Browsing by Author "Ackerson, Chris, committee member"
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Item Open Access Epidithiodioxopiperazines: synthetic studies of (+)-chetomin and (-)-sporidesmin A(Colorado State University. Libraries, 2012) Welch, Timothy R., author; Williams, Robert M., advisor; Rovis, Tomislav, committee member; Wood, John L., committee member; Ackerson, Chris, committee member; Thamm, Douglas H., committee memberThis dissertation documents efforts toward the asymmetric total syntheses of the natural products (+)-chetomin and (-)-sporidesmin A. Synthetic methods have been developed to efficiently construct the dioxopiperazine core of both molecules. Additionally, a simple epidithiodioxopiperazine has been synthesized to demonstrate a general method for the addition of a sulfur bridge to a dioxopiperazine ring. The work described herein, while not totally successful, provides a basis for future completion of the asymmetric total syntheses of these two epidithiodioxopiperazines and other related fungal metabolites.Item 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 memberGenetic 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.Item Open Access Gallium nitride high electron mobility transistors in chip scale packaging: evaluation of performance in radio frequency power amplifiers and thermomechanical reliability characterization(Colorado State University. Libraries, 2017) Shover, Michael Andrew, author; Collins, George, advisor; Chandrasekar, Venkatachalam, committee member; Chen, Thomas, committee member; Ackerson, Chris, committee memberWide bandgap semiconductors such as Gallium Nitride (GaN) have many advantages over their Si counterparts, such as a higher energy bandgap, critical electric field, and saturated electron drift velocity. These parameters translate into devices which operate at higher frequency, voltage, and efficiency than comparable Si devices, and have been utilized in varying degrees for power amplification purposes at >1 MHz for years. Previously, these devices required costly substrates such as sapphire (Al2O3), limiting applications to little more than aerospace and military. Furthermore, the typical breakdown voltage ratings of these parts have historically been below ~200 V, with many targeted as replacements for 50 V Si LDMOS as used in cellular infrastructure and industrial, scientific, and medical (ISM) applications between 1 MHz and 1 GHz. Fortunately within the past five years, devices have become commercially available with attractive key specifications: GaN on Si subtrates, with breakdown voltages of over 600 V, realized in cost effective chip scale packages, and with inherently low parasitic capacitances and inductances. In this work, two types of inexpensive commercially available AlGaN/GaN high electron mobility transistors (HEMTs) in chip scale packages are evaluated in a set of three interconnected experiments. The first explores the feasibility of creating a radio frequency power amplifier for use in the ISM bands of 2 MHz and 13.56 MHz, at power levels of up to 1 kW, using a Class E topology. Experiments confirm that a DC to RF efficiency of 94% is easily achievable using these devices. The second group of experiments considers both the steady state and transient thermal characterization of the HEMTs when installed in a typical industrial application. It is shown that both types of devices have acceptable steady state thermal resistance performance; approximately 5.27 °C/W and 0.93 °C/W are achievable for the source pad (bottom) cooled and top thermal pad cooled device types, respectively. Transient thermal behavior was found to exceed industry recommended maximum dT/dt by over 80x for the bottom cooled devices; a factor of 20x was noted with the top cooled devices. Extrapolations using the lumped capacitance method for transient conduction support even higher initial channel dT/dt rates. Although this rate of change decays to recommended levels within one second, it was hypothesized that the accumulated mechanical strain on the HEMTs would cause early life failures if left uncontrolled. The third set of experiments uses the thermal data to design a set of experiments with the goal of quantifying the cycles to failure under power cycling. It is confirmed that to achieve a high number of thermal cycles to failure as required in high reliability industrial systems, the devices under test require significant thermal parameter derating to levels on the order of 50%.Item Open Access Measuring dissolution rates and interfacial energetics of monolayer molybdenum disulfide electrodes in electrochemical systems(Colorado State University. Libraries, 2023) Toole, Justin R., author; Sambur, Justin, advisor; Henry, Chuck, committee member; Ackerson, Chris, committee member; Field, Stuart, committee memberMeeting carbon zero goals within the next few decades requires advances in energy conversion efficiency, and hydrogen fuel is believed to be a key part of the solution. Photoelectrochemical (PEC) devices can contribute to a renewable-based energy portfolio by directly producing storable chemical fuels. The electrode is a key component that determines what is thermodynamically and kinetically possible for a given PEC device. Unfortunately, semiconductor