Effects of Variable Oligosaccharide Lengths on the Infectious Nature of Chronic Wasting Disease Prion Proteins
Matt Breeggemann - Biology
The rate of a disease causing prion to convert a normal prion may depend on terminal sugar moieties on the oligosaccharides of the infectious protein. This project used two different insect cell lines that differed in their abilities to fully glycosylate proteins. Lectin affinity chromatography was used to confirm the lack of terminal sialic acid in the cell line that was unable to produce full-length oligosaccharides. Protein misfolding conversion assays were used to determine any differences in the rates of conversions between the fully glycosylated and partially glycosylated prions. These results may reveal insight into the mechanism for conversion of normal prions into disease causing ones by displaying the role of oligosaccharides in the conversion process in both animal and human prion diseases.
Birds as Reservoir Hosts for West Nile Virus in Montana
Victoria Dettman - Biology, Honors Scholars Program
West Nile virus (WNV) was first observed in the United States on the east coast in 1999. WNV is an arbovirus, meaning that it is stored in the tissues of arthropods and is then transmitted via a bite to vertebrates who become infected. Birds are vertebrates, and they then serve as vector reservoir hosts for the virus. They can transmit the virus to other vertebrates as well as back to arthropods. It was through birds that WNV quickly spread across the country, and we need to evaluate which birds are likely to transmit the virus and what living conditions they prefer. In this study, four regions of Montana were chosen for mosquito collection and bird observation. At each region there were at least two trap sites, one having canopy cover and the other open sky. Data were collected via both auditory and visual observations of birds. The results of this study indicate that Passerine bird species were found more often overall in Non-Canopy areas as well as regionally around Helena.
Clinical Outcomes of Gamma Knife Stereotactic Radiosurgery for Neurological Disease
Ameer Elaimy - Biology
Stereotactic radiosurgery (SRS) is a form of radiation therapy that delivers a focused, highly conformal dose of radiation to a single intracranial volume, while minimizing damage to the adjacent nervous tissue. SRS ensures precise radiosurgical localization by immobilizing the patient’s skull in a specified fixed position and in turn precisely aiming a high dose of radiation at the neurological target. SRS can be delivered to the patient via 3 therapeutic devices: Gamma Knife (GK) radiosurgery, linear accelerator based treatment, and a cyclotron-based proton beam. Published reports have not found statistically significant differences in terms of clinical outcomes when analyzing patients treated with either radiosurgical device. The GK is a cobalt-60-based machine, with 201 separate 4 to 18 mm collimator openings that emits multiple gamma rays that converge on a target specified by computer planning. This thesis describes the clinical outcomes of patients treated with GK radiosurgery at Gamma Knife of Spokane and Cancer Care Northwest for metastatic brain tumors, vestibular schwannomas, movement disorders, and trigeminal neuralgia. This thesis will not address specific treatment recommendations for other neurological disorders that may be treated using GK radiosurgery.
An Unconventional Approach to Undergraduate Research on the Micro Scale Abroad: Use of miRNA Inhibitors to Probe Signal Transduction Involvement of Lactobacilli in E-cadherin Expression at the University of Münster
Ashtin Jeney - Biology, Honors Scholars Program
The DAAD, German Academic Exchange Service, is a well esteemed organization that promotes advancements in higher education and works to establish internationalization of German universities. Their RISE program was one of many that invites natural science students from around the world to engage in progressive research as well as culturally integrating experiences. The Institute of Infectiology is part of the Center for Molecular Biology of Inflammation (ZMBE) at the University of Münster and their mission is to further understand relationships between pathogenic microorganisms and their targets. Contemporary research has been increasingly interested in the intestinal microbiota that has been directly related to human health. Probiotic bacteria prove to be vital components of the commensal gut microbiota and function as therapeutic treatments of inflammatory bowel diseases. E-cadherin is a component of the adherence junctions in epithelial cells that is known to be upregulated by the presence of various probiotics, to included Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus gasseri, and Lactobacillus acidophilus. This study aims to further investigate the roll of these species of Lactobacilli within the transcriptional regulation of E-cadherin by use of miRNA inhibitors of the SNAIL transcription factors. Further insight to the specific function of probiotics in gut health may lead to their more efficient use as therapeutic agents. Results did not clearly define the function of any of the Lactobacilli to work directly through either SNAIL transcription factors. Still, results did uphold the benefits of the Lactobacilli species and opened up opportunities for further analysis. Additionally, this project accomplished its purpose of the student learning new laboratory techniques such as cell and bacterial culture, coincubation and transfection, RNA isolation, cDNA preparation, and qualitative RT-PCR practices.
