Browsing by Author "Snow, Christopher D., committee member"
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Item Open Access Cloneable' nanoparticles: identification and utilization of metal reducing enzymes as biological electron microscopy tags(Colorado State University. Libraries, 2019) Butz, Zachary J., author; Ackerson, Christopher J., advisor; Nelson, James R., committee member; Snow, Christopher D., committee member; Santangelo, Thomas J., committee memberThe ability to image individual proteins in biological systems has yet to be realized. The identification and utilization of 'cloneable' nanoparticles (cNP), i.e. genetically encoded tags capable of forming in situ inorganic nanoparticles from soluble inorganic precursors is the focus of this dissertation. The long-term goal of this project is to produce GFP analogues that can then be used in electron microscopy, light microscopy, and correlative light/electron microscopy. The first chapter of this dissertation explores a metal reducing enzyme capable for converting soluble inorganic materials to insoluble (nano)particulates. Glutathione reductase-like metalloid reductase, GRLMR, was first isolated from Pseudomonas moraviensis stanleyae and characterized. GRLMR was identified as not only being able to reduce the precursor selenodiglutathione to produce Se⁰ nanoparticles but was also capable of increasing a host cells resistance to 10-fold that of the cell sans GRLMR. The structure of the enzyme was then predicted using Phyre² and related to other glutathione reductases to determine possible residues important for its inherent activity. In the second chapter a dodecapeptide was identified using phage display for its ability to bind to Se⁰ nanoparticles produced by GRLMR. Fusing this peptide to the C-terminus of GRLMR resulted in unexpected enzymes characteristics. Only when concatenated to GRLMR, the Se0 binding peptide conveyed increased size control of nanoparticle product over a wide range of substrate not seen with GRLMR alone. The peptide facilitated greater affinity between the enzyme and the nanoparticle product as well. Finally, presence of the peptide on GRLMR was also able to increase the enzyme's kinetics for precursor reduction. Raman spectroscopy was used to characterize which residues on the peptide were responsible for the interaction between the peptide and the nanoparticle surface. The third chapter explores the application of GRLMR as a cNP. A cNP tag containing two concatenated copies of GRLMR and two Se⁰ binding peptides was constructed and fused to the polymerizing protein FtsZ for expression and studies in native activity. Variants of the tagged FtsZ were isolated and studied in vitro or observed in vivo. In vitro studies resulted in filaments decorated with Se⁰ nanoparticles that could be observed with and without formal staining with uranyl acetate. Images resulting from in vivo studies indicated that both the tag and FtsZ were able to function to produce filaments within cells of high contrast. The fourth chapter isolates and characterizes a Te-reducing enzyme identified from screening environmental isolates collected throughout the Colorado Mineral Belt. A specific isolate, R. erythropolis PR4 possessed resistance to a broad range of metal and metalloid species. Specifically, R. erythropolis grew exceptionally well in up to 4.5 mM of TeO3²⁻ determined by broth microdilution. The lysate from the bacteria was also incubated in different metals and metalloids to identify any proteins with metal reductase activity. Mycothione reductase, a glutathione reductase analogue was characterized with Te-reductase activity. Mycothione reductase was then isolated and characterized and could form Te⁰ nanoparticles and bundled fibers. Although mycothione reductase was able to reduce SeO3²⁻, when the enzyme was incubated with TeO3²⁻ and an excess of SeO3²⁻ the resulting particulate had a mole ratio in favor of Te.Item Open Access Development of single cell shape measures and quantification of shape changes with cancer progression(Colorado State University. Libraries, 2018) Alizadeh, Elaheh, author; Prasad, Ashok, advisor; DeLuca, Jennifer, committee member; Munsky, Brian, committee member; Snow, Christopher D., committee memberIn spite of significant recent progress in cancer diagnostics and treatment, it is still the second leading cause of death in the United States. Some of the complexity of cancer arises from its heterogeneity. Cancer tumors in each patient are different than other patients. Even different tumors from one patient could differ from each other. Such a high diversity of tumors makes it challenging to correctly characterize cancer and come up with the best treatment plan for each patient. In order to do that, a complex combination of clinical and histopathological data need to be collected. This