STRATA
STRATA, the STudent Research, Academic, and Talent Archive, is a collection of selected Fort Lewis College student work, including undergraduate research, senior seminar papers, published works, conference presentations, and other creative and artistic projects. Search by name, subject, title, or academic department.
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- Determination of the Secondary RNA Structure and Its Importance to the HTLV-1 Pro-Pol Frameshift Site
- Ribonucleic acids (RNAs) are a group of macromolecules that encode genetic information. Primary RNA structures can fold into various complex secondary structures, which are important for numerous cellular functions. Many single-stranded RNAs serve as the genetic material for several viruses, such as the Human T-Cell Lymphotrophic Virus Type I (HTLV-1). Five to ten percent of the time, HTLV-1 infections result in leukemia. HTLV-1 is a retrovirus that uses cis-acting regulatory elements in RNA to prompt a frameshift during viral mRNA translation, allowing for the translation of genes in alternative reading frames that are critical to viral infectivity. The pro-pol frameshift site is the second frameshift site in the virus' genome and is defined by two signals: a slippery sequence and an RNA structure downstream of the slippery site. In order to target HTLV-1, it is important to experimentally determine RNA structures that contribute to its pathogenic characteristics. The RNA structure downstream of the slippery site is proposed to be a pseudoknot (a structural motif formed when the nucleotides in the loop of a stem-loop base-pair with nucleotides downstream of the structure). So far no structural evidence supporting or refuting this structure has been published. The goal of my work is to determine the RNA structure in the HTLV-1 pro-pol frameshift site. Thus far, I have designed RNA constructs containing the HTLV-1 frameshift site, predicted their structures, and successfully cloned the DNA that codes for the RNAs into a puc19 vector. I have also transcribed and purified one of these RNAs. Future experiments will include chemical modification of the RNAs using selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) to determine which regions of the RNA structure are base-paired and which regions are not and secondary RNA structure prediction using SHAPE data and various computer programs.
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- Determining Potential Glacial Refugia for Ostrya Knowlyonii Across the Southwester Us as Inferred Through Chloroplast Haplotype Analysis
- Knowlton Hop-Hornbeam (Ostrya knowltonii) is a small understory tree that is a member of the ancient Arcto-Tertiary flora extending across the Northern Hemisphere. According to the fossil record Ostrya once had an almost continues distribution in North America. Today Ostrya knowltonii exhibits a highly disjunct distribution across the southwestern United States and northern Mexico likely as a result of past climatic conditions. We investigated patterns of genetic relatedness and variation among populations across the region to uncover phylogeographic patterns relevant to past distribution and migration of the species. Ten populations of Ostrya were sampled with DNA from five individuals from each population extracted. Two non-coding chloroplast DNA regions, psbA-trnH and ndhF-rpl32 were amplified using the polymerase chain reaction. The sequences from the two chloroplast DNA plastid regions were then analyzed and reduced to haplotypes projected in geographical space. Four haplotypes were identified in psbA-trnH and five haplotypes in ndhF-rpl32. Similar geographic patterns were observed for both regions. The eastern portion of the range in southeastern NM and western TX shared identical haplotype structure. Similar haplotypes were also seen in the Colorado River drainage and adjacent areas of northern AZ. Slight haplotype differentiation was noted into southeastern UT, central AZ, and Chihuahua in northwestern Mexico. The diversity of haplotypes is principally fixed in individual populations pointing to long-term isolation. Limited haplotype sharing in the region of highest population abundance points to persistence of ancestral haplotypes within individual refugial regions.