The Development of an Efficient Immuno-Slotblotting Technique to Quantify the Effects of Protein Disulfide Isomerase on Prion Protein Misfolding Spencer Johnson - Biology
The objective of this project was to develop an efficient immune-slotblotting technique that could be used as a quantitative assay in measuring the effects of Protein Disulfide Isomerase (PDI) on prion protein misfolding in Chronic Wasting Disease (CWD). Chronic Wasting Disease is a transmissible spongiform encephalopathy that is horizontally transmitted amongst families of cervids, which includes elk, deer, and moose. The infectious agent in CWD is a misfolded prion protein that comes into contact with other prion proteins, causing them to misfold. Previous observations have suggested that disulfide bond rearrangement may be an important step in the misfolding process. However, previous attempts to quantify the amount of prion protein using an immune-slotblot technique have encountered problems. The technique developed here uses limiting dilution, immune-slotblot analysis through the use of a Bio Dot SF Microfiltration apparatus. The immuneslotblot analysis is found to be more sensitive than conventional immunoblotting and was run following a methanol precipitation of protein to maximize results. This technique can be used to measure large amounts of prion proteins using an immuneslotblot. Also, a technique involving Proteinase K digestion of smaller samples of prion protein was found to be effective at efficiently detecting
smaller samples of prion protein. This technique can be used to measure the effect of PDI on the misfolding of CWD prion proteins.
Investigation of West Nile Virus Infection Rates in Culex tarsalis at Medicine Lake Wildlife Refuge and Ninepipe Wildlife Refuge 2011
Joseph Maricelli - Biology
This study focused on the infection rate of West Nile virus in two Culex tarsalis mosquito populations in Montana. Medicine Lake Wildlife Refuge, MLWF, and Ninepipes Wildlife Refuge, NWR, Cx. tarsalis populations were chosen due to the variance
in past viral incidence. Viral incidence at MLWR was thought to be higher due to higher infection rate in the Cx. tarsalis population possibly from favorable environmental factors. This study suggests, however, that high viral incidence may be a result of high Cx. tarsalis population as a porportion of the total mosquito population. Samples were homogenized and purified for RNA using a series of centrifugations through a specialized filter. A RT-PCR and Taqman assay then determined viral presence. No positive samples were collected from either collection site; a pooled infection rate program utilizing a 95% confidence interval determined there was no statistical difference in infection rates. Future studies can build upon this research by increasing sample number, recording differing temperatures and precipitation levels at both sites, and incorporating a temporal study in addition to the spatial analysis.
The Effect of Reactive Oxygen Species on Tiopronin-mediated Collateral Sensitivity in Multidrug Resistant Cancer Cells
Travis Marshall - Biology, Honors Scholars Program
Multidrug resistance (MDR) has become a major obstacle to chemotherapeutic cancer treatments. MDR is often a result of an up-regulation and overproduction of an ATP-binding cassette (ABC) transporter following chemotherapy. The phenomenon called collateral sensitivity (CS) is associated with MDR cancers and has shown implications to alleviate this obstacle in vitro. CS agents selectively kill MDR cancer cells overnon-MDR cancer cells, often by up-regulating the overproduced ABC transporters. A common ABC transporter that is over-expressed in MDR cancer cells is P-glycoprotein, P-gp. The orphan drug tiopronin has been previously shown to mediate CS in a non-P-gp dependent manner. This means that the CS of tiopronin may be due to another ABC transporter or possibly not from an ABC transporter at all. I attempted to understand the mechanism of tiopronin’s CS capabilities and found that reactive oxygen species (ROS) play a major role in tiopronin-mediated CS. Specifically, superoxide (O2-) has little effect on the CS of tiopronin while hydrogen peroxide (H2O2) is imperative for tiopronin-mediated CS. However, H2O2 cannot display CS by itself, indicating that H2O2 is likely an intermediate necessary for tiopronin’s CS capability but is not the direct cause of CS. Further research should be conducted to investigate the mechanism of tiopronin-mediated CS.
