Download Life and Earth Sciences Senior Research Abstracts May 2006 Abstracts 2006

Life and Earth Sciences
Senior Research Abstracts
May 2006
Abstracts 2006
Freweine Berhane
Truncated EBV-encoded dUTPase induces enzymatic activity similar to the full-length
protein
Epstein-Barr virus (EBV) is an oncogenic gamma herpesvirus, which encodes for numerous
enzymes that are involved with viral DNA replication. We have recently demonstrated that the
EBV-encoded deoxyuridine triphosphate nucleotidohydrolyse (dUTPase) induces immune
dysfunction that is independent of its enzymatic activity.
Analysis of purified preparations of the EBV-encoded dUTPase demonstrated the presence of
two proteins species; one with a molecular weight of approximately 31 kDa, which represents
the full-length protein and a second species with a molecular weight of approximately 25 kDa,
which we believe represents a N-terminal truncated form of dUTPase. Using molecular biology
techniques, such as polymerase chain reaction, we have constructed and cloned a portion of the
of the EBV-encoded dUTPase gene, which lacks 114 nucleotides from the 5” end. The protein
expressed from this cloned gene was purified using affinity chromatography and was shown to
be approximately 25 kDa and to possess functional dUTPase activity. Studies are presently being
performed to determine whether the truncated dUTPase also causes immune dysfunction.
Jennifer A. Berry
The effects of diet on microbial populations in yellow-bellied slider turtle, Trachemys
scripta
Herbivorous vertebrates use microbial gut fermentation to digest plant material because they lack
endogenous enzymes needed to break down cellulose. Reptiles are the poorest studied group of
vertebrates that use microbial symbionts. The purpose of this study was to describe the microbial
gut population in yellow-bellied slider turtles, Trachemys scripta, and to determine how
microbial populations change with diet. This is especially important to understand in growing
reptiles that need to maximize digestion for survival and future reproductive success. Fifty T.
scripta hatchlings were obtained from a breeding farm in Louisiana. Their guts were inoculated
with microbial symbionts by feeding feces from wild adult turtles. They were maintained on the
following five different diets with ten turtles per diet: 100% duckweed; 79.1% duckweed/ 20.9%
cricket, 60.6% duckweed/ 38.4% cricket, 33.8% duckweed/ 66.2% cricket, and 100% cricket.
These diets represent plant to animal ratios turtles experience as they ontogenetically transition
between carnivory to herbivory. It was hypothesized that microbial species composition will
differ between diets. Turtle feces were collected twice a week to analyze microbial population.
EMB plates specific for gram positive bacteria and PEA plates specific for gram negative
bacteria were used to anaerobically grow colonies in candle jars. Isolated colonies were tested
for enzymatic activity using Enterotubes, which test metabolic products. Eight dominant
bacterial species were found, five of which were members of the Megasphaera genus. This is the
first finding of this species in an ectotherm. Further analysis is needed to determine the species
of the remaining three. Slight changes in bacterial populations among diets indicate gut flora
changes as diet changes from plant to animal.
Deborah D. Carter
The effects of smoking and secondhand smoke exposure on forced vital capacity and peak
expiratory force at college age
Chronic obstructive pulmonary disease (COPD) is the fifth leading cause of death in the world
but the disease does not present in youth. Lung function increases with lung growth until the late
teens, begins to plateau, and finally declines near the age of 25 years. However, there is a small
subgroup of those who smoke or were subjected to secondhand smoke exposure early in life who
experience an exaggerated decline of lung function that presents later in life at approximately the
fifth decade. Study of the early effects of smoking and early exposure to secondhand smoke
would determine which individuals are susceptible to respiratory impairment, and early detection
of this impairment would enable preventive measures to be applied. This study was performed
to determine whether any change in static and dynamic lung function could already be identified
at college age in students exposed to first hand or secondhand smoke, when compared with nonsmoking students. Pulmonary function was tested both before and after exercise in a volunteer
non-asthmatic population of Otterbein non-smokers, second-hand smokers, and active smokers
by static and dynamic spirometry. This measures the size of the lungs and the magnitude of the
air flow within the pulmonary airways, and is the standard way to evaluate lung function in
health, and to detect respiratory disease. Results showed trends toward decreased lung function
in smokers when compared with non-smokers: second hand smokers had intermediate values.
However there was no statistical significance of the variance of the mean values, possibly due to
the small sample size of smokers. These data suggest but do not prove that the effects of smoking
or secondhand smoke exposure early in life have adverse effects on lung function that can be
detected at college age.