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- Determining Structures That Control Groundwater Flow in San Juan Mountain Karst Aquifers Using Fluorescent Dye Tracer
- Communities of the San Juan Mountains in Southwest Colorado continue to grow. With this growth, demand for water is increasing. Many of these communities are located on top of karst aquifers. Karst aquifers are unique because they can develop caverns and underground streams, which affect the direction of ground water flow. In addition, contaminated surface waters can quickly enter and move through karst aquifers, jeopardizing the quality of drinking water for mountain communities. Therefore, it is important to understand the structural features which dictate the direction of groundwater flow in the area. The study area is located 41 miles north of Durango, CO on Highway 550, south of Andrews Lake. Above Andrews Lake is a stream which disappears underground (insurgent cave stream) into a small cave (swallow hole). Insurgent cave streams present a unique opportunity to research karst groundwater flow. The purpose of my study is to determine the structural controls of groundwater flow by observing springs for changes in water discharge and by injecting 15 grams of sodium fluoroscene, fluorescent dye, into the insurgent cave streams and then analyzing spring water samples for the fluorescent dye with a spectrofluorimeter. The results from the study were then compared to the orientation of regional folds, faults, and bedding planes to determine which structures promote the flow of groundwater in local mountain karst aquifers. The study found that bedding planes were the regional control for the formation of conduits. This was ascertained by observations of a highly karstified spring system wherein water discharge rates varied with the water recharge rates going into the insurgent cave stream. The spring and the insurgent cave steam flows into the same limestone unit in which the spring flows out of. Unfortunately, fluorescent dye was not detected in the spring samples. It is believed that more dye was needed and therefore the result of the dye tracer study was inconclusive. Yet, the qualitative evidence suggests that conduits form within limestone units in the Lower Hermosa Group, which are confined to the bedding plane.
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- Determining the Basement Fault Structure of the Hogback Monocline in Northwest NM Using Fracture Patterns and Geomechanical Modeling
- Monoclines on the Colorado Plateau are well understood in cross-section, but changes along strike are poorly understood. The Hogback monocline, on the Navajo Nation in northwest New Mexico, has multiple bends in map view. This study used geomechanical modeling (T7) and field data (fracture patterns) to explore the possible geometries of faults that created the bends. My research group hypothesized that under the Hogback monocline there are two possible basement fault systems a S-bend fault, two parallel faults (simple relay) These different models should show differing fracture patterns based on differing stress and strain directions. In the field, we measured fracture sets along the monocline in the Cliff House Sandstone. We observed four sets: one parallel to the strike of bedding, two sets oblique to the strike of bedding, and one parallel to bedding dip. Fracture geometry changes as bedding orientation changes, notably around the bends. Slickenlines on some oblique fractures suggest that they are strike-slip shear fractures rather than joints. We built two scenarios in T7 (elastic dislocation modeling, based on rock properties and structure to predict deformation). Each scenario was designed to match the geometry observed in map and cross-section view. The monocline has three bends: southern (changing strike from 014° to 057°), middle (changing from 057° to 018°), and northern (changing from 018° to 055°). Bends connecting straight segments are 2750 m long and are offset by 1650 m (northern bend) and 1900 m (southern bend). Throw on the faults was determined to be 180 m via cross sections. We tested each scenario with different boundary strain conditions and shortening directions and compared predicted fracture patterns to those observed in outcrop. Understanding timing and distribution of fractures is important for predicting the movement of oil, gas, and water, and for assessment of local subsurface risks. Any persons wishing to conduct geological investigations on the Navajo Nation including visiting the sites described in this study must first apply for and receive a permit from the Minerals Department – Telephone (928) 871-6587.
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- Determining the Secondary Structure HTLV Pro-Pol Frameshift Siten Using NMR and Native Gel Analysis
- Sheet1 Explore Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that targets CD+ T-cells in humans. Within its RNA genome, it has two open reading frames that are only accessed through programmed ribosomal frameshifting (PRF). PRF is defined by a directed change to the ribosome's reading frame during the process of translation. Each PRF sites consist of two RNA elements: a heptanucleotide slippery sequence followed by a downstream RNA structure. In retroviral frameshift sites, this RNA structure is typically a pseudoknot. The frequency of frameshifting is specific to the frameshift site. Additionally, this frequency is important for maintaining appropriate molar ratios of proteins translated from the original and alternative reading frames. HTLV-1's second frameshift site is located within the pro and pol overlap. This site contains a slippery sequence, spacer region, and a RNA structure. The RNA structure is predicted to be a pseudoknot. Pseudoknot structures occur when nucleotides in the loop segment of a stem loop base-pair with nucleotides downstream from the base of the stem. The goals of this work were to use nuclear magnetic resonance (NMR) and native gel analysis to determine the pro-pol frameshift site RNA structure. In addition to the wild-type RNA, three other constructs were used to evaluate the secondary structure and folding of the predicted pseudoknot. Two of these constructs have mutations in the loop and downstream nucleotides that eliminate base-pairing in the pseudoknot formation. Currently, the stem loop RNA has been purified and its purity and folding examined. Additionally, template and non-template sequences for the three pseudoknot RNAs (WT, PKM1, and PKM2) were cloned into the Puc19 plasmid and transformed into E. coli.