electrode efficiency can come at the cost of chemical stability. Also, the energetic description of an ultra-thin semiconductor electrode at the liquid interface is unclear. Here, we studied molybdenum disulfide (MoS2), a promising two-dimensional (2D) semiconductor, to improve understanding of interfacial energetics and electron transfer. The overarching hypothesis of this work is: if we quantitatively measure band energies of this 2D material, then we improve understanding of electron transfer efficiency and rates for involved chemical reactions. Knowledge from this research informs new ways to reduce solar energy conversion losses and may improve control over chemical reactions. Our experimental approach is to make in situ optical measurements while changing two key variables: (1) the electrode applied voltage (E), and (2) the liquid redox electrolyte environment (E0'). This thesis is organized into six chapters. Chapter 1 motivates semiconductor photoelectrochemistry as a viable approach for solar energy and chemical fuel production. Following the chronology of key scientific advances over the past few decades, Chapter 2 delves deeper into the established principles of semiconductor photoelectrochemistry, the unique properties of monolayer MoS2, and the current state of the field for making in situ optical measurements in an electrochemical cell. This chapter concludes with open questions that are addressed in Chapters 3 – 5. In Chapter 3, the stability of MoS2 is tested by literally pushing the semiconductor to its anodic decomposition limit. The crucial results are identification of the MoS2 dissolution onset potential (ED) and its thickness-dependent dissolution rates. Additional insights pertain to the long-term stability differences between monolayer and multilayer material. Chapter 4 includes the most noteworthy results wherein we develop a method to quantitatively measure the electronic band gap of monolayer MoS2 using a relatively simple optical setup. For the first time, we use an all-optical approach and many-body theory to report an abrupt change in potential-dependent band gap energies of monolayer MoS2 under electrochemical conditions. Chapter 5 summarizes preliminary work investigating how redox couples in the electrolyte may tune the optical signature of a monolayer MoS2 electrode. Finally, Chapter 6 concludes the thesis with suggestions for subsequent investigations available based on the expertise and resources within the Sambur group at Colorado State University.Item Open Access Orthogonal pair-directed codon reassignment as a tool for evaluating the factors affecting translation in E. coli(Colorado State University. Libraries, 2018) Schwark, David, author; Fisk, John, advisor; Ackerson, Chris, committee member; Kennan, Alan, committee member; Peebles, Christie, committee member; Snow, Chris, committee memberProteins are polymers of amino acids that are essential for life, central to cellular function, and have applications in fields ranging from materials science to biomedicine. Proteins in nature are composed of 20 amino acids with limited variability in size and chemical properties. Expanding the genetic code to contain non-canonical amino acids (ncAAs) that contain functionalities not contained in nature is a powerful strategy for probing and extending the properties of proteins. Current in vivo systems for expanding the genetic code have focused on using an engineered orthogonal aminoacyl-tRNA and aminoacyl tRNA-synthetase pairs (tRNA/aaRS) to direct incorporation of ncAAs at amber stop codons. In order to further expand the genetic code to 22 or more amino acids, additional codons must be targeted for reassignment to ncAAs. The genetic code is degenerate; 18 of the 20 canonical amino acids are encoded by more than one codon. Breaking the degeneracy of the genetic code by orthogonal pair directed sense codon reassignment is one pathway to genetic codes of 22 or more amino acids. However, orthogonal pair directed sense codon reassignment is hampered by a limited understanding of the relative importance of the factors that affect the translation of proteins. Here, we describe the repurposing of two commonly used orthogonal pairs from Methanocaldococcus jannaschii (M. jannaschiiI) and Methanosarcina barkeri (M. barkeri) to measure the in vivo reassignment efficiency of 30 different sense codons to tyrosine in E. coli with a simple fluorescence-based screen. The suite of sense codon reassignment efficiencies identified multiple promising codons for reassignment to ncAAs that have not been previously identified. Importantly, every sense codon was partially reassigned to tyrosine when either orthogonal tRNA/aaRS pair was used. Sense codons reassigned to tyrosine with high efficiency may be used directly for reassignment to ncAAs, and any sense codon with measurable reassignment to tyrosine may be improved through directed evolution. The sets of in vivo sense codon reassignment also revealed that E. coli are broadly tolerable to a large number of amino acid substitutions to tyrosine throughout the proteome. The codon reassignment efficiency measurements also enabled an analysis of the in vivo importance of local codon context effects, tRNA abundance, aminoacylation