Honors Thesis Abstracts
Exploring the Role of Dctn2 in the Trafficking of GluR2- containing AMPA Receptors
Mark Barnett - Biology
AMPA receptors are post-synaptic receptors that play a role in thinking and learning. Although their function is understood, the trafficking and regulation of AMPA receptors in neurons remains unclear. The goal of my research was to determine whether Dynactin Subunit 2 (Dctn2) affects the trafficking of AMPA receptors through the regulation of the AMPA receptor subunit, GluR2. To accomplish this, Dctn2 was amplified via PCR, ligated into the mammalian expression vector pcDNA 3.1, and transfected into Human Embryonic Kidney (HEK) 293 cells, NT2 cells and neurons. Immunostainings and Co-immunoprecipitations were performed on the transfected cells to determine protein localization and if Dctn2 associates with GluR2. Unfortunately, the immunostainings and co-immunoprecipitation assays did not yield definitive results, thereby preventing a clear understanding of the role of Dctn2 in AMPA receptor trafficking.
Investigating the Role of Olfm1 in the Trafficking of GluR2-containing AMPA receptors
Caroline Cardenas - Biology
Neurons have the ability to either increase or decrease the strength of their connections with other neurons. This property is known as synaptic plasticity and is thought to influence higher cognitive functions such as learning and memory. Decreases in cognitive abilities as well as neurological disorders can be attributed to decreased synaptic plasticity. The variable expression of GluR2 containing AMPARs has been linked to changes in synaptic plasticity. The mechanism governing the trafficking of GluR2-containing AMPARs to synapses is not fully understood and was the main focus of my study. I hypothesized that Olfm1 (Olfactomedin 1) plays a key role in the trafficking of GluR2-containing AMPARs to the synapse. HA-tagged Olfm1 was successfully cloned into the mammalian expression vector pcDNA3.1. Western blot analysis verified Olfm1 protein expression in both transfected HEK293 cells as well as endogenous expression in NT2 neurons. GluR2-GFP protein expression was seen in transfected HEK293 cells, but we were unable to confirm endogenous expression in the NT2 neuron cultures. Co-immunoprecipiation experiments were also performed to study the interaction of Olfm1 and GluR2, but the tests remain inconclusive. Therefore, further testing is needed in order to accept or reject my hypothesis that Olfm1 plays a role in the trafficking of GluR2-containing AMPARs.
The Associations Between Large Mammal Abundance, Elevation, and Tick Capture Rate in the Big Belt Mountains
Sean Condon - Biology
Associations Between Large Mammal Abundance, Elevation, and Tick Capture Rate in the Big Belt Mountains Abstract The Rocky Mountain wood tick, Dermacentor andersoni, is the primary tick vector of human pathogens like Colorado tick fever and Rocky Mountain spotted fever in the Rocky Mountain Region. Drag sampling was conducted to investigate the association between large mammal abundance, elevation, and tick capture rate in the Big Belt Mountains near Helena, Montana during suspected peak tick activity in May and June. Soil temperature and type, climate, humidity, aspect, slope, and the availability of hosts have been shown to be factors that determine tick distribution. Multiple factor regression, using elevation as a covariate, found that even after accounting for elevation, the relative large mammal abundance was significantly negatively associated with the number of ticks observed per hour. It seems likely that overgrazing by large mammals, such as deer and elk, causes a trophic cascade that negatively affects small mammals and, therefore, tick populations, due to a lack of vegetation. The majority of observed ticks were found at elevations between 1323-1761 m. It is unclear exactly why the majority of ticks are found within a specific elevation range, but it may be determined by climatic conditions found within a particular elevation range such as average daily maximum temperature and humidity.
Investigating the Role of PrP and Amyloid Beta Proteins in AD and Other Protein-Misfolding Disease
Steven Cornish - Biology
Prion diseases are very rare, neurodegenerative diseases caused by misfolding of the Prion protein. The pathologies created by the misfolded protein are remarkably similar to those of Alzheimer’s disease. This paper provides a comprehensive study of the genomic and proteomic similarities between Prion proteins and several proteins implicated in Alzheimer’s disease (AD). While genomic similarity has been proven, studies are unclear as to whether any genomic mutations in PRNP affect the progression of AD. Studies have definitively proven that Prion protein interacts on a molecular level with several proteins implicated in AD. These interactions require further study to definitively prove whether they speed or slow AD progression, but current evidence indicates that Prion protein interaction will lead to faster progression of AD in vitro.