Andria M. Coe
The effect of dietary energy source and time of adaptation on muscle glycogen
concentrations in the horse
It has been shown possible to manipulate muscle glycogen concentration in the equine with diet
and exercise. However, because of differences in exercise regimen, diet, and time of adaptation
used in previous studies, it is not clear how glycogen concentrations are affected by the
proportion of energy from carbohydrate and fat sources. The goal of this research was to
determine the effects of energy source and time adaptation on glycogen concentration in muscles
at rest in the horse. Six mature mix-breed horses were used for this study (mean body weight:
(590.7kg± 28.72kg). Horses were not in work for one month prior and during the experiment.
The horses were randomly allocated to a group to receive one of two diets (Phase A). One diet
was high in fat and fiber (FF; Purina Ultium, St. Louis MO) and another was high in starch and
sugar (SS; Buckeye. Dalton OH). A crossover design was used, so after a period of 4 weeks, and
a 2 week washout period, the treatments were reversed (Phase B) so that each horse eventually
received both diets. Muscle biopsies were performed on all horses at week 0 (prior to starting the
diet), and weeks 2 and 4 of each diet to determine muscle glycogen content. Muscle glycogen
concentrations were higher when horses were fed either concentrates compared to the start of
the trial (P<0.05). Furthermore, at weeks 2 and 4, muscle glycogen concentrations were higher in
horses fed FF diet compared to SS. In conclusion, a fat and fiber diet can increase muscle
glycogen concentrations in the resting horse, but further research on blood chemistry would need
to be done to find the metabolic mechanisms.
Samantha M. Coe
The effect of diet and time of adaptation on glycemic and insulinemic responses in horses
Glucose and insulin metabolism in horses may be impaired in conditions such as growth,
exercise, obesity, gestation, pituitary dysfunction, laminitis and aging. The objective was to
determine how the horses’ glycemica and insulinemic responses change according to the feed
type and the time of adaptation. Seven mature horses under no physical conditioning were fed
either a concentrate high in starch and sugar or high in fat and fiber. A cross-over design was
used with an initial washout period when horses were fed only hay. A washout period was also
used when switching the two diets. The horses consumed each diet for four weeks and at week
0, 2, and 4 a glycemic and insulinemic response test was performed. For the test, a baseline
sample was taken after which the horses were allowed to eat a test meal. Samples were then
taken continuously via a jugular catheter at post feeding, 30, 60, 120, 180, 240, and 300 minutes.
Plasma was analyzed for glucose concentrations and serum was analyzed for insulin
concentrations. There was no effect of diet or time on the horses’ body weights. In addition,
there was no effect of time of adaptation on glucose or insulin concentrations following
consumption of the test meal. In general however, glucose and insulin concentrations tended to
be higher when horses consumed the diet high in starch and sugar compared to the diet high in
fat and fiber. It is likely that there were not a large enough difference in the dietary energy source
to evoke a time of adaptation response in this group of horses. Furthermore, it is possible that
had this study been continued longer, changes due to dietary adaptation would have been
observed.
Amy D. Corbett
The post-acute effect of alcohol on muscle fatigability and systemic arterial blood pressure
in young male athletes
Alcohol and athletics have been linked together since the beginning of American sports
competitions. Alcohol is the most commonly consumed drug amongst athletes. Some athletes
believe that alcohol aids in athletic performance by reducing pre-game nerves or by forcing the
player to work harder to overcome a hangover on the day after drinking. In contrast, studies have
shown that alcohol carries a hefty number of harmful effects on health and performance both
while drinking and long term after years of high alcohol consumption. However, not much
information is known concerning the post-acute (medium term) effect of alcohol breakdown
byproducts of alcohol (acetaldehyde and oxygen radicals) on the athlete’s body. A group of
volunteer 18-24 year old Otterbein College male athletes similar in fitness level were tested for
the blood pressure, grip strength and endurance two days per week over a four week period
during the athletic season. Midweek testing was intended to follow no alcohol consumption, and
weekend testing was intended to follow self-chosen alcohol consumption. Results showed some
initial abnormally high blood pressure readings, but no significant upward trend with alcohol
consumption was seen. Grip strength showed there was a major decrease (an mean value of
290.00 kilogram seconds) with no alcohol consumption to 114.28 kg/seconds with 11 to 15
alcohol beverages) in overall strength due to an increase of the number of drinks the night
before. The conclusion is that even the ‘post-acute’ effect of alcohol greatly impacts the
physiology of athletes.