level, tRNA modifications, and codon-anticodon binding energy in determining translational fidelity. Quantitative sense codon reassignment efficiency measurements showed that the process of translation is highly balanced and both tRNA abundance and aminoacylation efficiency do not appear to be dominant factors in determining translational fidelity. Furthermore, quantitative measurements of amber stop codon reassignment efficiencies to tyrosine with the orthogonal M. jannaschii pair revealed that local codon context is an important factor for orthogonal pair directed amber stop codon reassignment.Item Open Access Regioselective functionalization of pyridines and other azines(Colorado State University. Libraries, 2021) Boyle, Benjamin T., author; McNally, Andrew, advisor; Chen, Eugene, committee member; Ackerson, Chris, committee member; Montgomery, Tai, committee memberPyridines and diazines serve as cores in pharmaceuticals and are common motifs in organomaterials and ligands. Selective functionalization of these motifs is of importance for discovery and optimization of new bioactive molecules. Pyridine functionalization is of interest for synthetic chemists, and despite modern advances in derivatization challenges and limitations remain. Chapter one focuses on the impact of pyridines and diazines in the pharmaceutical industry. Classical and modern methods for C-H functionalization are discussed. Chapter two describes work on phosphorus ligand-coupling for bisazine synthesis using a three-stage protocol. This method provides an alternative to traditional metal-catalysis for bisazine synthesis and can be applied to drug-like fragments. This work is expanded on in chapter three by using a tandem SNAr-ligand-coupling strategy for bisazine synthesis, in particular 2,2'-bipyridines. Chapter four describes a facile SNAr reaction by phosphines on iodopyridines. A bis-salt N-phopsphonium pyridinium is the key intermediate and provides a broad scope of reactivity. Chapter five shifts towards functionalization of the 3-position of pyridine through a Zincke-type intermediate. By exploiting a ring-opening-functionlization-rearomatization strategy a selective halogenation of pyridines was achieved. The Zincke imine intermediate is also viable for other functionalizations and provides products directly from the C-H bond on the initial pyridine.Item Open Access Transformation of soil organic matter in forest fire impacted watersheds elucidated by FT-ICR mass spectrometry(Colorado State University. Libraries, 2022) Bahureksa, William, author; Borch, Thomas, advisor; Farmer, Delphine, committee member; Ackerson, Chris, committee member; Heuberger, Adam, committee memberSoils provide numerous ecosystem services that are essential to life on Earth, including food security, water filtration and purification, and infrastructure for biodiversity. Soil properties (e.g., soil productivity, moisture retention, structure and aggregation, and nutrient supply) that facilitate these services depend on the soil organic matter (SOM), which can be dramatically impacted from ecosystem disturbances such as wildfires. Wildfires can provide benefits to an ecosystem through the cleaning of the forest floor, soil nourishment, and the removal of competitive underbrush. However, wildfires have grown in frequency and severity around the world, and there is great interest in resolving changes to SOM composition under wildfire conditions to secure water resources and recover fire-affected areas. In the following work, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was critically evaluated for the analysis of SOM. Data processing methods for FT-ICR MS were investigated to improve compositional analysis. Laboratory-simulated and field-based burn samples were collected and used to investigate changes to water-soluble fractions over progressive series of fire intensity, burn severity, and burn extent gradients. FT-ICR MS currently achieves the highest mass resolving power in the world, which makes it suitable for the study of complex mixtures with tens of thousands of compounds that are separated by mass on the order of a few electrons. Recent strategies for SOM characterization by FT-ICR MS are critically reviewed, with emphasis on SOM sample collection, preparation, analysis, and data interpretation. Importantly, the range of structures, functionalities, and mass means no technique achieves "complete" characterization, and methods used for processing and visualizing FT-ICR MS spectra can influence representation and interpretation of data. The complexity of DOM and influence of post-data processing was demonstrated by studying the effect of peak-picking threshold (3σ, 4σ, 5σ, and 6σ) on a Suwannee River Fulvic Acid standard measured by a custom 21 tesla FT-ICR mass spectrometer. Applying a 3σ peak-picking threshold revealed an additional 13,000 peaks that could be assigned compared to a 6σ peak-picking threshold with a difference of only 12 ppb root-mean-square mass error. Furthermore, isobaric overlaps differing by as little as the mass of an electron are identified up to m/z 1000, and 18O and 17O isotopologues were assigned for the first time in DOM at 3σ. Ecosystem recovery after wildfires in forested watersheds depends on revegetation and soil