Population Genetics of the Tick, Dermacentor andersoni, in Montana, based on the Mitochondrial 16S Gene
Seth Dotson - Biology
The tick Dermacentor andersoni is a vector for Colorado tick fever, Rocky Mountain spotted fever, Powassan encephalitis, and bovine anaplasmosis. Under the Infectious Disease Ecology Research Program at Carroll College, the protocols for developing a West Nile Virus, carried primarily by the mosquito vector Culex tarsalis, distribution map are being applied to D. andersoni. A portion of the 16s ribosomal mtDNA was PCR amplified using the D16S5 and 16S+1 primers for 105 D. andersoni spread across five sites in western Montana. There were nine different haplotypes found in this study. This is lower than the 14 reported haplotypes found in a similar study done near Lake Como in Montana. The statistical analysis indicated there was no statistical difference of haplotype distribution among the populations. Also, there were no more than two haplotypes that matched when comparing the current study, a study in Canada, and the two other studies done in Montana. However, the usefulness of the 16s ribosomal mtDNA in differentiating between sister species was demonstrated.
Using Glycosylated Hemoglobin and Heat Shock Protein 70 as Thermal Biomarkers in North American Pikas (Ochotona princeps)
Steven Edmonds - Biology
Pikas (Ochotona princeps), high altitude lagomorphs, are potentially one of the first mammals to be directly affected by global warming. Population decline has been observed in pika populations in Nevada and California. The cause of their decline is unknown but several studies suggest that heat stress, especially at lower altitudes, is a contributing factor. In Montana, populations are potentially stressed in the same way. One hypothesis is that direct thermal stress is causing population decline. This study looks at heat shock protein 70 (HSP70) and glycosylated hemoglobin levels in high and low altitude pika populations as biomarkers for thermal stress. Pikas were trapped from Gold Creek (1962m) and Vista Point (2832m) Montana. Blood was extracted and separated into plasma and packed blood cells. ELISA high sensitivity tests were used for HSP70 quantification. Chromatography techniques, using manimophenylboronic beaded gels, were used to measure the percent of glycosylated hemoglobin. A strong positive correlation was observed between HSP70 levels and weekly average temperatures prior to capture. Due to the inconsistent nature of the glycosylated hemoglobin results, these assays are unable to support oxidative stress as a factor caused by direct thermal stress. Future work includes attaining a larger sample size, locating more trapping sites, further temperature data collection, and refining techniques to assay for glycosylated hemoglobin before definitive conclusions can be made.
Comparison of West Nile Virus Infection rates in Culex Tarsalis and exposure rates of horses in Montana using RT-PCR and ELISA
Sarah Fitzpatrick - Biology
Horses are more susceptible to West Nile virus (WNV) than human beings. In addition, for many ranchers in Montana, horses are their livelihood. In Montana, the only risk assessment tool for WNV is mosquito surveying. Testing horses across the state for WNV may contribute to a better estimate of high-risk areas. Also, since the numbers of human and horse cases are similar on a yearly basis, testing horses may predict the human risk. In this study, Culex tarsalis infection rates were compared with horse exposure rates in Montana. Mosquitos were collected from 46 sites on a weekly or biweekly basis. Collected mosquitoes were sorted, homogenized, and run through an RNA-extraction. WNV in mosquitoes was detected using RT-PCR. Horse serum was collected from the Helena area and analyzed for NS1 and envelope IgM and IgG antibodies to determine WNV exposure and/or vaccination. Three counties had positive C. tarsalis pools for WNV; the state infection rate was 0.108%. Two unvaccinated horses had positive IgM WNV antibodies. However, due to borderline results and vaccination contamination a horse exposure rate could not be calculated to make a comparison with the mosquito vector infection rate. Since horse positives occurred in an area where no positive pools were found, horse surveillance may be critical for detecting WNV hot zones.
