Publications by Colleges and Departments (MSU - Bozeman)
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Item Beta-glucans and Anthocyanins in Barley for Human Food(Montana State Univeristy, 2017-04) Sickler, Nathanβ-glucans and anthocyanins hold nutritional value and are present at relatively high concentrations in barley. Purple, blue, and black barley were tested for the sake of improving beer and feed production. With the use of reagents and spectrophotometry, it was possible to measure the concentrations of each line of barley. Anthocyanin pigments because of antioxidant activity protect from many illnesses, such as cardiovascular disease. β-glucans can assist with human illnesses such as cancer and diabetes. A pH diffusion method was used in order to extract anthocyanins from ground barley. The obvious sign of the presence of anthocyanins was a pink-reddish haze in an acidic solution. β-glucans concentrations were measured in a photometric analyzer. There were significant differences between purple, blue, and black barley concentrations. Results showed increased concentrations of anthocyanins and β-glucans in purple and blue barley where black barley lacked in these nutritional qualities. Quantitative concentrations were compared to qualitative picture scans of each barley line to examine the colors of the seed coat. It was determined that select purple and blue barley had the highest concentrations of anthocyanins and β-glucans. Crossing these lines with low protein lines would introduce the idea of producing beer with added health benefits. Future research would be done to ensure anthocyanin and β-glucan molecules would be present through a malting process.Item How Aqp3b Influences Convergent Extension Through Noncanonical Wnt Signaling(Montana State University, 2017-04) See, KaitlynAquaporin-3b, Aqp3b, is an aquaglyceroporin, a membrane water channel, that is present during gastrulation and various other stages of development. Gastrulation organizes cells, via convergent extension, into germ layers, which will later form different body tissues. During gastrulation, cells fold into the embryo, then merge by convergent extension to form the long body axis. These cell movements are regulated by noncanonical Wnt signaling, an intercellular signaling pathway that controls the migration and polarity of tissues. When Aqp3b is inhibited using a morpholino oligonucleotide (MO), convergent extension does not occur properly, suggesting a link between Aqp3b and noncanonical Wnt signaling. To assay these defects, we use the Keller tissue explanting method to observe convergent extension. Our goal is to determine which parts of the Wnt signaling pathway are influenced by Aqp3b. We conducted rescue experiments by inhibiting Aqp3b and injecting an RNA or DNA construct of several proteins involved in Wnt signaling. Successful with rescue Dvl1ΔDix and Dvl2ΔDix constructs indicated that Aqp3b is involved in noncanonical Wnt signaling, since DvlΔDix acts in all noncanonical Wnt signaling. Further, Aqp3b acts through the Wnt/Ca2+ subpathway, indicated by rescue by PKC, and through a branch of the Wnt/PCP pathway, indicated by successful rescue with RhoA but not with Rac1. Aqp3b does not directly affect the Wnt/Ror2 pathway. In conclusion, I have demonstrated that the ability of Aqp3b to influence convergent extension is dependent on noncanonical Wnt signaling, specifically the Wnt/Ca2+ pathway and the RhoA branch of Wnt/PCP pathway. I am collecting additional data to ensure statistical significance.Item Effect of coal particle size on microbial methanogenesis in the presence of oxygen(Montana State University, 2017-04) Park, RitaThis project is a sub-experiment within the DOE-funded Microbially Enhanced Coal Bed Methane (MECBM) project, which studies methanogenesis in shallow coal beds of the Powder River Basin (PRB) located in southwestern Montana. The proposed research will observe the effects of coal particle size on: (1) methane production yield, (2) microbial diversity, (3) dissolved oxygen (DO) concentration in the coal bed methane (CBM) water, and (4) bioavailability of organics from the coal. Microbial CBM has a strong potential as an energy resource worldwide, but the exploitation of production wells with oxygenated surface waters may impact in situ microbial diversity and thus the methanogenic potential of coal beds. This experiment will provide further information on the oxygen-scavenging capacity of coal particles in CBM water, and subsequent impact on the anaerobic microbial methanogenesis. Cultures containing coal and CBM water from the PRB coal beds will be set up in triplicate glass bottles sealed with a rubber septum to allow for regular sampling. Coal of 2 particle sizes will be obtained with sieves: small (0.075 < 0 < 0.85 mm) and large (18.3 < 0 < 28.3 mm). These cultures will be treated with a methane-producing inoculum from a PRB coal bed; coal degradation and methane production will be monitored over time. This experiment will evaluate methanogenesis rates as a function of coal particle size and of subsequent DO levels in the CBM water. Results will provide a better understanding of the subsurface energy potential of MECBM.Item Molecular Epidemiology of Clostridium difficile