microbial communities and is therefore limited by the availability of nutrients. The remaining nutrients and substrate available for microbes depends on specific wildfire intensities and are poorly understood. To investigate SOM byproducts during heating and mechanisms that contribute to pyrogenic organic matter (pyOM) formation and mobilization, water-extractable organic matter was extracted from soils heated at discrete temperatures using laboratory microcosms. Relative to the unburnt control, dissolved organic carbon and nitrogen increased at ≥150°C and decreased when ≥450°C. Nitrogen-containing species predominated mass spectra at temperatures >150°C, and mass difference-based analysis suggested that products formed during heating could be used to model transformations along the Maillard reaction pathway. To investigate the short-term impacts of burn extent on water chemistry and dissolved organic matter (DOM) in fire-affected watersheds, streams originating from catchments of low, moderate, and high burn extent within the area of the Cameron Peak Fire of 2020 were sampled before, during, and after the first large rainstorm following the fire. Water chemistry parameters (DOC, TDN, turbidity) for moderate and high burn extents streams tended to increase during the storm and decrease following the storm in high burn extent streams. Fluorescence indices indicated that low/moderate burn extent streams exhibited an increase in microbially-derived residues compared to high burn extent. While a substantial portion of DOM species between every stream were common between each event and included labile and aromatic residues during the storm, the low burn extent exhibited the most unique aromatic features after the storm. When chlorinating stream samples to simulate drinking water treatment, the total DBPs were greater in streams of moderate/high burn extents compared to low burn extent. When DBP concentrations were normalized to DOC, the DOM introduced during the storm resulted in fewer DBPs, suggesting the increase in DBP formation is due to increased DOM loading overall rather than increased reactivity of the DOM. In total, the work presented here contributes to the mechanistic understanding of the residues produced during SOM heating that can be mobilized and impact water chemistry in fire-affected watersheds.Item Open Access Understanding the role of prion-like domains in ribonucleoprotein granule dynamics(Colorado State University. Libraries, 2019) Boncella, Amy Elizabeth, author; Ross, Eric, advisor; Kennan, Alan, advisor; Peersen, Olve, committee member; Ackerson, Chris, committee memberRibonucleoprotein (RNP) granules are membraneless organelles, comprised of RNA-binding proteins and RNA, that are integrally related with the cellular stress response. Stress granules and processing bodies (p-bodies) are the two primary types of RNP granules that reversibly assemble upon stress. Interestingly, many of the proteins that localize to stress granules and p-bodies contain aggregation-prone prion-like domains (PrLDs). Furthermore, mutations in the PrLDs of a number of stress granule-associated proteins have been linked to various neurodegenerative diseases, leading to the idea that aggregation-promoting mutations in these PrLDs cause stress granule persistence. Altogether, these finding suggest an important role for these domains in the dynamics of these assemblies. In order to gain a greater understanding of how PrLDs contribute to RNP granule biology, I have taken two different approaches. The first was to investigate how aggregation-promoting mutations affect stress granule and p-body dynamics. I introduced various aggregation-promoting mutations into the PrLDs of different stress granule and p-body proteins and assessed the ability of these granules to disassemble, hypothesizing that these mutations would cause RNP granule persistence, as is observed in disease. Interestingly, despite successfully increasing the aggregation propensity of these PrLDs, stress granules and p-bodies do not persist and can efficiently disassemble after stress relief. Given that aggregation-promoting mutations in PrLDs of RNP granule proteins fail to cause granule persistence, I took a second, less targeted approach towards understanding the roles of these domains in RNP granules. I focused on investigating how PrLDs are recruited to RNP granules by screening a set of PrLDs for ability to assemble into foci upon stress. Interestingly, many PrLDs are sufficient to assemble into foci upon various stresses, with robust recruitment to stress granules upon heat shock. Furthermore, several compositional biases are observed among PrLDs that are and are not sufficient to assemble upon stress. Using these biases, we have developed a reasonably accurate composition-based predictor of PrLD recruitment into heat shock-induced stress granules, which has been further validated using rational mutation strategies. This predictor is reasonably successful at predicting whether a PrLD will assemble into stress granules upon stress. Additionally, scrambling of PrLD sequences does not disrupt recruitment to stress granules. Together, these results suggest that PrLD localization to stress granules is based on composition rather than primary sequence.