Search for potential replication origins in Borrelia burgdorferi.

Fellow- Daniel Belz
Mentor- Dr. Scott Samuels

Abstract

Shuttle vectors, which allow transfer of DNA between species, can be valuable genetic tools. To be able to replicate in a given organism, they must have an origin of replication recognized by that organism. This project involved creating a plasmid useful for testing sequences that may have the ability to act as replication origins in Borrelia burgdorferi, the spirochetal bacterium that causes Lyme disease. The origin of the LE1 bacteriophage, which replicates in the spirochete Leptospira biflexa, was tested, but found not to replicate in B. burgdorferi. In addition, a genomic library of B. burgdorferi was constructed to search for replication origins.
 

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Inhibition of a-Ketoglutarate Dehydrogenase due to Arsenite Exposure

Fellow- Erik Bergquist
Mentor- Dr. Brooke Martin

Abstract           

            a-Ketoglutarate dehydrogenase (KGDH) was inhibited with increasing arsenite exposure.  Activity loss was linked to modification of the sulfhydryl groups on lipoic acid, the essential cofactor of the KGDH E2 subunit.  Modification of lipoic acid by arsenite was found to be irreversible causing 25% enzyme inhibition as low as 250ppb As(III).  To verify lipoic acid modification, anti-lipoic acid antibodies and mass spectroscopy were performed enabling the confirmation of a mechanism for arsenite induced oxidative stress.
   

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Effects of vegetation and ramp modification on small- and medium- sized animal use of culverts as travel corridors.

Fellow- Erin Bohman
Mentor- Dr. Kerry Foresman

Abstract

Culverts are becoming recognized as important small- and medium-sized animal corridors to connect wildlife populations separated by roads, highways and railways.  However, many factors influence their effectiveness, including various dimensions of the culverts themselves, fences near the culverts, and vegetation cover at the entrances.  In this paper, I investigated four culverts and the effect of vegetated entrances on the entry and crossing of animals in these culverts.  Also, I studied the effects of modifying an experimental ramp inside one of the four culverts.  This was done by tying a uniform surface over the original metal mesh surface.  This ramp was originally installed by the Montana Department of Transportation as an experimental method of promoting animal travel through the culvert even if it is occupied by water and debris.  A wide range of small- and medium-sized mammals used the culverts, although the meadow vole (Microtus pennsylvanicus) did not travel through them even though they were the most abundant species along the roadside.  No conclusions were feasible in this study, however several inferences can be drawn from the gathered information. 

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Effects of Aphthona species and integrated management on leafy spurge and native plant communities of Mount Sentinel

Fellow-Cynthia Buckalew
Mentor- Marilyn Marler

Abstract

Euphorbia esula L., commonly known as leafy spurge, is an invasive weed that has displaced native plant species throughout the western United States.  While a great deal of research has addressed methods of managing leafy spurge, few studies have investigated responses of native plant communities to these various control methods.  This research project has two portions, which are both continuations of studies initiated during the summer of 1999 and continued through the summer of 2000. The first portion will study how the use of flea beetles to control leafy spurge affect native plant species and leafy spurge populations already established on Mt Sentinel.  I expect that release sites will show a slight decrease in leafy spurge density and a slight increase in native plant species density.  These results should continue over the years while the flea beetle populations establish.  The second portion is to continue research on the effects of mowing, grazing, flea beetles, reseeding, and herbicides, alone and combined, on leafy spurge and the establishment of native plant species.  I expect there will be lower leafy spurge density in the plots with combined treatments than the plots with only one treatment.  To monitor both portions I will count the number of leafy spurge stems (frequency) and measure percent cover of leafy spurge, bare ground, and all other plant species in the data plots.  This will allow for a long-term analysis of the plant community responses.  This research will help us understand if intensive management practices are achieving restoration objectives in addition to controlling this invasive plant.
 

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Analysis of Red-backed vole, Clethrionomys gapperi, Movement Patterns in Relation to Grounded and Elevated Fallen Logs.