infection(Montana State University, 2017-04) Kills Pretty Enemy, TrisheenaClostridium difficile is the most commonly acquired nosocomial pathogen in the United States. Of all patients on antibiotics, 10-25% will develop C. difficile infection (CDI) and some of these may develop a severe complication, known as pseudomembranous colitis. The overall goal of this project was to understand C. difficile epidemiology at the Bozeman Deaconess Hospital (BDH). Prompt detection of pathogenic strains with molecular biology techniques can aid in the rapid intervention of CDI cases. In this project, we were given de-identified specimens (stool samples) from patients who were suspected to have CDI. Specimens were plated onto selective media and presumptive single colonies of C. difficile were isolated. I verified through PCR that isolates were indeed C. difficile and that they carried genes encoding for at least one of the C. difficile toxins, TcdA and TcdB. I also helped differentiate between strains using PCR ribotyping, which is a general genotyping method for C. difficile. However, the presence/absence of toxin genes and PCR ribotyping are not highly discriminant methods to differentiate between strains. To do so, I used a technique called multi-locus variable-number tandem-repeat analysis (MLVA) to detect sub-groups within individual PCR ribotypes. Due to inconsistent results with MLVA, we then used a technique called High Resolution Melt Curve Analysis (HRMCA) to differentiate between strains. A fluorescent dsDNA dye, called SYBER green, was included in the initial PCR and was detected when released from the melting DNA. Using HRMCA, we could quickly and accurately discriminate between strains of the same PCR ribotype.Item Arizona Amphibole Asbestos Induces Autoimmunity and Fibrosis in Mice(Montana State Univeristy, 2017-04) Kaupish, Zoie; Stair, CalebAsbestos is a well-known carcinogen that contributes to autoimmunity and other health consequences. Libby Amphibole (LA) asbestos was a contaminant of vermiculite mined near Libby MT for decades, leading to asbestos diseases not only in mine workers, but in the entire community. Amphibole asbestos fibers in Arizona (AzA) have recently been discovered, but their health impact is unknown. The goal of this study was to determine whether these environmental fibers induce autoimmune and fibrotic responses at a very low dosage. Seven months after exposure, blood, urine and lungs were collected from mice. Serum was used to determine autoantibody (ANA) levels and T helper cytokine responses. Urine was used to measure protein excretion, suggesting kidney involvement. Results revealed ANA levels were statistically significant with positive results with AzA. Also, all three Th-17 cytokines were shown to have increased levels in treated mice that were statistically significant above controls. Urine analysis indicated significant amounts of excreted proteins by treated mice. Using the right lungs, a Total Collagen Assay was performed to determine the presence of fibrosis. The results determined a statistically significant increase in treated mice. Therefore, our results show that the AzA poses a serious health risk, even in small doses.Item Temporal Dynamics of Microbial Communities in Four Yellowstone National Park Hot Springs(Montana State Univeristy, 2017-04) Harnish, AnnetteThe microbial communities of Yellowstone National Park (YNP) thermal features have been well-characterized, and have provided a wealth of information about thermophilic microbial community ecology. However, there has been little study of how these communities change in composition over time. The objective of this project is to use culture-independent methods to characterize the microbial community composition of four acidic YNP hot springs with weekly sampling over a six-month sampling period spanning multiple seasons. Analyses will be conducted using high-throughput 16S rRNA sequencing to determine community composition and quantitative PCR to determine population abundances in tandem with geochemical analyses, in order to assess how and why microbial communities respond to spring geochemical fluctuations over time. In addition to the three springs sampled continuously over six months, emphasis will be placed on microbial community succession in a newly formed hot spring that appeared during the course of sampling to provide insight into the establishment and succession of thermal feature microbial communities. I hypothesize that seasonal variances in climatic factors, especially precipitation, will have a pronounced impact upon hot spring communities due to the mixing of surface waters with subsurface hydrothermal waters. Preliminary molecular and geochemical results will be presented in context of temporal dynamics of the system.Item The Evolution and Mechanisms of Type 1 CRISPR Systems(Montana State Univeristy, 2017-04) Forsman, BrittneyBacteria and Archaea have adaptive RNA-guided immune systems called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR associated) that provide protection against invading genetic elements. There are currently five reported CRISPR types comprised of at least nineteen subtypes that encode for a different crRNA-guided surveillance complex. The Type 1-E