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Browsing by Author "Argueso, Juan Lucas, committee member"

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    Assessment of water quality, toxicity and treatment strategies downstream of NPDES oil and gas produced water discharges intended for beneficial reuse
    (Colorado State University. Libraries, 2019) McLaughlin, Molly Cook, author; Borch, Thomas, advisor; Blotevogel, Jens, advisor; Argueso, Juan Lucas, committee member; Mouser, Paula, committee member; Sale, Tom, committee member
    Produced water is the largest waste stream associated with oil and gas operations. This complex fluid contains petroleum hydrocarbons, heavy metals, salts, naturally occurring radioactive materials (NORMs) and any remaining chemical additives. In the United States, west of the 98th meridian, the federal National Pollutant Discharge Elimination System (NPDES) exemption allows release of produced water for agricultural beneficial reuse if it is of "good enough quality." Due to the complex and variable composition of produced water as well as the variations in permit effluent limits and treatment approaches, the downstream impacts of NPDES produced water releases are not fully understood. The goal of this dissertation was to determine if the current NPDES produced water permit effluent limits are adequate and if not, to identify additional steps that can be taken to improve water quality. As a first step towards this goal, a detailed chemical and toxicological analysis was conducted on a stream composed of produced water released for agricultural beneficial reuse. Over 50 geogenic and anthropogenic organic chemicals not specified in the effluent limits were detected at the discharge including hydrocarbons, halogenated compounds, and surfactants. Most were removed within 15 km of the discharge due to volatilization, biodegradation, and sorption to sediment. Additionally, the attenuation rate increased substantially in a wetland downstream of the discharge point. Tens of inorganic species were also detected in the watershed, including many sourced from produced water. In contrast to organic chemicals, the concentrations of most inorganic species increased downstream due to water evaporation. This included contaminants of concern such as boron, selenium and total dissolved solids (TDS). An assessment of regulatory health thresholds revealed that eight of the organic species detected at the discharge were listed by the U.S. Environmental Protection Agency (EPA) and the International Agency for Research on Cancer (IARC) to be known, probable or possible carcinogens. Mutagenicity of this water was assessed using a yeast mutation assay that analyzed copy number variation (CNV) duplications, CNV deletions, forward point mutations and reversion point mutations. These mutations are established as having a role in human disease, including cancer. Higher rates of mutation were observed at the discharge point and decreased with distance downstream. This correlated with the concentrations of known carcinogens detected in the stream including benzene and radium. Mutation rate increases were most prominent for CNV duplications and were higher than mutation rates observed in mixtures of known composition containing all detected organic carcinogens in the discharge. In addition, samples were evaluated for acute toxicity in Daphnia magna and developmental toxicity in zebrafish (Danio rerio). Acute toxicity was minimal, and no developmental toxicity was observed. Finally, in response to the observation that attenuation of organic chemicals increased in wetlands, constructed wetlands downstream of three different NPDES produced water discharges, including the discharge of focus in the chemical and toxicological analysis, were evaluated for their viability to polish produced water. The results showed that wetlands are effective at attenuating commonly used non-ionic surfactants, as well as a commonly used biocide. Attenuation was not only due to degradation, but also accumulation in sediments. Sediment accumulation has the potential to limit the lifetime of the wetlands or increase the frequency with which sediment must be excavated. The results of this dissertation identified multiple improvements that can be made to NPDES produced water regulations. Current regulations apply to the discharge site only. This dissertation shows that downstream changes in water quality must be considered to adequately evaluate potential impacts of produced water discharges, as exemplified by the increasing concentrations of inorganic species downstream. Secondly, toxicological results showed that chemical analysis alone is insufficient to assess impacts of these releases and that a thorough assessment of chronic toxicity is necessary to fully assess produced water for beneficial reuse. Current regulations require acute toxicity testing, but no assessment of chronic toxicity. Finally, prior to widespread implementation of constructed wetlands for produced water treatment, additional research is needed to assess the impact of oil and gas chemical additives on the maintenance schedules of these systems, as well as the long-term impact to soil health. If these waters can be reused safely and economically, many stakeholders stand to benefit. If this practice is expanded prematurely, the quality and health of water, soil, crops and downstream users could be negatively impacted. The research contained in this dissertation is one step in a life-cycle analysis of the costs, impacts and benefits associated with oil and gas extraction.