Fellow- Heather Davis
Mentors-  Dr. Don Christian

Abstract

Red-backed voles are associated with fallen logs and coarse woody debris. They prefer habitats that include these structural components, and are thus often used as indicators of forest health in the context of understanding forest management practices. Although they have been shown to utilize logs in a higher proportion than proportionately available, a question arises as to the nature of that use. Are they actively selecting for the logs, perhaps for some benefit provided (such as cover)? Or are they merely showing such a disproportionate use because their movement is being conscripted by a funnel-like effect of the log? In this study l examined the portions of vole trail associated with elevated and grounded logs in attempt to quantify their movement patterns. An elevated log, by definition, cannot constitute a barrier or a funnel. In order to determine selection versus conscripted movement, I examined the comparative tortuosity, approach and departure angles between the two log position categories. I tested the null hypothesis that log position does not affect movement pattern. This study showed that movement patterns were similar between both categories of logs, in all areas measured. This fails to reject my null hypothesis and does not support the assertion that logs are acting as vole “chutes”.

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Analysis of a New Selectable Marker for the Lyme Disease Bacterium, Borrelia burgdorferi

Fellow-  Kristi L. Frank
Mentor- Dr. Scott Samuels

Abstract

The genetic system for Borrelia burgdorferi currently has only three selectable markers, and only one is widely used.  In an effort to develop more antibiotic resistance markers for this system, we constructed an E. coli-B. burgdorferi shuttle vector that carried a spectinomycin and streptomycin resistance gene (aadA gene) fused to a strong Borrelia promoter and investigated its potential to confer antibiotic resistance in B. burgdorferi strain B31.  High levels of resistance to both spectinomycin and streptomycin were measured.  Transformants formed colonies in solid medium under selection with streptomycin or with both antibiotics used simultaneously.  This hybrid spectinomycin and streptomycin resistance cassette will be useful as a selectable marker to probe gene function in B. burgdorferi. 

Monitoring and Prediction of Malaria Outbreaks

Fellow- Jennifer Harlow
Mentor- Dr. Steve Running

Abstract

Malaria is caused by infection with Plasmodium species parasites, which are transmitted to humans through the bite of anopheline mosquitoes.  Vector-borne diseases such as malaria are highly influenced by spatial and temporal changes in the environment, which are introduced mainly, but not exclusively by climate variability.  Climate has been established as an important determinant in the distribution of vectors and pathogens. The purpose of my research was to study relationships between the social, climatic and economic variables, which currently best describe malaria transmission.   There are few well-established methods for assessing the impacts on health due to climate change, since complex ecological systems involved are difficult to model and measure. Many factors other than climate, such as land-use, migration of people, and errors in water management influence the emergence of vector-borne diseases. Satellite observations allow a new method of health assessment due to the wide availability of climate data collected.  Satellite data serves as an important source of continuous global information that can be used to monitor disease outbreaks. My objective is to use satellite data to look for relationships between climate fluctuations and malaria epidemics without overlooking important socio-economic factors that also may contribute to increased incidence of disease.

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Antenna morphology and pheromone-based mate location in Polyphemus moths.

Fellow-Erin Heydenreich
Mentor- Dr. Doug Emlen

Abstract

Mate location in moths is accomplished by long distance detection of low concentrations of pheromones emitted by females upon emergence.  Pheromone based mating systems have been suggested to be under strong sexual selection (Phelan, 1997).  There are several aspects of moth biology that suggest that larger body size and larger antennae will be selected for in males.  In this study I predicted that polyphemus (Antheraea plyphemus: Family Saturniidae) males able to successfully locate females would have proportionately larger antennae than males sampled from the population at random. Captive, reared males represented the base population and males caught with captive females represented the “successful”, or sexually selected, population.  Wild males were captured using female-baited traps in Mclay Flats of the Blue Mountain Recreation Area, the Rattlesnake Recreation Area, and Rock Creek, in Missoula, Montana. Neither absolute nor relative antenna size differed between “successful” (i.e. baited) and control males, suggesting weak or absent sexual selection for antenna size in this population.  Study of moth pheromones is a useful tool in monitoring populations and has important implications for agriculture and conservation.

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Bacterial Fingerprints from the Feces of Snowshoe Hare Represent A Non-Invasive Sampling Technique and a Tool for Conservation Biology.