dissertation provides the evidence that the shape of the cells can be used in conjunction with other methods for a more reliable cancer characterization. In this study, experimental studies, numerical representation of the cell shape, big data analysis methods, and machine learning techniques are combined to provide a tool to better characterize cancer cells using their shape information. It provides evidence that cell shape encodes information about the cell phenotype, and demonstrates that the former can be used to predict the latter. This dissertation proposes detailed quantitative methods for quantifying the shape and structure of a cell and its nucleus. These features are classified into three main categories of textural, spreading and irregularity measures, which are then sub-categorized into nine different shape categories. Textural measures are used to quantify changes in actin organization for the cells perturbed with cytoskeletal drugs. Using the spreading and irregularity measures, it is shown that the changes in actin structure lead to significant changes in irregularity of the boundary of a cell and spreading of the cell and nuclei. Using these methods, the shape of retina, breast, and osteosarcoma cancer cells are quantified and it is shown that the majority of cells have similar changes in their shape once they become cancerous. Then, a neural network is trained on the shape of the cells which leads to an excellent prediction of class of cancer cells. This study shows that even though cancer cells have different characteristics, they can be categorized into clinically relevant subgroups using their shape information alone.Item Open Access How stress affects rice: a characterization of the rice transcriptome during single and simultaneous abiotic and biotic stresses(Colorado State University. Libraries, 2019) Cohen, Stephen Philip, author; Leach, Jan E., advisor; Argueso, Cristiana T., advisor; Snow, Christopher D., committee member; Antunes, Mauricio S., committee memberEnvironmental stresses, both abiotic and biotic, are large contributors to pre-harvest crop loss. Abiotic stresses, such as drought, salinity, non-optimal temperature and others, are non-living factors in the environment that have a negative effect on plants. Biotic stresses are biological factors that can harm plants, including pathogens, pests and competition from other plants. With climate change increasing the incidence of abiotic stresses and the constant pressures of pests and pathogens, it is critical to world agriculture that varieties of plants broadly tolerant to stresses are developed. For this, it is necessary to understand how plants respond to multiple simultaneous stresses. The goal of this work is to characterize the stress response of the global staple food plant rice. Here, I present the results of two comprehensive transcriptome studies. In the first, I characterize how the rice transcriptome changes in response to simultaneous heat stress and infection by the bacterial pathogen Xanthomonas oryzae (Xo). Xo includes the causal agent for the economically important bacterial blight disease of rice, Xo pathovar oryzae (Xoo). Bacterial blight is more severe during abiotic stresses such as high temperature and drought. Most rice resistance (R) genes that target Xoo lose function at high temperature; however, function of the R-gene Xa7 is enhanced when the host is subjected to abiotic stresses. Understanding why Xa7 is more effective during heat stress gives insight into host processes that are important during combined abiotic and biotic stresses. The major finding of this study was that the abscisic acid (ABA) pathway is a node of cross-talk in the interactions between heat stress and pathogen attack, during both susceptible and resistant interactions. In the second comprehensive study, I characterize how the rice transcriptome is universally regulated by all stresses. Understanding universalities in rice stress response transcriptomes provides insight into how plants endure a wide variety of stresses in the field. To explore the universal rice transcriptome response, I developed a custom workflow to analyze publicly available RNA-Seq data from rice stress response studies, including the abiotic stresses drought, salinity, heat and cold, and the biotic stresses bacterial leaf streak, bacterial blight, rice blast, and two viral diseases. From this study, I concluded that the rice stress response is a robust system with many overlapping features. This core response includes down-regulation of photosynthetic processes and up-regulation of downstream signaling of the hormones ABA, salicylic acid and jasmonic acid. Within this dissertation, I present networks of gene regulation in four major rice responses: (1) response to a susceptible interaction with Xo during high temperature, (2) response