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    Investigating the regulators of cytoplasmic dynein
    (Colorado State University. Libraries, 2019) Dilsaver, Matthew, author; Markus, Steven, advisor; Di Pietro, Santiago, committee member; DeLuca, Jennifer, committee member; Argueso, Juan Lucas, committee member
    Organization of the cell is a dynamic and complex process that is often underappreciated. To accomplish this, cells use motor proteins to move different cargo to their destination. Cytoplasmic dynein is one such motor protein that uses filaments called microtubules as tracks. However, there is only one cytoplasmic dynein to accomplish over forty tasks. To achieve this, the cell uses a complex array of cofactors and regulators to specifically control dynein. But the role of each of these cofactors and regulators in poorly understood. To better understand how dynein is regulated we turn to budding yeast that provides a simplified system where dynein only has one known function, this is to position the spindle in the division plane between two dividing cells. Localizing dynein is extremely important. One regulator of dynein is Pac1 which was recently found to also activate dynein motility in vitro. Pac1 works to localize dynein to microtubule plus ends where it can interact with dynactin and Num1. Ndl1 is known to interact with Pac1, knockouts of Ndl1 led to a mild phenotype mimicking a dynein knockout. But how Ndl1 functions is poorly understood. Dynactin is an essential regulator of almost all dynein's tasks in humans and dynein's only role in yeast. Without dynactin, dynein cannot reach Num1 patches at the cell periphery and pull the spindle. In this study we sought to better understand dynactin and Ndl1's role in dynein regulation using in vitro single molecule assays where the activity of dynein can be recorded. Initial attempts to purify dynactin for these assays failed. We then developed a cell lysis assay to study dynactin and other proteins role in dynein regulation. We found in preliminary results that dynactin increased dynein activity. We also attempted to use a protein known as Num1, that is essential to dynein localization and interacts with dynactin, to purify the dynein-dynactin complex. Preliminary results showed that this complex was motile, indicating an intact complex. We also found that Ndl1 can bind motile dynein and increase run length using in vitro assays. We also were able to determine that Pac1 cannot bind dynein and Ndl1 at the same time indicating that there is a release mechanism for Pac1 from Ndl1 to bind dynein. We were able to map Ndl1's binding site to the N terminus of the dynein accessory chain Pac11. Then we tested to see if Ndl1 influence on Pac1-dynein interaction and found that Ndl1 was able to increase Pac1 comigrating with dynein in these assays. This work has opened new strategies for studying the regulators of dynein as well as better determined the interaction between Ndl1, dynein and Pac1. Further work will determine how each of these proteins affect dynein activity.
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    Spn1, Spt4, Spt5, and Spt6 preserve chromatin structure over promoters and open reading frames
    (Colorado State University. Libraries, 2024) Tonsager, Andrew Jordan, author; Stargell, Laurie A., advisor; Hansen, Jeffrey C., committee member; Santangelo, Thomas, committee member; Argueso, Juan Lucas, committee member
    The eukaryotic chromatin landscape presents formidable nucleosomal barriers for processes that require access to DNA, such as transcription. These barriers are overcome through the action of many factors, including histone chaperones Spn1, Spt5, Spt6, and FACT and transcription elongation factor Spt4. However, it is poorly understood how each contributes to this process. To ascertain the role that these factors play on preserving chromatin structure over the genome, this thesis has utilized micrococcal nuclease digestion followed by sequencing (MNase-seq) to analyze chromatin protections in the yeast genome in cells expressing numerous mutant alleles of these factors. Extensive characterization of MNase-protected fragments in a wide range of sizes established that the essential histone chaperone Spn1 preserves both nucleosomal and subnucleosomal structures over both promoters and open reading frames across the genome. Additional analyses from existing MNase-seq datasets demonstrated the extent to which Spn1 and other RNAPII-associated factors maintain nucleosome features over genes of varied characteristics. The study of factors described in this thesis is performed in living cells, which have been genetically modified to express mutant alleles of chromatin factors. This thesis also describes a course-based undergraduate research experience (CURE) developed to introduce upper-level biochemistry students to techniques in yeast genome engineering in an authentic research setting.