Mapping the Abundance of Culex tarsalis to Canopy Closure in Montana
Benjamin McIsaac - Biology
In Montana, West Nile virus (WNV) was first recorded in 2002, and by 2003 there were 222 reported cases statewide. Currently, WNV still causes high fever, vomiting, muscle weakness, convulsions and paralysis in over 1,000 Americans each year. The primary vector of WNV differs spatially across the United States, with Culex pipiens as the primary vector in the east and Culex tarsalis contributing to the majority of human cases in the west. Different primary vectors suggest that the geographical limitations of Cx. pipiens and Cx. tarsalis may be related to ecological factors. In the present study mosquitoes were collected fromthree different locations in Montana using CDC light traps. Elevation, distance to water and canopy closure were measured at each location. Total mosquito abundance and Cx. tarsalis abundance were compared with the independent environmental characteristics. Although no correlations were linked to total mosquito abundance, Cx. tarsalis displayed preferential habitat patterns. Elevation and location were shown to impact the presence of the primary western vector. Knowledge on environmental conditions conducive to the primary western vector can be used in primary prevention efforts to decrease incidence of human cases and advance subsequent undergraduate research of WNV at Carroll College.
Species Identification of Ovine-Feeding Mosquitoes in Southwestern Montana
Whitney Miller - Biology, Honors Scholars Program
In order to contribute to research on Cache Valley virus, an ovine virus that leads to fetus malformations and abortion, this study investigated the specific species of mosquitoes that bloodfeed on domestic sheep. Nine sheep were separated into three groups of three and were contained in a constructed trap one night per week for seven weeks (July-August 2011) in a rural area in southwestern Montana. Mosquitoes that entered thesheep-baited trap during the evening were then collected for species identification and bloodfed status the following morning. This study also examined the effectiveness of two different insecticide treatments. The three groups of sheep consisted of a control group, a group with Python insecticide ear tags, and a group with a permethrin pour-on. Populations of bloodfed mosquitoes were found in each of the 16 mosquito species collected from the sheep-baited traps. The species collected from the sheep-baited traps were then compared to species collected from a CDC light trap located near the study site. The data collected from this study showed that in the sheepbaited traps Ochlerotatus increpitus, Ochlerotatus idahoensis, and Aedes vexans were the top three most prevalent species and in the CDC light trap, Ochlerotatus dorsalis, Aedes vexans, and Ochlerotatus idahoensis were the top three most prevalent species. These results help to direct further investigations of potential vectors for Cache Valley virus.
Spatial Risk Assessment of West Nile Virus in Montana Based upon Temperature Effects on Culex tarsalis
Brian Murphy - Engineering
Spatial and seasonal West Nile virus (WNV) transmission risk was assessed throughout Montana with Geographic Information System (GIS) models based on the temperature threshold below which virus development will not proceed in Cx. tarsalis. The model used maximum, minimum, and average daily temperature data; a degree day modeling derivation that produced West Nile virus development units (WDU); and varying time-scales throughout June, July, August, and September. A temperature of 12.6 °C maintained for 107.7 degree days (dd) was utilized as the point of zero virus development and was based on the extrinsic incubation period (EIP) or the average amount of time between a female Cx. tarsalis imbibing an infectious bloodmeal and transmitting the virus. The models were produced to evaluate transmission risk on monthly scales and multi-month additive scales that utilize accumulated monthly WDUs.Overall, the models based on average temperature data showed higher correlation to Cx. tarsalis prevalence and West Nile virus human infection than their corresponding maximum or minimum temperature based WDU models. The models also showed an increasing risk of virus transmission
throughout Montana from west to east and an absence of transmission in areas of cooler daily temperatures. By the end of September about 82% of the state was determined to be viable for West Nile virus transmission while approximately 87% of the state was determined to be at risk of virus transmission by the end of August when peak risk occurs. Further improvements to the present models may arise from utilizing additional Cx. tarsalis trapping data from varying years or by incorporating population and travel density throughout state counties in order to better depict county counts of West Nile virus human infection. In brief, this study assesses the thermal and temporal effects on WNV transmission throughout Montana and
provides a guide for WNV prevention.