Fellow-Erin Inkley
Mentor- Dr. W.E. Holben

 Abstract

Using new and developing research in microbiology, the microbial DNA from the feces of snowshoe hares (lepus americanus) was analyzed to determine the structure of the microbial communities.The objective was to test the hypothesis that molecular analysis of GI tract microbial communities can be used as a non-invasive sampling technique for population dynamics studies with snowshoe hares. DNA isolated from hare fecal pellets was used for analysis in which partial 16S rDNA genes were amplified using PCR. The resulting mixture of PCR products was subsequently separated using Denaturing Gradient Gel Electrophoresis (DGGE).  This technique produces microbial community “fingerprints” which were used to analyze and compare the similarities and differences in the microbial populations in the fecal pellets of snowshoe hares.  Comparisons were done between pellets in a single individual, between individuals in a single population, and between two distinctly separate populations. Pattern matching analysis using GelCompare software generated a dendrogram of the DGGE results indicating the relatedness of the bacterial communities and their corresponding hare populations. Differences were detected between individual hares and between the two distinct populations.  For example, two individual hares from a single site (Cold Creek) showed distinct similarities within their populations yet also had distinct differences between each other.  On the final dendrogram they are branched together in the Cold Creek group.  When two geographically separated populations (Cold Creek and Tally Lake)were compared they showed larger and distinct differences between populations.  Collectively, these data indicate that this approach has potential to resolve not only geographically discrete populations but also individual hares within a single population.
 

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System for Genetic Manipulation of ialB promoter from Bartonella bacilliformis, a regulator associated with virulence

Fellow- Chad Lang
Mentor- Dr. Mike Minnick

Abstract

Bartonella bacilliformis is a bacterium that causes Oroya fever, and verruga peruana in humans.  The illness is found in South America, mainly in the valleys of the Andes Mountains.  The bacterium uses arthropod-mediated transmission and invasion of human red blood cells as its parasitic strategy. Life-threatening symptoms occur, characterized by acute fever, malaise, and extreme hemolytic anemia.  Although what is happening to the cells is clear, little is known about the (ialB) gene and how it is regulated.  Bartonella Baclliformis, the model in the study, thought only to be in higher elevations, was recently thought to have spread to some lowland areas, making scientists concerned that the bacterium is invading new territories.  In Specific Aim 1 our goal is to examine the (ialB) promoter, a regulator for a known virulence factor found in B. bacilliformis, and study (ialB) genes involvement in the invasion of human red blood cells by transformed E. coli pCJL100.  Specific Aim 2 will examine whether or not temperature plays a role in the expression of (ialB) and if it is up-regulated or down-regulated at different degree of temperature.  In Specific Aim 3, we will determine the significance of temperature fluctuations, which are a common event in bacteria transmitted from an arthropod to a human.  The CAT reporter gene will be tested for by Elisa, tests will give us an easy way to measure the (ialB) promoter’s activity in response to temperature changes.
 

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Soil Aggregate Formation: determining the timeline for aggregate formation using a glacial chronosequence.

Fellow- Jacqueline Papez
Mentor- Dr. Matthias Rillig

Abstract

Although soil aggregation is known to be an important element in soil stability, little is known about the processes and organisms involved in the formation of aggregates from non-aggregated soil.  Grinnell Glacier in Glacier National Park provided an ideal setting for examining the relationships between glomalin and water stable aggregates over recently uncovered soils.  Glomalin was found to increase linearly from the low nearest the glacier to the highest level at the samples taken at the greatest distance from the retreating glacier.  Aggregate water stability increased stepwise, with stability remaining low until the 70-m point.  This suggests that the beginning processes of soil aggregation require more than glomalin alone can provide, likely in the form of microbial secreted matrices. 

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Investigation of Microbial Transport in Subsurface Aquifers

Fellow- Mike Reed
Mentor- Dr. William E. Holben

Abstract

Bacteria have been proposed as a possible solution to facilitate the effective clean-up of contaminated subsurface areas around the United States.  Bacterial metabolism can degrade toxins in the subsurface, converting it into non - harmful organic material; this is the process of bioremediation.  The focus of this research is to study bacterial transport potential --the ability for bacteria to move to all areas of contamination-- and to determine the factors that cause the bacteria to colonize in these areas at an aquifer field site in Oyster, Virginia.  Specifically, my project was to attempt to relate bacterial transport behavior to colonization and attachment of bacteria to sediments, which has not been successfully accomplished to date for any in situ field injection of bacteria.  The in situ injection involved injecting a number of bacteria into the field site only to return later to take samples of the water and sediment.  The sediment and water samples are then shipped back to the University where they are analyzed by RT-qPCR in the lab.  The laboratory technique involves an analysis of the number of template molecules in the reaction.  This concentration can be translated to the actual number of bacteria at that sample site.  This data is then transferred to a spreadsheet program where it can be compared and contrasted to data from other sites to determine migratory patterns of the bacteria.  It must be determined whether bacteria injected into the subsurface can migrate to contaminated areas for bioremediation to be effective.