Bryan M. Duncan
Relationships within the Index of Biotic Integrity (IBI): what the subdivisions of the IBI
suggest about the water quality of the Scioto River watershed
Stream quality assessment metrics, such as the IBI, document the ecological integrity of the
stream. They are methods of depicting existing water quality and of documenting both positive
and negative changes in our most valuable resource, freshwater. In addition, stream quality
metrics can help to identify the underlying causes of water quality degradation and in this way
assist in the preservation of the wildlife within a stream. The IBI scores from each of seven
tributaries of the Scioto watershed (including the Scioto River, Big Darby Creek, Big Walnut
Creek, the Olentangy River, Alum Creek, Mill Creek, and Whetstone Creek) were examined by
dissecting the metrics into their subdivisions (metrics associated with Species Composition,
Trophic Structure, and Fish Health) and comparing these subdivisions to water quality data such
as nutrient loading, metals, streamside deforestation, etc. All data were extracted from Ohio
EPA water quality assessment documents, and so all data were collected by using methods
standardized by Ohio EPA. The streams selected for the current study represented excellent,
good and fair condition streams of the Scioto River watershed. Our data showed a strong
correlation between the Species Composition subdivision and the overall IBI score (r2 = 0.6975).
In addition, poor Fish Condition scores were found to be positively correlated with metals,
organics, and nutrients found in these streams (r2 = 0.8050). Low QHEI scores (Qualitative
Habitat Evaluation Index) related to low Tropic Structure scores. These data suggest the
majority of the problems associated with the Scioto River watershed are related to urbanization,
wastewater treatment plants and industrial waste. Understanding these relationships between
species, habitat and water quality is important for preserving as well as repairing our watersheds.
Lenora M. Evans
Quantitative analysis of cytotoxic T-cell responses to H-2D CT26.WT colon carcinoma cells
The immune system is composed of many interdependent cell types that collectively protect the
body from pathogenic infections and the growth of tumor cells. This particular study focused on
the latter, and set out to quantitatively determine the effects of the immune system in response to
an injection of tumor cells; specifically, lysis of CT26.WT (H2D) cultured colon cancer cells by
CD8-T cells that were isolated from murine immune systems. This was measured using a
colorimetric cytotoxicity assay. The cancer cell line initially was grown and diluted by a factor
of 10. Approximately 10^6 CT26.WT colon cancer cells were injected into the hind quarters of
BALB/c (H2 D) and C57BL6 (H2 B) mice. Spleen cells from both mice were harvested and
introduced to the assay. Although lysis was expected in the C57BL6 sample, the microplate
reader revealed none. It is hypothesized that no effector cells were introduced into the assay, and
therefore no lysis occurred. Further research was completed to optimize the target and effector
cell numbers. A high absorbance should have been seen in the wells containing lysis buffer and
target cells and low absorbance in those wells containing only target cells; however the
absorbance level data from the plate reader reported the numbers to be in the same range. This
suggested that the target cells in the wells without lysis buffer were undergoing spontaneous
lysis. Future research will focus on optimizing culture conditions to prevent spontaneous lysis.
Matthew B. Fosnaugh
Microbial diversity in metal contaminated soils of Kilgore Farm, Delaware Co., OH,
evidence for arsenic tolerance
The Kilgore Farm, located in Delaware Co., OH, was home to ammunition manufacturing
facilities during World War II. The destruction and burial of wastes took place on the property in
a marked ‘burn pit’. Our interest in the site was to determine the microbial diversity of the burn
pit and to screen for bacteria with tolerance to the metal contaminants present in the soil. Soil
samples were taken from three sites within the burn pit and two sites out of the pit with the same
soil type. From each site, samples were taken at a depth 12”, 24” and 40”. Soils were digested
with acid for inductively coupled plasma mass spectrometry analysis (ICP-MS) to determine the
metal composition in the samples. Genomic DNA was isolated from the samples using an
isolation kit from Epicenter®. Bacterial universal primers were used with Escherichia coli DNA
and the correct PCR conditions for amplification of the bacterial DNA from soil were
determined. In addition, bacteria capable of metabolizing heavy metals, such as arsenic were
identified on plates with concentrations of arsenic ranging from 100 ppm to 1000 ppm. Arsenic
tolerance was shown at 500 ppm from soil depths of 24”, and 40” compared to control plates
with no growth on arsenic plates. Studying the transport and chemical pathways of the identified
bacteria will aid in future research of bioremediation of site. By determining arsenic
concentrations at various soil depths, we can determine the types of bacteria that are tolerant to
arsenic, with the ultimate goal of removing hazardous metals from the site.
David A. Kimberly
Relationship between Anuran species richness and abundance and pond water quality
The global decline of amphibians is of widespread concern, and water quality may play an
important role. The goal of this study was to assess the relationship between water quality
variables and anuran communities in Central Ohio pond habitats. During the 2005 breeding
season (April, May, June, July), anurans were surveyed 10 times in 21 ponds in Franklin County,
using the Ohio Frog and Toad Calling Survey protocol. Ponds were classified as either retention,
wooded, and non-wooded. Anuran calls from each site were recorded for three minutes. Species
richness and abundance were determined by listening to the recorded choruses, identifying
anurans to species, and then assigning a score of 0 - 3 depending on calling amount.