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- Determining the Secondary Structure of the HTLV Pro-Pol Frameshift Site Using Shape Chemical Probing
- Human T-cell Leukemia virus (HTLV) is a retrovirus that infects adult CD4+ T-cells. Production of infectious virions is dependent on two programmed changes in reading frame during translation. These changes are triggered by specific sequences and structures within the HTLV ribonucleic acid (RNA) genome. In the context of retroviruses, frameshiting is frequently used to initiate the translation of viral enzymatic proteins. HTLV's second frameshift site is positioned within the overlap of the pro and pol open reading frames. This site contains two RNA elements: a slippery sequence, UUUAAAC, and a predicted pseudoknot structure. Pseudoknot's are composed of stem-loops that have their loop nucleotides base-paired to downstream RNA. To date, no experimental evidence has confirmed the stable formation of the HTLV pro-pol frameshift site pseudoknot structure. The goal of this work is to use chemical probing methods to determine the pro-pol frameshift site RNA structure. Specifically, selective 2'-hydroxyl acylation and primer extension (SHAPE) will be used to identify areas of base-pairing within the frameshift site RNA. During these experiments, the N-methyl-isatoic anhydride (NMIA) chemical does not acylate nucleotides involved in stable base-paring interactions. Therefore, the pattern of RNA modification will reveal which nucleotides within an RNA are base-paired or single-stranded. Computational programs can be paired with SHAPE data to predict the secondary structure.
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- Development in Navajo Nation
- The purpose of this study was to demonstrate why Native American communities, and in particular the Navajo Nation, were targeted for power plants and other health-harming mineral extraction schemes. I proposed that the Navajo Nation was targeted because of the need for economic development. Environmental racism was also another factor I looked at. My findings concluded that economic development did play a huge role in the instigation of power plants. But other factors such as neocolonialism also contribute to the exploitation of Native American land and resources. Neocolonialism is a factor that heavily influences decisions on development in third world countries. With this new factor, I included other similarities between third world countries and the Navajo Nation regarding development to illustrate the difficult transition to a better standard of living due to the same factors affecting third world countries, which also explains the occurrence of health hazardous industries on the Navajo Nation.
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- Development of Q-Pcr Technique for Determining Bartonella Infection in Wild Deer Mice (Peromyscus Maniculatus)
- Sin Nombre virus (SNV) is a North American hantavirus that causes Hantavirus Pulmonary Syndrome (HPS) in humans, an acute and rapidly progressing disease with high (~40%) mortality rates. Deer mice (Peromyscus maniculatus) are the primary mode of human HPS infection; humans become infected with SNV via inhalation of virus particles shed through deer mouse excrement. It is unknown whether chronic infection with SNV impacts the ability of deer mice to respond to other immune challenges. This has significant implications for the deer mouse - SNV system, as deer mice with reduced immunocompetence may shed greater amounts of SNV, increasing the likelihood of human SNV infection. Thus, our objective was to develop a protocol in which we could quantify Bartonella, a bacterial pathogen that is endemic in wild rodents, through Q-PCR. Similar to SNV, Bartonella has low pathogenicity, but requires that deer mice mount a chronic, low-level immune response post infection. For the development of a Q-PCR assay, we are using the forward and reverse primers BhC5781p (5'-GGGGACCAGCTCATGGTGG-3') and BhCS1137n, (5'-AATGCAAAAAGAACAGTAAACA-3' respectively. Q-PCR trials were completed using serum and whole blood; however, we were unable to isolate and amplify Bartonella DNA using serum and whole blood alone. DNA isolation appears to be a necessary step to detect the presence of Bartonella DNA in the deer mouse whole blood samples. The further development of this protocol will allow us to determine the extent to which SNV and Bartonella infections are correlated. This research will ultimately improve our ability to predict human patterns of infection with zoonotic pathogens.