system of Escherichia coli relies on a surveillance complex called Cascade (CRISPR-Associated Complex for Antiviral Defense) and a nuclease/helicase, Cas3. Similarly, the Type 1-F system of Pseudomonas aeruginosa relies on a surveillance complex called Csy (CRISPR system yersinia) and the trans-acting nuclease, Cas2/3. The Type 1-F system is unique, because it contains a fusion of the Cas2 and Cas3 proteins into a single polypeptide. In most Type 1 systems, Cas2 and Cas3 are separate proteins that are involved in adaptation and interference, respectively. When the surveillance complex of a Type 1 system binds to target DNA, it recruits the nuclease to degrade the invader DNA. My aim is to determine if the Cas2/3 protein from P. aeruginosa can complement the activity of Cas3 from E. coli.Item Metabolic preferences of a chemoautotrophic thermophilic microorganism(Montana State Univeristy, 2017-04) Fernandes Martins, Maria ClaraHigh-temperature chemotrophic hot springs in Yellowstone National Park are inhabited by microorganisms that subsist on varied substrates supplied primarily by water-rock interactions. Hydrogen oxidation is a particularly important metabolism for many such microorganisms and helps fuel chemoautotrophic growth. Hydrogen-oxidizing microorganisms (hydrogenotrophs) living in YNP are capable of coupling this activity to the reduction of oxidants, including oxygen and ferric iron. We’ve observed seemingly contradictory metabolic preferences carried out by a chemoautotrophic, thermophilic, hydrogenotroph in the ‘Roadside East’ hot spring (pH 3.0, 82.4°C). Sediment-associated communities from the source of this spring are dominated by a species of Metallosphaera (80% of the total community) which has the genomic potential for hydrogen oxidation with [NiFe]-hydrogenases. Additionally, we have shown the ability of this organism to oxidize hydrogen in laboratory cultures. However, net microbial hydrogen oxidation is not measured in situ in this spring, but we can observe the presence of iron oxides in this spring. The Metallosphaera species present in this community is seemingly conducting iron oxidation coupled with oxygen reduction instead of hydrogen oxidation, a process which yields less energy. Here I present preliminary work aimed at investigating why this microorganism is using iron oxidation instead of hydrogen oxidation, with both metabolisms requiring oxygen, often limited in hot springs. Results will be discussed in light of the ability of a putative Metallosphaera isolate from Roadside East to conduct both metabolisms. Further, results will be discussed from laboratory competition experiments determining metabolic preferences when both iron and hydrogen are available.Item Targeted joint therapy using TAT peptides in Osteoarthritic cartilage(Montana State Univeristy, 2017-04) Dinkel, SabrinaOsteoarthritis (OA) is a debilitating disease that affects millions of Americans each year. It is characterized as a loss cartilage, the smooth tissue lining human joints. Currently, there is no cure for OA and the only treatment is an invasive and painful knee or hip replacement. Due to this, there is a need for a novel drug therapy to be developed. The objective of this study is to characterize the diffusion and uptake of TAT Protein Transduction Domain (TAT-PTD), a positively charged, small peptide. TAT-PTD has been tested in FDA clinical trials and has been shown to be a safe and effective drug delivery vehicle. We hypothesize the charge difference between positively charged TAT-PTD and negatively charged cartilage will increase the efficacy of drug delivery to cartilage. In this study, samples of OA cartilage were placed into a solution containing fluorescently labeled TAT peptides. I expect the study will show limited results because there is a diminished negative charge in OA cartilage due to the loss of negatively charged proteins. This will then decrease the attractive force between the TAT-PTD and cartilage. Multiple other studies have also had the same results. These studies have found that positively charged molecules have a decreased uptake in OA affected cartilage. Therefore, similar results could be expected in this OA model system. The impact of this data is to better understand how positive charge can affect the efficacy of drug delivery to OA cartilage. In addition, the data provides information on how positively charged molecules could be used to enhance drug delivery.Item Mouse TLR4 vs. Human TLR4(Montana State Univeristy, 2017-04) Beauchene, JulianaThe overall goal of the project is to understand the immune response of Toll-like receptor 4 (TLR4) in mice that express the human TLR4 gene compared to mice with the mouse TLR4 gene. Research concerning TLR4 has high potential to lead to the modification and transformation of vaccines we use today. The hypothesis is that human TLR4 has an amplified TRIF-biased response compared to mouse TLR4. A TRIF biased response is optimal in vaccine production because the TRIF adaptor protein has minimal inflammatory response. The moving target of creating vaccines on ever changing viruses is a challenge. TLR4 research has the potential to create vast improvements in vaccines we use today and improve biomedical and public health.