Simultaneous with calling surveys, data were collected on several water variables, including pH,
conductivity, dissolved solids, and water and air temperatures. Water samples were collected
from each pond four times to measure nitrates and phosphates. These data provide the first
quantitative data on the relationships between anuran communities and water quality parameters
in Central Ohio ponds. Species richness was not significantly related to nitrate (p = 0.503), or
phosphate (p = 0.873). However, a significant negative relationship was found between species
richness and conductivity (p = 0.001) and dissolved solids (p < 0.001). Species richness was also
negatively correlated with pH (p = 0.001, R2 = 0.69). As pH became more alkaline, species
richness decreased. The majority of previous pH studies focused on the effects of acidification
of anuran biology. My data suggest a negative relationship between anuran communities and
alkaline pH. Future studies should be conducted on the effects of alkaline pH on anuran biology.
Phillip J. La Susa
Contaminants of emerging concern: detection of organic wastewater contaminants
(OWC’s) in Alum Creek Water Reclamation Facility (ACWRF), Westerville Drinking
Water Facility, Alum Creek, Scioto River, and Big Darby Creek
Water treatment has been left unaltered for almost the past century and with society now using
many different types of products, drinking and waste water treatment facilities are failing in
ridding all of the contaminants present in surface and wastewater. It is true that many substances
are being placed into the water that normally is not found in the natural world. Many of these
“emerging” contaminants that are being found in the environment today are not regulated. With
virtually all of these contaminants coming in contact with water as a pathway of distribution into
the natural world, the Environmental Protection Agency (EPA) regulates approximately eighty
contaminants in drinking and surface water. Because many of these contaminants are not
regulated does not mean that they are not potentially harmful to humans as well as to the natural
world. Only recently has society begun to place an interest and awareness of major pollutants in
our environment. Within the past few years, there has been a great interest in the destiny of
pharmaceuticals and hormones that are dispersed into the aquatic environment. Many
hypotheses have been made about the fate of these compounds as they are transported throughout
our water supply but analytical methods were not able to detect the low levels of these
contaminants. Therefore experimentation upon this question of pharmaceutical fate as well as
the ultimate destiny of other compounds that society places into the ecosystem could not be
efficiently tested. By utilizing dichloromethane and solid phase micro-extraction as sampling
preparation techniques in correlation with gas chromatography and mass spectrometry, detection
of OWC’s was efficiently found. Linalol, Squalene, and D-limonene as well as other
pharmaceuticals, endocrine disruptors and industrial pollutants were found to be present in local
water concentrations.
Alana M. Moczydlowski
Determination of the efficacy of decontamination procedures against trace microbial and
nucleic acid contamination
Decontamination of water and food supplies is a largely explored area of concern; however,
there is a lack of information regarding complete microbial and nucleic acid decontamination in
forensic biological safety cabinets. Although common methods exist for microbial and nucleic
acid decontamination, the efficacy of such methods is an area of particular interest to scientists
conducting procedures where even slight contamination by either a viable organism or its genetic
material poses a threat to the validity of experimental results. In addition, when dealing with
organisms that may pose a potential health threat to scientists, complete and thorough
decontamination is desired in order to prevent exposure of workers who come into contact with
these organisms. In this experiment, MS2 phage and Bacillus thuringiensis v. kurstaki (B.t.k.)
were used to test the efficacy of several proposed decontamination procedures by way of PCR
(for nucleic acid decontamination) and standard microbial plating (for viability of the selected
organism). All of the proposed decontamination procedures provided a significant reduction in
contamination, and two of these procedures proved effective in complete decontamination (0
viable organisms, 0 gene copies present through PCR). Treatment with both bleach and a
proprietary detergent, DNA Erase™, in either order followed by an isopropanol rinse and 15
minutes of ultraviolet irradiation proved to be effective in providing complete decontamination
of a surface. These two procedures were thus concluded to be efficient means of complete
decontamination of both RNA and DNA contamination, while also eliminating the viability of
viruses/phages, vegetative bacterial cells, and bacterial spores.
Amber K. Murphy
Associative effects and transit time in juvenile freshwater turtles, Trachemys scripta
During development, freshwater turtles experience an ontogenetic shift from carnivory to
herbivory. As they transition between diets, they consume mixed diets of different plant and
animal ratios, which is known to produce associative effects in adults. Associative effects occur
when diet items interact with each other so total energy gained is more or less than predicted
based on gains from pure diets. In this study, hatchling slider turtles, Trachemys scripta, were
used to determine the presence of associative effects in juveniles and whether their cause is
related to differences in transit time between diet items. It was hypothesized that adding plant
material to an animal diet could increase transit time, and therefore digestibility. Conversely,
adding animal material to a plant diet could decrease transit time and digestibility. Fifty
juveniles were divided into five groups and maintained for six weeks on either: 100%
duckweed, Lemna minor, 100% cricket, Acheta domesticus, or one of three mixed diets
containing 33.8% duckweed and 66.2% cricket, 60.6% duckweed and 39.4% cricket, and 79.1%
duckweed and 20.9% cricket, dry mass basis. Growth rates and transit times were measured.