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- Development of a Cloning Procedure for Triose Phosphate Isomerase, Phosphofructokinase, and Transaldolase Genes from Acidobacterium Capsulatum
- Acidobacterium capsulatum was discovered in 1991 as the first species of the phylum Acidobacteria. It is a bacterium found in soils with an uncanny ability to withstand extremely polluted and low pH environments. It is ubiquitous in soil and aquatic environments, suggesting that A. capsulatum serves an important role in these ecosystems. Interestingly the annotated genome of A. capsulatum suggests that the organism is missing aldolase, an enzyme known to play a crucial role in glycolysis. To better understand the way A. capsulatum utilizes glucose, we began the process of developing cloning and expression vectors for three key enzymes involved in glucose metabolism. These enzymes are triose phosphate isomerase (TIM), phosphofructokinase (PFK), and transaldolase (TA). These three enzymes were chosen because they are the most likely candidates for catalyzing alternate routes for glucose utilization by A. capsulatum. Phosphofructokinase is an enzyme normally involved in regulating standard glycolytic pathways. It phosphorylates fructose-6-phosphate to fructose-1,6-bisphosphate, which takes place immediately before aldolase splits fructose-1,6-bisphosphate into two three-carbon molecules. Triose phosphate isomerase interconverts between glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP), the two three-carbon molecules produced by aldolase. Transaldolase catalyzes the aldol condensation of fructose-1,6-bisphosphate and erythrose-4-phosphate (E4P) into sedoheptulose-7-phosphate (S7P) and GAP. They were examined to see how their metabolic functions compared to those of well-understood organisms with fully functioning glycolytic pathways. To further study this pathway, cloning experiments were designed using a TOPO Cloning Kit. The genes that encompass these three enzymes were amplified using PCR. The amplification of these genes will lead to future cloning experiments that will help determine the role and regulation of these enzymes in A. capsulatum.
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- Development of a Lab Testing Protocol to Assess Ski Mountaineering Performance
- Ski mountaineering is a popular winter recreation sport that is quickly gaining importance and recognition in the competitive world. Competitive ski mountaineering is set to be included in the 2020 Youth Olympic Games and in the Olympics as a test sport. Ski mountaineering is a timed race done on an established track using light equipment designed specifically for the sport. Research is lacking on most aspects of the sport, especially the physiological variables that influence race performance. The purpose of this study was to develop an exercise testing protocol that simulates a ski mountaineering race. There were lab and field portions of this study. For the lab portion, subjects completed a ten stage graded exercise test on a large treadmill with roller skis that consisted of five-minute stages starting at 2 mph and 12% grade and increasing speed by 0.2 mph and grade by 2% each stage. The field test was a race simulation and involved two skinning and one bootpack intervals at race pace. Each interval consisted of 600 feet of vertical elevation gain with an average gradient of 20% for the two skinning intervals and 30% for the bootpack interval. VO2 and heart rate were taken continuously throughout both tests using a Cosmed K5 portable metabolic cart. Lactate and rate of perceived exertion were taken at the end of each stage during the laboratory test and at the end of each uphill interval during the race simulation. Pearson’s R-Test was used to examine the relationship between the lab and field results. There were strong significant correlations between lab and field results in terms of VO2 and heart rate, indicating that the developed lab test accurately predicts performance in a ski mountaineering race. The lab test can be used to stratify ski mountaineers into skill levels, which will allow for the inclusion of varying skill levels and a higher volume of athletes in ski mountaineering races and the development of athlete-specific training programs as a result of the specific physiological data the test provides.
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- Development of an Off-Road Vehicle for the SAE Baja Competition
- Baja SAE is an intercollegiate engineering design competition for undergraduate and graduate engineering students. The object of the competition is to simulate real-world engineering design projects and their related challenges. Each team is competing to have its design accepted for manufacture by a fictitious firm. The students must function as a team to design, engineer, build, test, promote and compete with a vehicle within the limits of the rules. They must also generate financial support for their project and manage their educational priorities. Each team's goal is to design and build a single-seat, all-terrain, sporting vehicle whose structure contains the driver. The vehicle is to be a prototype for a reliable, maintainable, ergonomic, and economic production vehicle which serves a recreational user market, sized at approximately 4000 units per year. The vehicle should aspire to market-leading performance in terms of speed, handling, ride, and ruggedness over rough terrain and off-road conditions. Performance will be measured by success in the dynamic events which include rock crawl, acceleration, hill climb, and endurance events. Along with the dynamic events, the success of the vehicle will be determined by static events including the design report and presentation. Using the expertise provided by the Fort Lewis Engineering department, the 2015 Baja SAE team was able to design the entire vehicle from scratch. The design has been inspired by universities which have competed in the event for many years, yet the originality of the vehicle has been the overriding motivation behind the design. The team's focus was directed towards ensuring that the vehicle would do well in each dynamic event rather than designing around a specific event. It was essential that each member of the team worked closely with one another in order to overcome the difficulty of an integrated project.