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    Sub-cellular localization of the PenA β-lactamase in Burkholderia pseudomallei
    (Colorado State University. Libraries, 2013) Randall, Linnell B., author; Schweizer, Herbert P., advisor; Borlee, Brad R., committee member; Argueso, Juan Lucas, committee member
    Burkholderia pseudomallei, a Gram-negative soil bacterium found in tropical regions, is the etiologic agent of melioidosis. B. pseudomallei is intrinsically resistant to many antibiotics, and melioidosis treatment involves prolonged antibiotic therapy. PenA, a chromosomal beta-lactamase in B. pseudomallei, confers resistance to many beta-lactams. Point mutations in penA leading to PenA amino acid changes can cause resistance to ceftazidime and amoxicillin-clavulanate, which can result in treatment failure. Typically beta-lactamase enzymes are found in the periplasm of Gram-negative bacteria. Previous studies have shown that PenA is secreted via the twin arginine translocase (Tat) system, but failed to demonstrate periplasmic localization. The purpose of this study was to determine the sub-cellular localization of PenA in B. pseudomallei. Using alkaline phosphatase as a periplasmic marker, we optimized a method for extracting periplasmic proteins from B. pseudomallei. Through subcellular fractionations, immunoblotting, and colorimetric enzyme assays, we have shown that PenA does not localize to the periplasm. Rather, it is present in a detergent-soluble fraction of the cellular membranes. Further experiments including site-directed mutagenesis, metabolic labeling with 14C-palmitate, globomycin treatment, and mass spectrometry indicate that PenA is likely a lipoprotein with post-translational lipid modification of the cysteine residue at position 23. This work implicates PenA as the first example of a beta-lactamase that is a Tat-secreted lipoprotein, and provides a better physiological understanding of an important antibiotic resistance mechanism in B. pseudomallei.
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    Uncovering mechanisms driving variation in mutation rates in organellar and nuclear genomes
    (Colorado State University. Libraries, 2024) Waneka, Gus, author; Sloan, Daniel B., advisor; Lockridge Mueller, Rachel, committee member; Argueso, Juan Lucas, committee member; Stenglein, Mark, committee member
    Mutations are changes to DNA sequences which drive evolution by supplying raw genetic variation for natural selection to act upon. At the same time, mutations tend to have negative fitness consequences and are the source of genetic diseases. Such costs and benefits of mutation create opposing forces of selection on mutation rate modifiers, which are alleles (typically in DNA repair genes) responsible for increases or decreases to mutation rates. Essentially all eukaryotes possess at least two genomes: the nuclear genome (nucDNA) and the endosymbiotically derived mitochondrial genome (mtDNA). Photosynthetic plants and algae additionally possess endosymbiotically derived plastid genomes (cpDNAs). Together, the mtDNA and cpDNA are referred to as organellar genomes. Chapter 1 of this dissertation provides a framework for understanding how DNA repair machinery and mutation rates have evolved in complex eukaryotic cells. Chapters 2 and 3 focus on specific repair pathways active in organellar genomes. Finally, Chapter 4 shifts focus to understand how environmental perturbations in expression level impact mutation in plant nuclear genomes. Repair of organellar genomes is conducted by nuclear-encoded genes that are translated in the cytosol and targeted to the organelles. In terms of evolutionary history, organellar repair machinery is a mosaic network of bacterial-like repair genes, which came into the cell with the organelles, and nuclear repair genes that have been co-opted for organellar function. In some cases, repair proteins are targeted to both the nucDNA and mtDNA (and/or cpDNA in plants) to perform similar functions. This is the case as for many base excision repair (BER) proteins, which identify and remove of chemically damaged bases. In contrast, organellar repair arsenals are thought to lack canonical mismatch-repair (MMR) and nucleotide excision repair (NER), which are both important repair pathways in nuclear genomes. Instead, the diverse eukaryotic lineages have adopted unique strategies for organellar genome maintenance. As a result, there is a tremendous diversity in mtDNA mutation rates, which show over a 4000-fold variation across eukaryotes. Interestingly, much of this variation is driven by the extremely low point mutation rates plant mtDNAs. In addition, plant organellar genomes are more recombinationally active and plant mtDNAs are structurally