Growth of turtles on the 20.9% cricket diet reflected a negative associative effect, attributed to
the shorter mixed diet transit time relative to the 100% duckweed diet, allowing less time for
fermentation. No associative effects were found in the other two mixed diets. The absence of
positive associative effects formerly observed in adults may be attributed to the especially high
digestibility of animal matter in juveniles previously documented.
Melissa A. Oxley
The effects of geotextile tubing on hatching and emergence success of marine turtles,
Caretta caretta and Chelonia mydas, on the Archie Carr National Wildlife Refuge,
Melbourne Beach, Florida
Female marine turtles migrate to their natal nesting beach biannually to nest. The Archie Carr
National Wildlife Refuge, Melbourne Beach, Florida is home to the highest density of
loggerhead sea turtle, Caretta Caretta, nesting in the western hemisphere. The beach and is also
home to a growing population of green sea turtles, Chelonia mydas. The 2004 hurricane season
was particularly destructive at Melbourne Beach due to four powerful storms hitting the state,
which washed away a significant amount of beach sand. This study was done to determine the
effects of the Federal Emergency Management Agency’s (FEMA) placement of Geotextile
tubing, on marine turtle emergence and hatching success. Geotextile tubing is a form of beach
armory placed beneath sand to protect housing and other structures from the devastating affects
of beach erosion. We performed daily beach surveys, marking nests on the Geotextile tubing
and in adjacent control areas. After the incubation period (approximately 60-65 days) was
complete, inventories were performed by removing the clutch from the nest and counting the
number of hatched eggs, unhatched eggs, and dead hatchlings found in the nest. These data
allow us to determine hatchling success (eggs hatched divided by total clutch size) and
emergence success (number of hatchlings that leave the nest divided by total clutch size) for each
nest. Geotextile tubing on the Archie Carr National Wildlife Refuge did not have a significant
effect on the hatching and emergence success of C. caretta or C. mydas, although more studies
should be conducted in the upcoming years to verify these results.
Jeanne M. Pearson
Environmental mastitis associated with the bacterial counts in wood chip bedding
Environmental mastitis is defined as an intramammary infection with organisms whose natural
reservoir is the external cow environment. Control of environmental mastitis is of the utmost
importance to dairy farms, and research is being done to determine the various factors which
contribute to it. Throughout 2005, gram negative and gram positive bacterial counts were
estimated for fresh and used sawdust bedding at a university dairy. Used bedding samples from
the free-stall barn and the dry-cow barn were obtained monthly. Average temperature, average
maximum temperature and average relative humidity were recorded and compared to bacterial
counts. Additionally, bacterial counts were compared to the risk of new cases of clinical mastitis.
No relationship was found between weather variables and unused bedding bacterial counts.
Gram-negative bacteria from used bedding in free-stall and dry-cow barns were correlated to
average and maximum temperature, but not to average relative humidity. No relationship was
found between weather variables and gram-positive bacteria from these areas. Gram-negative
bacteria counts were correlated with mastitis cases more so than gram-positive bacterial counts.
These findings were of great importance because they allow quantization and estimation of an
important disease outcome as a function of environmental bacterial loads, and offer insight into
factors limiting control of environmental mastitis.
Adam R. Rothermel
Mus musculus antibody response to CT26.WT colonic cancer tumerogenisis
Over the past few years, clinical oncology and cancer research have established that the
promotion of an infected subject’s immune response to a malignancy can serve to slow tumor
growth and metastasis, subsequently decreasing mortality. Furthermore, it has been shown
clinically that increasing the subject’s tumor-antigen specific antibody serum concentration via
Intravenous Immunoglobulin (IvIg) can have the same beneficial effects. However, there was a
recent publication showing Balb/c mice relieved of their B cell or antibody production
capabilities during development having much lower rates of mortality and tumorogenisis upon
inoculation with cancer cells than the wild type mouse. A subsequent publication reveled that
infected B cell absent mice showed increased mortality and tumerogenisis after intravenous
injection of tumor-antigen specific antibodies. This research project was developed to further
explore the specificity of the Mus musculus immune response to CT26.WT colonic carcinoma
and ultimately establish whether in vivo tumor-antigen specific antibody inhibition could serve to
retard tumor development. The immediate goal of this study was o identify the spectrum of
antibodies responding to the tumor. The study used Balb/C mice inoculated with CT26.WT
cancer cells. After 2 weeks of subcutaneous tumor development the mice were euthanized and
spleens were extracted. Total RNA was isolated from the spleen tissue for immunoglobulin
transcriptome analysis. Immunoglobulin heavy chain constant region specific primers were used
in combination with reverse transcription and the polymerase chain reaction (RT-PCR) to
successfully detect the presence of an antibody mediated immune response. Next,
immunoglobulin heavy chain variable region degenerate primers were used to amplify all
immunoglobulin variable region transcripts. The data support the hypothesis that the antibody
response to the tumor includes both IgM and IgG antibody isotypes and that it is polyclonal. The
current focus of the research is precise quantification of the antibody response through the
utilization of denaturing gradient gel electrophoresis (DGGE) to determine clonality and DNA
sequencing to determine the specific sequence of the anti-tumor antibodies.