Microsatellite Analysis Comparing the Genetic Variation of Culex tarsalis Populations across Montana
Kellie O’Rourke - Biology
West Nile Virus (WNV), first detected in Montana in 2002, is transmitted by the mosquito Culex tarsalis in Montana. The purpose of the WNV project at Carroll College is to produce an infection risk assessment map for the state of Montana, created by determining the factors that affect the prevalence of the virus across the state. The objective of this specific study is to add one layer to the risk assessment map by using microsatellite analysis to compare the variation of genetic structure of differing populations of Cx. tarsalis across Montana. I hypothesized that the genetic differences (as measured by values) between pairs of populations would be lower if the populations are connected by waterways and are near in distance to each other. By inference, populations separated by large distances or by geographic barriers should have higher values. PCR amplification of three microsatellite loci for 12 populations was followed by high-resolution gel electrophoresis using the QIAxcel Advanced System to determine alleles present for each individual. Number of alleles per locus, null allele frequencies, and FST values were calculated using FreeNA and FSTAT. The number of alleles per locus ranged from 8–12. Null allele frequencies ranged from 0–0.38. values ranged from 0.005–0.39. Presence of null alleles and missing data made analyses difficult. values do not allow for either rejection or support of the hypothesis.
Isosorbide and Chytridiomycosis in Panamanian Golden Frogs (Atelopus zeteki)
Olivia Rolando - Biology, Honors Scholars Program
Global amphibian decline is an established problem, first noted over 40 years ago. Without an obvious cause beyond the natural factors, such as habitat loss, disease has risen as a reasonable explanation. Chytridiomycosis is an infectious disease targeting amphibians, namely frogs, in Central and South America. The fungus, Batrachochytrium dendrobatidis, has been shown to be inhibited by natural mixes of antimicrobial peptides (AMPs) found on the backs of susceptible and resistant frogs. A unique case, Atelopus zeteki, does not show the same use of AMPs. Rather, a heterocyclic diol, isosorbide, seems to be involved in their immunological response to B. dendrobatidis infection. Using liquid chromatography mass spectrometry, gas chromatographymass spectrometry, flame ionization detection, and chytrid bioassays, isosorbide was determined and suggested to function in an antimicrobial manner in A. zeteki. The lowest concentration exhibiting antimicrobial properties was 250 mM isosorbide against chytrid zoospores. The case study of A. zeteki is illustrative of how future infectious diseases’ activity could be quenched before such drastic losses occur. Additional research is needed to identify the antimicrobial properties of isosorbide in a quantitative manner and for determining whether the frog or the topical bacteria on the frog are synthesizing this compound.
Biochemical Analysis of Insect Camouflage in Manduca sexta, the Tobacco Hornworm
Kevin Semmens - Mathematics
Manduca sexta (Tobacco Hornworm) larvae exhibit diet-induced adaptation, demonstrating green coloration in their natural environment but becoming a pale blue when placed on a lab diet lacking plant material. I hypothesized that the color change observed in the Tobacco Hornworm was induced by the dietary intake of a plant pigment of orange or yellow color (specifically ß-carotene). The pigment ß-carotene is highly hydrophobic, so I hypothesized that it was traveling to the skin via a lipoprotein transport pathway. Hornworms were reared and then bled to extract hemolymph. Ultracentrifugation was used to float out the lipoproteins from the hemolymph. Lipoprotein isolation was confirmed by gel electrophoresis. The lipoproteins isolated from plant-fed insects did indeed contain a bright yellow pigment. UVvisible spectroscopy supported ß-carotene as the pigment being carried by the lipoproteins and responsible for inducing the insect’s color change.
Defining the Cytotype and Persistence Concepts in Black Flies (Diptera: Simuliidae)
Jeanna Van Hoey - Biology
Y-linked chromosomal inversions promote reproductive isolation in black flies. Time to fixation to total sex-linkage varies among incipient species. Persistence from year-to-year of chromosome types at any location is largely unstudied. Therefore, I analyzed the polytene chromosomes of a large sample (n = 800) of black fly larvae of the Simulium arcticum complex from the Little Blackfoot River (LBR) in western Montana to determine: 1) the extent of linkage of inversions to the Y chromosome and 2) persistence of taxa from year-to-year and compared these data to those of previous analyses, which were based on smaller sample sizes. I hypothesized that the large sample in 2011 would support previous observations.Thirteen Y chromosome types were described in 2011, whereas only nine types had been described previously. This study suggests that the IIL-10, IIL-18, IIL-38, and IIL-51 inversions are Y-linked at the LBR and that they chromosomally define cytotypes (taxa diverging from a common ancestor), which deserve more detailed study. Moreover, persistence was observed since all previous inversions, with the exception of IIL-30, IIL-35, and IIL-84 were found in 2011.