to a resistant interaction with Xo during high temperature, (3) core response to abiotic stresses and (4) core response to biotic stresses. Common among all of these pathways are the pathways upstream and downstream of the plant hormone ABA. ABA-related processes are universally up-regulated by abiotic and biotic stresses, and are only repressed during the enhanced Xa7 response at high temperature. Because ABA signaling is critical for stress response, we need a thorough understanding of how genes in the ABA response network interact to most efficiently improve rice to be tolerant to multiple and simultaneous stresses. The gene networks I have characterized can be integrated with genome and transcriptome data from stress-tolerant rice varieties. By having a complete understanding of the rice stress response, we can develop an informed approach for developing new varieties of rice that are resistant to stress.Item Open Access Mechanistic investigations and ligand development for rhodium catalyzed [2+2+2] and zinc catalyzed [4+2] cycloadditions(Colorado State University. Libraries, 2013) Dalton, Derek M., author; Rovis, Tomislav, advisor; Wood, John L., committee member; Kennan, Alan J., committee member; Rappé, Anthony K., committee member; Snow, Christopher D., committee memberDescribed herein are mechanistic studies and ligand development for Rh(I) catalyzed [2+2+2] cycloaddition reactions of alkene tethered isocyanates and exogenous alkynes. A mechanistic hypothesis has been proposed and supported through experiment. Novel perfluoroaryl Taddol phosphoramidite ligands were developed based on the mechanistic hypothesis. Improvements in product and enantioselectivity were found using the perfluoroaryl Taddol phosphoramidite ligand, CKphos. This catalyst system was studied by NMR, X-ray and DFT calculations. Rh(I)-C6F5 and Co(-1)-C6F5 interactions were found in the course of studying the CKphos catalysts. The Rh-CKphos catalyst system was used in the synthesis of the tricyclic core structure of the cylindricine and lepadiformine alkaloids. Finally a Zn(II)-catalyzed [4+2] cycloaddition of 1- azabutadienes and nitro olefins was discovered and developed as an efficient and selective means to synthesize tetrahydropyridines.Item Open Access Metabolic engineering and elucidation of the terpenoid indole alkaloid pathway in Catharanthus roseus hairy roots(Colorado State University. Libraries, 2016) Sun, Jiayi, author; Peebles, Christie A. M., advisor; Snow, Christopher D., committee member; Pilon-Smits, Elizabeth A. H., committee member; Fisk, Nick, committee memberCatharanthus roseus (Madagascar periwinkle) produces many pharmaceutically important chemicals such as vinblastine, vincristine, serpentine, and ajmalicine. They are synthesized through the highly branched and complex terpenoid indole alkaloids (TIA) pathway in C. roseus. Among these TIAs, vinblastine and vinblastine, which are solely extracted from C. roseus, are the efficient anti-cancer drugs widely used in the clinic. However, due to the low accumulation of these TIAs within the plant and the industrial infeasibility of production using chemical synthesis, the market price of these drugs still remain high, and the production is inconsistent. With the advanced knowledge of molecular biology, metabolic engineering and bioinformatics, building a robust and efficient alternative production platform by manipulating the TIA pathway has become a major trend and promising strategy in recent research. However, many biosynthetic enzymes in TIA pathway and the regulation of the pathway are still poorly understood which impedes the rational engineering of this plant for enhanced TIA production. This thesis first uses advanced high-throughput sequencing technology to study the global transcriptional alterations after overexpressing a rate-limiting enzyme anthranilate synthase (AS) in the pathway. This study helps to increase understanding of TIA regulation in this transgenic hairy root line from a broader perspective. Furthermore, transcriptome sequencing of this unique transgenic line under three different conditions (uninduced control, induced AS overexpression, and methyl jasmonate elicitation) is analyzed using hierarchical clustering. A 200 candidate transcripts set was identified for the pathway genes located around the tabersonine branch point. Six cytochrome P450 monooxygenase candidates are selected for the unknown tabersonine 6,7 epoxidase that can convert tabersonine to lochnericine in C. roseus hairy roots. Meanwhile, effort on genetic modification of C. roseus hairy roots for TIA production using two different strategies are reported here. The first strategy helps establish a transgenic hairy root line with significantly increased TIA accumulation of all measure alkaloids by co-expressing the positive transcription