Sarah R. Scalley
Adhesion of Pseudomonas aeruginosa and Staphylococcus aureus on the contact lenses and
lens cases of college-aged female students under different conditions of hygiene
The adhesion of bacteria, such as Pseudomonas aeruginosa and Staphylococcus aureus, to the
eye causes a chain of events to occur which may eventually lead to the loss of vision. These
events include bacterial contamination and the bacterial adhesion of those naturally present in the
eye to the contact lenses, invasion of the bacteria into the corneal stroma, which then releases
bacterial toxins that trigger the host's response to infection, which may result in vision loss. The
purpose of the project was to determine whether it is possible to reduce the number of adhesive
bacteria on contact lenses by changing hygiene measures alone. Volunteer subjects completed a
questionnaire describing their own personal eye and contact lens hygiene. Twenty-four female
subjects between the ages of eighteen and twenty-three were selected according to survey scores:
seventeen had poor contact lens hygiene and seven had good contact lens hygiene. Bacterial
swabs were taken from the contact lenses on the side that comes into contact with the cornea.
The bacteria were then grown on Tryptic Soy Agar plates and identified as Pseudomonas
aeruginosa and Staphylococus aureus by Enterotube II results. Ten subjects from the poor
hygiene group were asked to alter their hygiene to exhibit proper contact lens hygiene. The other
fourteen participants kept their same hygiene habits. Samples were collected and bacterial
colonies were counted every five days for twenty days total. Statistical significance was then
determined for the Pseudomonas aeruginosa and Staphylococcus aureus adhesion to contact
lenses and lens cases in the Otterbein community to show that the number of adhesive bacterial
colonies can be reduced with proper hygiene alone. The results of the experiment include
evidence that proper hygiene may reduce the number of adhesive bacteria on contact lenses.
Erin M. E. Scott
Creation of mutations in Helix IX of lactose permease in Escherichia coli, steps towards
characterization and implications for structure and function
The lactose permease symport protein in Escherichia coli is a model system for the study of
transport in bacteria. Based on the crystal structure deduced in 2003 it has been shown that eight
helices line the inner channel with four anchoring helices III, VI, IX and XII in contact with the
membrane. It is hypothesized that these hydrophobic outer helices function as stabilizers for the
inner helices and play an important role in the structural integrity of the protein. Site directed
mutagenesis was completed on two amino acids, S307K and A309E, in helix IX. The PCR
product was ligated with T-vector and successfully transformed with rubidium chloride. Once
the plasmid was isolated, a restriction digest was completed with the restriction enzyme SnaB1.
The desired 6.7 kilo base pair was cut from the gel to be ligated with wild type. The
complementary wild type fragment was not successfully isolated, and the cloning of these
mutations is ongoing. Previous work completed to analogous helices in lactose permease
suggests that the mutations made in helix IX will disrupt substrate transport into the cell
supporting the hypothesis that these helices are important in maintaining the structure of the
protein.