Incidence Modeling and Protocol for the Detection of Dirofilaria immitis in Montana
Ryne Dougherty - Biology
Heartworm, Dirofilaria immitis, is a parasite that infects dogs, coyotes, cats, and humans. D. immitis is transmitted from dog to dog through numerous mosquito vectors. Studies in Minnesota demonstrated the likelihood of Ae. vexans beingthe chief vector based on its frequent feeding habits and anatomy that are conducive for larvae growth. Therefore due to climate and geographical similarities, the most likely vector is Ae. vexans in Montana. Due to the climate dependent growth of D. immitis, the summers of 2007 and 2005 were sampled for mosquitoes. These mosquitoes were examined for D. immitis using a SY BR-Green real time PCR assay since the SYBR-Green flourophore requires less specialization of the real time machine and allows for broader use. Additionally, geo-referenced thermal data and GIS were used to build a risk assessment map for Montana. The development of the DNA extraction and SY BR-Green PCR protocol was successful and the results of the mosquito sampling suggest that D. immitis is nonexistent or at least extremely rare within the mosquito populations that were sampled. The risk assessment model supports the observed, negative lab results by suggesting that there are few locations in Montana where D. immitis could develop.
Using Population Models to Compare Strategies for Slowing Population Growth in Countries with High Fertility
Shannon Flynn - Biology for Secondary Education, Honors Scholar Program
Countries in the developing world continue to feel the effects of rapid population growth despite slowing growth in some regions. Due to limited funding and costs of fieldwork however, programs working to slow population growth have been hesitant to spend money to compare different methods. My study used computer modeling to create control models for Haiti and Niger, two countries with the highest fertility in their respective regions. Control models were compared to experimental models that took into account improvements in either education or family planning programs. The models revealed that in Haiti, helping women with no education achieve primary education would be the most effective method of reducing growth. In Niger, achievement of secondary education for all women would be most effective. These results may suggest that more emphasis should be placed on education as a strategy for slowing population growth in developing countries.
Cytogenetic, Molecular, and Array-based Analysis of a Complex Translocation found in a Patient Diagnosed with CLL
Shane Gallogly - Biology and Chemistry
Although a number of genes and chromosomal abnormalities have been associated with the presence of chronic lymphocytic leukemia (CLL), no oncogenes or critical tumor suppressor genes (TSGs) have yet been implicated in its onset. In this study, we describe a chromosomal abnormality existing in a subset of white blood cells from an individual experiencing early stages of CLL: a balanced rearrangement involving the p-arms of chromosomes 1, 3, and 6. This mutation being both unique and balanced suggests that the genes disrupted by the translocation breakpoints may be critical to the initiation of CLL. Microdissection techniques were used to physically isolate chromosomal breakpoint regions, which were then analyzed via array comparative genomic hybridization and results confirmed by fluorescence in-situ hybridization. The translocation breakpoints were narrowed to contain WAS protein family member 2 (WASF2), AT hook DNA binding motif (AHDC1), and Gardner-Rasheed feline sarcoma viral oncogene homolog (FGR) genes on chromosome 1, a TSG cluster on chromosome 3, and the UHRF1 binding protein 1 (UHRF1BP1) gene on chromosome 6. Earlier research found that the FGR gene and 3p21.3 TSG cluster have properties that may promote neoplastic tendencies during abnormal expression. In conclusion, we suggest one or more of these genes is a candidate for initial and/or early development of CLL. Future work will be focused on further narrowing the breakpoints of this rearrangement and studying cellular implications involved in abnormal expression of these candidate genes.