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The Quantification of Microbial Communities in Soils Associated with Centaurea maculosa Lam (Spotted knapweed)

Fellow- Alex Rodriguez
Mentor- Dr. William E. Holben

Abstract

Centaurea maculosa Lam (Spotted knapweed) is one of the most environmentally disruptive weeds in North America. Biological control measures have produced ambiguous results in the field, and greenhouse experiments have led a number of researchers to note the potential for an escalation in the spread of C. maculosa in response to the herbivorous activities of natural enemies released as agents of biocontrol. These observations contrast significantly with the effects of natural enemy attack on C. maculosa in its native environment, Eurasia, where the plant is ecologically integrated. Two explanations are offered: First, it is hypothesized that through introduction to a new environment C. maculosa has been able to escape soil pathogens that work, along with a suit of resident herbivores, to inhibit invasive proliferate in its native habitat. Second, studies indicate that in Montana soils C. maculosa may have the ability to alter the microbial community to favor its own growth at the expense of native grasses. One or both of these processes are potentially at play in the spread of this plant. The goal of this study has been to use molecular techniques to quantify differences in the microbial community structure between both foreign and domestic soils occupied by C. maculosa and soils not invaded by the plant.
 

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Structure and Function of a Surface Protein on Bartonella quintana

Fellow- Kate Sappington
Mentorr- Dr. Mike Minnick

Abstract

 Bartonella quintana has repeatedly emerged throughout history as a cause of infection among distinct and diverse populations across the globe.  The bacterium has been identified as the agent in trench fever, a disease responsible for one million deaths during World War 1.  A disease of the past, B. quintana has re-emerged as a public health concern after recent outbreaks have infected inner-city homeless people and patients suffering from AIDS.  Despite these characteristics, little is known about the pathogen.  The gram- negative cell wall associated with the bacterium consists of a multi-layered structure that contains proteins essential to the structure and function of the bacterium.  A prominent outer membrane protein has been identified to contribute to cell survival.  We hypothesize that this protein, which we have termed OmpA, serves as a porin.  Porins function to form channels in the cell membrane which aid in the uptake of cell nutrients, hence they play a significant role in cell survival and growth.  A recent study has attempted to characterize OmpA using biochemical and genetic approaches.  OmpA was isolated and used to generate monospecific antibodies against the OmpA protein. The antibodies were used to screen a library for the OmpA encoding gene. These data will be used to form a foundation to more fully explore OmpA’s hypothesized role in B. quintana’s survival and virulence. 

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Testing for Differences in Newborn Snowshoe Hare Survival

Fellow- Kevin Sterling
Mentor- Dr. Scott Mills

Abstract

Newborn survival is an extremely important and influential factor in population growth in snowshoe hare (Lepus americanus).  This study focused on the first 35-day survival rate of newborn snowshoe hares (leverets).  Radio-transmitters were attached to the newborn snowshoe hares at birth in order to monitor daily survival and movement.   The data collected in the field was tested against a priori models that reflect five related hypotheses:

1.      The 35-day survival rate of leverets depends on litter size.

2.      The 35-day survival rate of leverets depends on birth pulse number.

3.      The 35-day survival rate of leverets depends on habitat context.

4.      The 35-day survival rate of leverets depends on birth weight.

5.      Leveret survival is a function of time since birth

The models provided strong evidence that the first 35-day survival rate of leverts was dependent upon birth pulse number, birth weight, litter size, and time.  The hypothesis linking daily survival rate to habitat has not yet been tested.  
            

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PCR Amplification of Eukaryotic DNA from Snow: Developing Tools for Phlylogenetic Analysis of the Geographic Distribution of Snow Algal Populations.