unstable compared to the mtDNAs of other eukaryotes. Chapter 2 of this dissertation explores the mechanistic basis of low point mutation rates and recombination-mediated repair in plant organellar genomes. We performed high-fidelity Duplex Sequencing on a panel of Arabidopsis thaliana lines lacking specific organellar genome repair genes. We report large point mutation increases in mutant lines lacking MSH1, a mutS homolog that has been proposed to induce double-stranded breaks at the site of DNA mismatches, effectively shuttling such lesions into homologous recombination (HR) pathways that play important roles in plant organellar genome replication and repair. We see smaller point mutation increases in other mutant lines lacking RADA, RECA1 and RECA3. In addition, we generated long-read Oxford Nanopore sequencing to characterize repeat-mediated recombination in several of the mutants in the panel. Our findings provide valuable insights into the mechanisms driving the fascinating patterns of organellar genome evolution in land plants. The aforementioned lack of NER in organellar genomes is surprising given the importance of NER as a 'catchall' for repair of a variety of bulky DNA lesions in nuclear genomes. Chapter 3 focuses in on the fate of photodamage (an important type of bulky DNA damage) in organellar DNA. To do so, we leverage publicly available XR-seq datasets, which were generated to quantify and map active NER excision products in nuclear genomes following UV exposure. The taxonomic scope of chapter is expanded from plants to also include fungi (the brewer's yeast Saccharomyces cerevisiae) and animals (the model fruit fly Drosophila melanogaster). We find that mtDNA-derived XR-seq reads in A. thaliana and S. cerevisiae have distinct and repeatable patterns in terms of length and internal positioning of pyrimidine dimers (known targets of photodamage formation). These data mirror established patterns of NER-derived reads originating from the nuclear genomes, raising the exciting possibility that NER-like repair pathways may exist in for repair of photodamage in organellar genomes. The focus of chapter 4 shifts to understanding how environmental changes impact mutation in plant nuclear genomes. The textbook view of mutation and adaptation is that mutations occur randomly with respect to their environment-specific fitness consequences. However, this view of random mutation is challenged as evidence increasingly establishes a correlation between increased expression and decreased mutation via the coordination of transcription and DNA repair machinery at the molecular level. As a result of this correlation, intragenomic mutation rates likely vary with changing environments given that expression levels are environmentally labile. Therefore, certain genes may be predisposed to higher or lower mutation rates depending on the environment, though the magnitude and importance of this effect remains largely untested. A technical challenge in addressing these questions is that large scale plant mutation datasets are time and resource intensive to generate. A recent plant study relied on low frequency somatic calls from Illumina based shotgun libraries to generate a large number of mutations, but others report that most of these inferred mutations are sequencing errors. To overcome these challenges, we took a novel approach to measuring somatic mutations by using Duplex Sequencing to quantify mutations in targeted regions of the A. thaliana nucDNA. We identified a set of differentially expressed genes in plants grown at different temperatures, which we then targeted for mutation detection using hybrid capture. In addition to wild type (WT) lines, we also studied mutant lines deficient in BER and MMR to test if either of these pathways are responsible for the correlation between expression and mutation in plants. We found large point mutation increases in the MMR mutants compared to WT plants, which displayed surprisingly few mutations at either temperature. Though the small number of WT mutations precluded a meaningful comparison of expression and mutation in a WT background, this result if nonetheless valuable for establishing that the true frequency of somatic mutations in plants is indeed very low suggesting that previous estimates likely conflated Illumina based sequencing artifacts with mutations. Mutation rates vary by over three orders of magnitude across the tree of life. Much of this variation is captured in mitochondrial mutation rates. The chapters of this dissertation provide valuable insights into the molecular processes that drive mutation rate variation in eukaryotic genomes. Such mechanistic understandings are critical for advancing the broader field of mutation rate evolution.