Incidence of Salmincola californiensis in Westslope Cutthroat Trout in Relation to Brook Trout Density in the Lolo Creek Drainage
Bradley Grammens - Biology
The objective of my study was to discover if density of invasive Brook trout had an effect on the incidence of native Westslope Cutthroat trout infected by Salmincola californiensis. The study was conducted under a United States Fish and Wildlife collection permit while electro fishing in the Lolo Creek drainage in northwestern Montana. Streams in the Lolo Creek Drainage were sampled and trout densities and infection incidence were quantified. A Linear regression test was performed to test for significant associations. A significant negative correlation was observed between the Brook trout density and the incidence of Salmincola infection in Westslope Cutthroat. My study showed an invasive species aiding a native species. Brook trout are effective at reducing the amount of Salmincola infected Westslope Cutthroat trout.
West Nile Virus Presence and Blood-Feeding Behavior of Culex tarsalis in Wildlife Refuges and Management Areas in Montana
Kellie Kalbfleisch - Biology
West Nile virus (WNV) has been documented across the state of Montana since 2002. Humans, other mammals, and birds have been affected by this virus in a heterogeneous manner throughout the state. Correlating the feeding patterns of Cx. tarsalis, the principal WNV vector in the Western United States, with the presence of virus at Ninepipe, Freezeout, Benton, and Bowdoin wildlife refuges and management areas allowed for analysis of the link between competent avian hosts and the presence of WNV. Further, identification of avian species present in Cx. tarsalis blood-meals allowed for the analysis of the correlation between relative abundance of avian species and their incidence in blood-meals. Presence of WNV RNA in mosquito pools was measured using RT-PCR and TaqMan assay. Mitochondrial cytochrome b and cytochrome c oxidase I gene sequence analysis of DNA extracted from individual blood fed Cx. tarsalis mosquitoes was used for blood meal identification by the comparison of sequences of both the cytochrome b and cytochrome oxidase I gene fragments with the GenBank DNA database. The combination of WNV detection and blood meal analysis can be used to better understand the temporal relationship of viral presence or absence and seasonal feeding patterns of the WNV vector mosquito Cx. tarsalis.
Distribution of Bird Reservoir Hosts for West Nile Virus in Montana
Billy Mcelroy - Biology
West Nile virus (WNv) is an arthropod-borne flavivirus that was first detected in the United States in 1999. By 2004 it had spread to 48 states and to date it has been the source of greater than 29,000 cases of human infection. WNv is maintained through an avian-mosquito transmission cycle, with birds serving as the primary vertebrate hosts for the amplification of the virus. Previous studies have shown Passeriform birds to be the most virulent competent hosts for the virus, producing adequate viremia counts for continuation of the transmission cycle. I tested for associations between total bird abundance, Passeriformes abundance, and virulent competent bird species abundance with mosquito population data sampled across Montana. I found no temporal correlation between bird and mosquito numbers across the summer sampling period. However, I observed a positive correlation between passerine abundance and total mosquito numbers. These findings suggest that passerine abundance is associated with mosquito population numbers, which may result in an increased risk for WNv transmission.
Genetic Variation of West Nile Virus in Montana
Meghan Mckeown - Biology
West Nile Virus, an arbovirus classified in the genus Flaviviridae, presents a serious threat to humans and horses in Montana. The virus, first isolated in the West Nile district of Uganda in 1937, spread rapidly across the United States after its initial introduction to New York in 1999. In 2003 there were 222 human cases of West Nile. The overarching goal of the Carroll College West Nile Virus study is to provide a risk assessment map of the state of Montana. This will bring better awareness to the risk of West Nile infection based on geographic location and time. The specific goal of this project was to use microsatellite data of the mosquito vector to assess the genetic differentiation between study sites across the state of Montana and to evaluate if populations separated by greater geographic distance will have higher FST values than those located closer together. Multiplex and individual locus PCR methods were used to amplify five microsatellite loci (CUTC6, CUTC12, CUTD107, CUTD113, CUTD120). HE values for each population were obtained, and FST values were obtained for between population comparisons. My hypothesis that FST values increase as geographic distance increases was not supported. Harlem and Medicine Lake have a lower FST than Medicine Lake and Bowdoin, which are separated by a smaller geographic distance. Three factors-small sample sizes, high frequencies of null alleles, and deviations from Hardy-Weinberg equilibrium-are almost certainly confounding the data such that the expected patterns are not recognized.