Fellow- Nathan Stevens
Mentor- Dr. William Holben

Abstract

Red colored snow can be seen in the mountainous regions around the world every late spring or early summer.  Persisting or permanent snow packs provide a suitable habitat for a number of psychrophilic algae. Two common species causative of red snow are Chlamydomonas nivalis, the more widely recognized, and Chlainomonas rubra, the less formally known.   While it may seem feasible for the wind or animals (i.e. migrating birds) to transport snow algal spores considerable distances, suitable snow algal habitats are rare and widely separated.  Despite the limited habitats and chance dispersal, snow algae are common in every hemisphere on earth.  Relative isolation and limited dispersal of these organisms suggest that considerable genetic variation may exist between isolated populations.  This study focuses on genetic differences of several snow algal populations throughout Montana and Northern Wyoming.  By analyzing sequences within the internally transcribed spacer regions (ITS) of ribosomal DNA genetic differences can be found in closely related organisms. These differences will allow us to estimate spatial and perhaps historical dispersal patterns, and identify separate distinct populations based on their geographical distribution. Here it is shown that eukaryotic DNA can be amplified using PCR and eukaryotic specific primers.  This study, not yet finished, demonstrates the feasibility of using molecular strategies to isolate algal DNA sequences from snow samples, as opposed to culturing.  
 

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Effects of Nucleocapisid and Gag Protein on HIV-2 dimerization

Fellow- Colby Stoddard
Mentor- Dr. Stephen Lodmell

Abstract

Human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) cause similar disease in humans and are the etiological agents of the global AIDS pandemic.  Nucleic acid binding proteins are essential in viral replication processes such as dimerization of the viral genome and reverse transcription.  Viral gag and nucleocapsid (NC) proteins exhibit nucleic acid binding properties (1) and are desirable for studying protein-nucleic acid interactions.  This study focuses on the construction of gag and NC clones with and without a protease-coding region, also the overexpression of recombinant viral protein within bacterial cells is investigated.
 

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Characterization of the changing antibody response in wild cougars infected with feline immunodeficiency virus (FIV).

Fellow- Jesse Thompson
Mentor- Dr. Mary Poss

Abstract

Feline immunodefeciency virus (FIV) is a lentivirus belonging to the same family of viruses as HIV.  It is known that domestic cats are infected with this pathogen and experience a similar disease state as humans, characterized by a compromised immune system.  Although FIV is also known to infect wild felids, little is known about its pathology in these cats.  This study was conducted as a serological method to detect the prevalence of FIV infection in two populations of wild cougars in Yellowstone National Park and the Snowy Range, Wyoming.  Five FIV proteins could be detected by immunoblot:  gp105, p55, gp35, p25, and p15.  Serial dilutions were performed on longitudinal serum samples from infected individual’s to determine the titer at which a 50% reduction in viral protein recognition signal was produced.  Using titers to individual viral proteins, the hypothesis that an individual’s antibody response to FIV changes over time was explored. 
 

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Movement and Behavior of Long-Toed Salamanders (Ambystoma macrodactylum).

Fellow- Andrew Van Eck
Mentor- Dr. Colin Henderson

Abstract

In the Pacific Northwest salamanders comprise a significant portion of the ecosystem’s fauna.  Despite their ecological importance, few studies have accurately depicted habitat selection and movement patterns.  This dearth of knowledge has arisen from methodological limitations.  Due to their small size and fragile bodies, active marked recapture techniques, such as radio telemetry, do not exist for salamanders.  Without the ability to actively track salamanders, determining habitat characteristics and movement is extremely difficult, if not impossible.  Herein, I describe a method for active marked recapture with salamanders using harmonic radar.  This system contains advantages that radio telemetry lacks.  To begin with, the tag attached to the salamander of interest does not require a battery, which permits a lighter weight, increases durability, and decreases the hindrance upon movement.  In addition, harmonic radar is highly directional and capable of finding tags underground, beneath rocks and behind trees.  For these reasons, I used harmonic radar with long toed salamanders (Ambystoma macrodactylum).  I developed an active tracking technique that did not impede movement, feeding, or normal bodily functions.  In addition, I described summer home range and suggestive habitat characteristics for long-toed salamanders in the Swan Mountain range of Montana.

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Floral Wetness as a Source of Selective Pressure

Abstract

Flowers have the potential for wet petals nearly everyday due to rainfall or dewfall.  The collected water has the potential to flow back into the corolla of a tubular flower such as those common in Penstemon and Campanula.  There are many possible detrimental effects that could arise from floral wetness.  Water could affect pollen count by removing pollen from the anthers before a vector, such as a bee or hummingbird, has had a chance to visit the flower thus decreasing the reproductive capacity of the plant.  In this study, wettability and retention were used to investigate any differences in water interactions on the different surfaces of a tubular corolla.  The outside of the corolla had a greater wettability than the inside.