factor ORCA3 (AP2-domain DNA-binding protein 3) and a pathway gene strictosidine glucosidase (SGD) that is not controlled by ORCA3. Since C. roseus hairy roots do not produce detectable vinblastine and vincristine due to the absence of the vindoline pathway, the second strategy initiated the effort to introduce the pathway by engineering the first two enzymes in C. roseus hairy root. Overexpression of these two genes, tabersonine 16-hydroxylase (T16H) and 16-O-methyl transferase (16OMT), leads to the accumulation of the expected vindoline pathway intermediates 16-hydroxytabersonine and 16-methoxytabersonine but not vindoline. Interestingly, the overexpression of these two genes influences the root native metabolite levels, triggers the altered transcription of TIA genes, and leads to the production of two new unknown metabolites. Overall, studies in this thesis not only contribute new transcriptome information to current publicly available databases, but also facilitate elucidating the TIA pathway and its complex regulation. This thesis also provides a metabolic engineering approach to enhance alkaloid production in C. roseus hairy roots by simultaneously overexpressing ORCA3 and SGD. Genetic modification of T16H and 16OMT in C. roseus hairy roots promisingly leads to the production of vindoline pathway intermediates. It also emphasizes some potential complexities for the future attempts to express the full vindoline pathway in hairy roots.Item Open Access Phototunable block copolymer hydrogels(Colorado State University. Libraries, 2017) Huq, Nabila A., author; Bailey, Travis S., advisor; Kipper, Matthew J., committee member; Reynolds, Melissa M., committee member; Snow, Christopher D., committee memberThermoplastic elastomer (TPE) hydrogel networks, based on swelling of nanostructured blends of amphiphilic, sphere-forming AB diblock and ABA triblock copolymers, provide direct access to thermally processable plastics that exhibit exceptional elastic recovery and fatigue resistance even after hydration. In such two-component systems, the ratio of ABA to AB block copolymer (BCP) is used to control the resultant swelling ratio, system modulus, and overall mechanical response. This dissertation focuses on developing material strategies through which adjustment of such AB/ABA ratios, and thus the resultant properties, can be accomplished using light. The chapters within capture the manipulation of a photoreactive AB diblock copolymer micelle-like spheres to controllably generate ABA triblock copolymer and the network nanostructure in situ, both in the melt state and after dispersal in solution. This was accomplished using efficient photoinduced [4 + 4]cycloaddition (λ = 365 nm) between terminal anthracene units on a ω-anthracenylpolystyrene-b-poly(ethylene oxide) diblock copolymer precursor to produce the desired amount of polystyrene-b-poly(ethylene oxide)-b-polystyrene triblock copolymer. This direct, UV-mediated handle on tethering between adjacent micelles in the BCP matrix was found to be capable of controllably manipulating hydrogel material properties using (1) duration of irradiation, (2) hydration level and consequent micelle spacing upon exposure, and (3) photopatterning strategies to spatially direct swelling and mechanics. This level of control yielded an array of hydrogels, ranging from those irradiated in the dry melt to produce high-modulus, elastic materials suited for fibrocartilage repair and replacement, to moldable or injectable precursor solutions irradiated into soft, conformally shaped TPE hydrogels ideal for use in high contact applications such as wound healing. The development and scope of this versatile new photoactive BCP system is enclosed.Item Open Access Pump-free magnetophoresis for improved point-of-care diagnostics(Colorado State University. Libraries, 2022) Call, Zachary D., author; Henry, Charles S., advisor; Reynolds, Melissa M., committee member; Dandy, David S., committee member; Snow, Christopher D., committee memberInfectious diseases are one of the largest health burdens for low-income countries and claim millions of lives every year. The loss of life in low-income countries is largely due to the lack of access to preventative healthcare and appropriate diagnostic testing. Several health agencies have recognized the need for improved diagnostics to reduce the burden of infectious diseases. The following works described in this thesis are focused on improving the capabilities of point-of-care (POC) testing to improve patient healthcare. Microfluidic devices are a popular approach for diagnostics because they offer reduced assay times, reduced sample volume, and are small (<10 cm). Additionally, microfluidic devices can be used with magnetophoresis to improve sensitivity and specificity. However, traditional microfluidic devices have difficulty