Colleen M. Shaver
Role of mitogen activated protein kinase/nuclear factor – κB signaling in increasing
Acrolein-Induced Matrix Metalloproteinase (MMP) 9 mRNA
Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United
States and is largely caused by cigarette smoking. Acrolein, a component of cigarette smoke,
targets the respiratory system leading to emphysema and excess mucus production - hallmarks of
COPD - but the mechanisms are poorly understood. Recently our laboratory found that acrolein
increases the release and activation of metalloproteinases (MMPs) in NCI-H292 cells, a
pulmonary carcinoma cell line, leading to the activation of epidermal growth factor receptor
(EGFR) in a ligand-dependent fashion. Acrolein also increases the transcription of MMP9 in
NCI-H292 cells. The purpose of my experiment was to examine the signaling pathways that
influence MMP9 after acrolein treatment. NCI-H292 human mucoepidermoid carcinoma cell
lines were used. Acrolein increased MMP9 mRNA in a concentration dependent manner (0.010.1 μM) followed by a decrease in MMP9 mRNA at higher concentrations (0.3-10 μM). Pretreating the cells with an EGFR inhibitor, AG1478, decreased the acrolein-induced or EGFinduced increase in MMP9 mRNA suggesting EGFR activation is required for MMP9
transcription. Activation of EGFR leads to signaling through downstream mitogen activated
protein kinase (MAPK). Pre-treating cells with a MEK inhibitor, PD98059, decreased the
acrolein-induced or EGF-induced increase in MMP9 mRNA. Pre-treating the cells with c-jun Nterminal kinase (JNK) inhibitor, SP600125 (1 µM) or p38 MAPK inhibitor, 2-(4-Chlorophenyl)4-(4-fluorophenyl)-5-pyridin-4-yl-1,2-dihydropyrazol-3-one (1 µM), had no effect on acroleininduced MMP9 mRNA increase. Pre-treating the NCI-H292 cells with phosphatidyl inositol 3
(PI-3) kinase inhibitor, LY 294002 or nuclear factor κ (NF-κB) inhibitor, evodiamine (0.5 µM)
decreased partially the acrolein-induced MMP9 mRNA increase. The data suggests that acrolein
increases MMP9 mRNA by activating EGFR and ERK 1/2, PI-3 kinase, and NF-κB with the
potential of cross-talk between the various signal transduction pathways.
Jillian A. Shellabarger
Engineering of a DNA construct for the study of Perilipin C function in steroidogenic cells
Perilipin C is predominately found on the surface of lipid storage droplets in steroidogenic cells.
Cholesteryl esters are stored within intracellular lipid droplets in steroidogenic cells, and
hydrolysis of these esters releases free cholesterol to be used in membrane and steroid hormone
synthesis. The sequence similarities between the perilipin A and C isoforms, suggest that
perilipin C (like perilipin A) serves a similar function as a protective coating of the lipid droplet
until phosphorylated by protein kinase A (PKA). Therefore, it may be possible to target perilipin
C for the regulation of hormone-related diseases. In this study, the roles of perilipin A and
adipose differentiation-related protein (ADRP) in cholesterol ester lipolysis were examined
through measuring their respective cholesterol and protein contents when treated with oleic acid
and cyclic adenosine monophosphate (cAMP) elevating agents. Previous studies have shown
expression of perilipin A increased triacylglycerol (TAG) storage in Chinese hamster ovary
(CHO) cells. Expression of Perilipin A also increased the storage of cholesterol compared to
control cells expressing ADRP. Treatment of CHO cells with oleic acid to increase TAG storage
also increased cholesterol concentrations, but only in cells expressing ADRP. Finally, treatment
of cells with lipolytic stimuli (Forskolin and IBMX) caused decreases in stored cholesterol in
perilipin expressing cells but not in cells expressing ADRP. We have also genetically
engineered a plasmid to code for the perilipin C isoform. In the future, this coding region will be
cloned into the 3XFLAG expression vector, and the effect of ectopic expression of perilipin C
in CHO cells on cholesterol storage may be examined using these same fluorescent cholesterol
assays. Overall, this data will lend insight into the function of perilipin A, ADRP, and perilipin
C, and could ultimately have significant impact on revealing the mechanisms involved in the
regulation of steroid hormone synthesis.
Jenna L. Stump
The effects of bone marrow derived human mesenchymal stem cells on ovarian carcinoma
growth rate and progression
The understanding of how fibroblasts interact directly with metastatic cancer cells is limited due
to the complexity of the in vivo tumor microenvironment. The in vivo environment contains
various cell types which play an influential role in the growth and survival of tumor cells,
making it difficult to dissect out the role of fibroblasts within the tumor microenvironment. To
gain a better understanding of how stromal fibroblasts directly interact with ovarian tumor cells
an in vitro, three-dimensional tumor growth assay (TGA) was used to assess the direct impact of
bone marrow derived fibroblasts on ovarian carcinoma cells. Fluorescent ovarian carcinoma
cells were grown in 3D tumor matrix alone or in the presence of fibroblasts. Bone marrow
fibroblasts were shown to enhance the growth rate of ovarian cancer cells in vitro using the 3D
TGA assay. To examine the in vivo impact of bone marrow fibroblasts, human ovarian tumor
cells were injected intra-peritoneal into athymic mice alone or in the presence of human bone
marrow fibroblasts. Tumors were dissected after forty days and analysis showed that human
bone marrow fibroblasts were incorporated into the growing ovarian tumors. Data from the
above mentioned studies suggests that fibroblasts integrate into developing ovarian tumors in
vivo and that tumor growth is enhanced by the presence of bone marrow derived fibroblasts.