Characterization of a Novel Mycobacteriophage via Sequence Analysis
Sarah LaPierre - Biology
Bacteriophages provide a relatively new area of scientific research. Approximately 1031 viruses on earth are capable of infecting bacteria and thereafter disseminating. Despite their widespread and pervasive nature, bacteriophages have been relatively unstudied. Not only do they provide a new class of organism but they also present potential usefulness to humans, including within the medical field. Bacteriophages represent a new tool in medicine’s ever-developing fight against bacterial infections. Notably, bacteriophages may offer a new weapon to battle infections that have developed resistance to multiple antibiotics. The prospect of bacteriophage use against infectious processes will require significant research which begins with a basic understanding of structure and function of the phages themselves.
Isolation and Analysis of the Novel Mycobacteriophage, CCP1
Kevin McNamee - Biology
Bacteriophages are the largest remaining body of undescribed biodiversity on the planet. The present study attempted to isolate and analyze the structure and genetics of a novel mycobacteriophage extracted from a soil sample. It was hypothesized that undescribed phages exist in the study area. A phage, named CCP1, was isolated from soil obtained in Helena, Montana, U.S.A. via serial plating of phage material taken from plaques formed in Mycobacterium smegmatis. Genetic and morphological analyses by restriction enzyme fragment length polymorphism (RFLP) analysis and transmission electron microscopy (TEM) respectively, suggest that CCP1 is a novel mycobacteriophage.
2016 Theses - Biology
Exploring GluR2-Interacting Proteins in Caenorhabditis elegans
John C. Brothers - Biology
The expression of Υ-3-hydroxy-5-Methlisoxaole-4-Propionic Acid Receptors(AMPARs) on the post-synaptic region of a neuron has direct impacts on many physiological functions of the brain, including learning and memory. Because AMPARs are Qirst assembled in the endoplasmic reticulum and later transported to the post-synaptic region of a neuron, understanding the mechanisms involved in AMPAR-trafQicking is crucial to understanding a variety of neuronal functions. The goal of this research was to investigate the role of the putative AMPAR-trafQicking proteins DLI-1, VDAC-1, SVOP-2, and DCN-2 on AMPAR-mediated behavior in the nematode Caenorhabditis elegans (C. elegans). Unfortunately, several technical issues prevented development of the constructs and behavioral assays needed to fully accomplish the goals of this study. Therefore, I am unable to discourse on the roles of DLI-1, VDAC-1, SVOP-2, and DCN-2 in AMPAR-mediated behavior in C. elegans.
Comparison of Capture Methods and Infection Rates for the Tick, Dermacentor andersoni, in Montana
Hanna Dotson - Biology
Montana is home to the tick Dermacentor andersoni which serves as a vector for Colorado tick fever. It is important to study ticks in the field to improve capture rates for the purpose of testing infection rates. To determine the most effective way to capture ticks, four separate capturing methods were assembled in two tick prevalent locations. These methods include AAAP pheromones, CO2, drag netting, and AAAP in combination with CO2. I hypothesized that the combination of AAAP and CO2 would yield the best capture results. I implemented a mark recapture study using fluorescent powder and hypothesized that CO2 and pheromones combined would provide the best population estimates. My findings indicate that drag net sampling and CO2 alone were the best tick attractants which could be due to pheromone amounts high enough to repel ticks rather than attract them. CO2 and drag netting had similar efficacy, whereas AAAP attracted no ticks throughout all trials. I developed an RT-PCR protocol to detect the presence of Colorado tick fever in D. andersoni and hypothesized that infection rates would be within the range of 10-25%. At Woodlake Campground and Dearborn the infection rate of Colorado tick fever was 12%. The infection rate at Beaver Creek drainage was 10%. My results suggest that drag netting, perhaps combined with CO2 , lead to higher capture rates and the most effective means of estimating population density. Also, my results show that the infection rate of ticks was within 10 12% at three sites in Lewis and Clark County.