translating to the POC because of tedious and expensive fabrication. Microfluidic paper-based analytical devices (µPADs) are a popular alternative to traditional microfluidics due to the natural capillary action through cellulose fibers and simple fabrication. µPADs are portable, low-cost, and do not require external instrumentation, making them ideal for POC settings. However, µPADs often suffer from poor analytical performance resulting in failing to translate to POC testing. In Chapters 2, 3 and 4 of this thesis, I described combining µPADs with magnetophoresis to improve the analytical performance without sacrificing the advantages of µPADs. Coupling magnetophoresis with µPADs is a novel approach and was not reported until the publication of chapter two. Chapter 2 of this thesis describes the first reported example of paper-based magnetophoresis. Magnetophoresis has always needed external pumps to drive flow, however we demonstrate the ability to perform magnetophoresis completely pump-free in a µPAD. We demonstrated the ability to detect E.coli at 105 colony forming units (CFU/mL) with a fluorescent label in a pooled human urine sample. Chapter 3, describes improvements to the device described in chapter two. The limit of detection was improved by three orders of magnitude and calculated at 4.67 x 102 CFU/mL in pooled human urine, which is below detection limits for commercial urinary tract infection tests. Colorimetric detection was used instead of fluorescence detection to eliminate any instrumentation needed and create an easy read-by-eye assay. Additionally, the device design was modified to incorporate a burst valve to generate more consistent laminar flow and simplify user-end steps. We envision this technology to be used a platform for future paper-based devices incorporating magnetophoresis for improved POC devices. In Chapter 4 of this thesis, we describe a new platform for microfluidic magnetophoresis that simplifies user-end steps further through a simple magnet sliding operation. Here we introduce a MagnEtophoresis Slider Assay (MeSA) for sequential binding and washing steps without the need for any external instrumentation. A competitive biotin assay and a sandwich immunoassay are demonstrated to display the functionality of this new platform. The limit of detection was calculated at 1.62 x 103 CFU/mL using colorimetric detection. The MeSA is extremely user-friendly, provides sensitive and rapid results (<15 min), and can be applied to a wide range of applications.Item Open Access Stochastic modeling to explore the central dogma of molecular biology and to design more informative single-molecule, live-cell fluorescence microscopy experiments(Colorado State University. Libraries, 2024) Raymond, William Scott, author; Munsky, Brian, advisor; Stasevich, Timothy J., committee member; Snow, Christopher D., committee member; Ben-Hur, Asa, committee member; Krapf, Diego, committee memberDespite being described nearly a century ago, the Central Dogma of Molecular Biology still harbors many intricacies and mysteries that scientists have yet to unravel. With the convergence of many multidisciplinary scientific advances such as stronger computing power, next-generation sequencing, machine learning, and single-cell and single-molecule experiments, cellular biologists have never had more investigative power. These complex methods often are used in tandem--necessitating a closer relationship between computational biologists, computer scientists, and bench top experimentalists. As practice of this emerging dynamic, my corpus of work spans multiple areas within computational and quantitative biology with the goal to facilitate better computational tools to interpret and design experiment. For my main work at Colorado State University, I have developed the open source Python package "RNA sequence to Nascent protein simulator," rSNAPsim, to simulate Nascent Chain Tracking experiments and used it as a backbone for an entire experiment simulation pipeline to check experiment design feasibility. The rSNAPsim software provides start-to-finish capabilities for model design, model fitting, and model selection so that experimentalists can fit a mechanistic model to the Nascent Chain Tracking single-mRNA translation experiments. Along with this main work, I have provided computational modeling efforts on live-cell data on the first two steps of the Central Dogma, DNA transcription and mRNA translation. For the final entry in my corpus, I have used my interdisciplinary skills acquired at CSU to do machine learning based ncRNA riboswitch classification and discovery within the human genome; This work provides the broader scientific community with a starting point for searching for this important secondary structure within humans, where it has not been described as of time of writing.