within NS3 helicase caused by the mutations. These simulations indicate that Motif V controls communication between the ATP-binding pocket and the helical gate. Motif V mutations T407A and S411A exhibit a hyperactive helicase phenotype leading to the regulation of translocation and unwinding during viral genome replication. Next, we utilized T407A and S411A West Nile virus (Kunjin subtype) mutants in cell culture and in vivo to probe the how these mutations play a role in pathogenesis and transmission of flaviviruses. Of the two Kunjin virus mutants, only S411A Kunjin virus was recovered. In cell culture, S411A Kunjin decreased viral infection and increased cytopathogenicity as compared to WT Kunjin. Similarly, in surviving Culex quinquefasciatus mosquitoes, S411A Kunjin decreased infection rates as compared to WT Kunjin, but S411A Kunjin infection increased mortality compared with that of WT Kunjin infection. Additionally, S411A Kunjin increased viral dissemination and saliva positivity rates in surviving mosquitoes compared to WT Kunjin. These data suggest that S411A Kunjin increases pathogenesis in mosquitoes. Overall, these computational simulation, biochemical assay, and virology data indicate that flavivirus NS3 helicase Motif V may play a role in the pathogenesis, dissemination, and transmission efficiency of Kunjin virus, not just regulation of translocation and unwinding during viral genome replication. The molecular level insights presented in this dissertation provide the fundamental research for understanding how to target specific regions of NS3 helicase for the advancement of anti-viral therapeutics.Item Open Access Thermoplastic electrode surface modifications for use as label-free electrochemical immunosensors(Colorado State University. Libraries, 2024) Martinez, Brandaise, author; Henry, Charles S., advisor; Reynolds, Melissa, committee member; Snow, Christopher, committee member; Tobet, Stuart, committee memberPoint-of-care (POC) testing has grown in popularity in recent years, though most common lateral flow assay (LFA) techniques lack sensitivity and are not quantitative. Electrochemical sensors are a promising alternative, specifically thermoplastic electrodes (TPEs) due to their electrochemical performance and durability while remaining inexpensive. TPEs have been used for a wide variety of applications, but their use as immunosensors has been limited due to difficulty with antibody immobilization. This work seeks to explore techniques for modifying TPE surfaces for use as label-free immunosensors. Chapter 2 examines common antibody immobilization techniques applied to TPEs and determines that the standard existing protocols are lacking. Passive adsorption, EDC/NHS coupling, and chitosan films are used to attach antibodies to the surface. It was found that while each are commonly used in immunosensor fabrication, they have drawbacks that make them unsuitable for TPE immunosensors. Passive adsorption results in unstable antibody attachments leading to inconsistent sensing. EDC/NHS crosslinking is prone to side reactions and again led to inconsistencies in detection of the antigen. Chitosan films were perhaps the most promising, but they passivated the electrode to the extent that detecting the antigen was limited. Chapter 3 moves towards the development and characterization of a new TPE surface modification using aryl diazonium grafting followed by click chemistry to biotinylate electrodes for easy antibody immobilization. A variety of electrochemical techniques and surface characterizations were used to examine the stepwise modification of the TPE surface. It was shown that click chemistry can be successfully used on TPEs to attach various moieties following aryl diazonium grafting. Ethynyl ferrocene was clicked to the surface resulting in a surface coverage (ΓFc) of (1.0 ± 0.2) × 10-10 mol∙cm-2, which is comparable to literature values for similar approaches on commercial carbon electrodes. Streptavidinated antibody was successfully attached as well with a clear change in electrochemical signal upon binding. The method is expanded in Chapter 4 with the use of heterogeneous modifications with multiple functions. The monolayer contains surface bound ferrocene to aid in electron transport, long polyethylene glycol (PEG) spacers to block nonspecific adsorption, in addition to the antibody immobilization point. The modified TPEs were used to successfully detect the nucleocapsid protein of inactivated SARS-CoV-2 virus in buffer solution as a proof-of-concept without the need for a label. The LOD was approximately 6 PFU/mL which exceeds many existing POC tests for COVID-19. The work here expands on the potential applications of TPEs with increased performance and durability over other carbon electrode immunosensors. Potential future directions to expand the sensing capabilities include multiplexed sensors, alternative electrode materials, and expanding to non-antibody based systems.