An Evaluation of Population Differentiation in Culex tarsalis in Montana Using COI Sequences
Jake Fiocchi - Biology
The Infectious Disease Ecology project at Carroll College has established protocols to successfully detect West Nile virus in Culex tarsalis, a species of mosquito that acts as a vector for this disease. The detection data combined with ecological factors have been used to produce a risk model capable of predicting where West Nile positive specimens will be detected annually. This specific project aimed to determine the relatedness of C. tarsalis from various regions across the state of Montana as this knowledge could aid in determining how West Nile virus infections may spread across the state. Cytochrome c oxidase I (COI) gene sequences were analyzed from specimens from eleven locations across the state. Analyses included population pairwise distances and FST calculations, an analysis of molecular variance, and a neighbor-joining phylogenetic analysis. The results of these analyses suggest the Montana populations of Culex tarsalis are not genetically differentiated from one another. This means either that the populations truly are panmictic or that the sequence used is not informative for this question. Due to the low sequence variation among individuals, it seems more likely that this sequence is not an informative one for this study.
Genetic Diversity in Dermacentor andersoni Populations in Western Montana
Ezekial Koslosky - Biology
Ticks have been made a top priority for analysis as disease vectors. Specifically the tick Dermacentor andersoni is a vector for Colorado Tick Fever, Rocky Mountain Spotted Fever, Bovine Anaplasmosis, and Powassan Encephalitis. This study was designed to assist in the utilization of the current protocols for a West Nile Virus vector distribution map in Montana and apply these to develop a D. andersoni distribution map. Samples were collected from across western Montana, and the 16S mitochondrial DNA gene was amplified. Six tick populations were tested, and there were sixteen different haplotypes found. However, when comparing these haplotypes there was no statistically significant difference with respect to the haplotype frequencies found between the populations, as well as no significant difference between the genetic diversity of tick populations on either side of the Continental Divide. This leads to the conclusion that either the genetic marker used is not an informative indicator between tick populations within 300 kilometers of one another, or that these tick populations are panmictic and therefore are not genetically isolated within the tested area.
Isolation and Characterization of a Bacteriophage The Flathead Lake Monster
Ian Lorang - Biology
It has been suggested that bacteriophages are the most abundant entities on the planet. The goal of this study was to isolate and characterize a novel phage. Few phages have been isolated, which provides this study a good chance to isolate a novel phage from Northwest Montana where a phage has never been isolated. Using plaque techniques, restriction digest, and phage enzyme tool a phage was isolated and named the Flathead Lake Monster (FLM) and is a novel phage based on results from the study. FLM had abnormally small plaque diameters and an unusually long
tail. Compared to literature on other isolated phage’s tail lengths, the FLM has the longest tail ever isolated using M. smegmatis as a host. This led to the investigation of a correlation between plaque diameter and phage tail length. Comparisons within our lab confirmed that there is a correlation. An additional question of this study was to see if tape measure gene length, which is highly conserved in all isolated phages, correlates with phage tail length. Genome analysis of the phage will help to answer that question for the FLM and possibly reveal genes that are unique.
Does Drainage Affect Diversity within the Simulium arcticum complex in the Clark Fork River?
Chance Stewart - Biology
Chromosomal rearrangements seem to play a role in the speciation process of Simulium arcticum. This is an exception to the current dogma of speciation in most animals. Two models have been proposed for dispersal of S. arcticum. One relies on females returning to their natal sites to lay eggs; the other states that females choose the best available environment to lay their eggs. Chromosomal diversity has been shown to remain the same at various sites each year. With this in mind, larvae were collected from Bearmouth along the Clark Fork River, analyzed in micromorphological detail, and compared with larval chromosomes from various other sites within the Clark Fork and Blackfoot River Drainages. The goal of this analysis was to determine whether or not drainage influences chromosomal diversity. It appears that drainage does influence chromosomal diversity within the S. arcticum complex in these two drainages although the influence is slightly different within each drainage. Within the Clark Fork River Drainage, it appears that the Garrison influences Bearmouth, however, the entering Rock Creek channel has a possessive influence over Turah and Bonner, which are downstream. The opposite influence is seen in the Blackfoot River Drainage where the Clearwater River appears to have no influence on downstream sites.