Koji Takeo
Characterization and quantification of intracellular lipid droplets by Raman microscopy
Raman microspectroscopy is potential effective technique identification, and quantifying the
contents of intracellular lipid, and cholesterols. Using purified lipids sample, and cholesterol:
tripalmitin, cholesteryl palmitate, and cholesteryl oleate, we are able to characterize for each
samples based on the unique peaks corresponding to different functional groups. Furthermore,
based on these unique peaks and their intensity we attempt to quantify the ratio for mixed
samples: cholesteryl palmitate and tripalmitin.
Based on the observation, the change in the intensity of peaks was directly corresponding to the
ratio of the mixed samples. Therefore, we hypothesize that Raman spectroscopy can be used to
quantify the intracellular lipids, or cholesterol contents within the cells.
Benjamin G. Van Allen
Directional quantification of macroinvertebrates in Big Walnut Creek, Ohio, USA
The goal of this study was to determine if macroinvertebrates colonize substrate only by drift
(flowing downstream in the current) or by other means. Knowledge concerning invertebrate
dispersion in streams is needed to understand their ecology and evolution, and could help
develop more accurate water quality testing methods. Macroinvertebrates are currently used to
determine compliance with water quality standards. During this study, Hester-Dendy plates
were used as bare substrate for macroinvertebrate colonization. Unmodified control plates were
placed vertically in the stream. Experimental plates were modified by attaching a shield that
blocked the plate from one direction. Each plate was positioned vertically by attaching it to a
concrete block, which maintained its position and orientation in the flowing water. Two plates
for each treatment (control, front-blocked, rear-blocked, and side-blocked) were attached to each
cinder block. Two sets of cinder blocks were placed in Big Walnut Creek three times from 15
April to 15 October 2005. Each set of plates was in the water for a period of six weeks. Results
suggest trichopterans and beatids colonized front blocked plates significantly less than control
plates (trichopterans, t=1.88 p<0.05, beatids, t=1.37 p<0.10). Stenonema did not significantly
favor any plate and chironomids favored rear blocked plates (t=-1.05 p<0.10). This trend
suggests that drift may play some role in the dispersal of trichopterans, beatids and chironomids
and that proper placement of Hester-Dendy plates requires consistency of orientation of the
plates. It also suggest that macroinvertebrates are affected by drift differently between species
and that drift is not the only important factor in macroinvertebrate dispersal.
Kacy D. Walton
Gingivitis and Integrin-Beta-2: a single nucleotide polymorphism association study
Gingivitis is described as a form of periodontal disease characterized by inflammation of the
gums along with minor bleeding. There are two contributing causes of gingivitis: poor hygiene
and genetic predisposition. Integrins are integral cell-surface proteins composed of an alpha
chain and a beta chain that are known to participate in cell adhesion as well as mediate cellsurface signaling. Integrins are involved in leukocyte function during immune and inflammatory
responses. The integin-beta-2 (ITGB2) gene produces a protein that is found in this category.
Recent research shows that genetic variation in the integrins may be a factor contributing to
gingivitis. The goal of this study is to determine whether there is an association between single
nucleotide polymorphisms in the ITGB2 gene and gingivitis. The recently published “HapMap”
of single nucleotide polymorphisms (SNPs), including those in the integrin beta-2 gene, affords
the opportunity for a case-control study designed to test the relationship between integrin beta-2
SNPs and gingivitis. A gingivitis index (GI) indicates the level of gingivitis that is present in the
mouth. The criteria include an evaluation of the gums by probing for a level of inflammation
and bleeding. The GI index used in this study is the Loe and Sillness model. A study of
Otterbein College students was recruited. DNA samples were obtained and GI indices were
determined. The polymerase chain reaction was used to amplify the region of DNA containing
ITGB2 SNP rs235326 in all 20 subjects. SNP genotyping of rs235326 was carried out
successfully and the SNP was detected by capillary electrophoresis. Currently SNP genotyping
of 20 subjects is now being carried out to search for associations between the rs235326 and
gingivitis.
Justin G. A. Whitehill
Transmission of the fungal pathogen, Sphaeropsis sapinea, to Austrian pines, Pinus nigra,
by the pine engraver beetle, Ips pini
Sphaeropsis sapinea is among the most common and widely distributed pathogens of conifers
worldwide and one of the most important limiting factors in the growth of Austrian pine (Pinus
nigra) in the Midwestern United States. Short-range dissemination of S. sapinea is attributed to
rain splash and moist wind, while information regarding the long range dispersal of the pathogen
is relatively unknown. The objective of this study was to determine whether or not the pine
engraver beetle, Ips pini, might be a vector of the pathogen onto Austrian pines. In summer 2004
and 2005, individuals of I. pini were collected with pheromone traps at two locations in central
Ohio (197 and 1017 individuals for 2004 and 2005, respectively). Individuals were screened for
the presence of S. sapinea, and phoresy rates were calculated based on the percentage of
recovery of S. sapinea. In the field, fresh logs of Austrian pine were baited with pheromone
lures, mechanically wounded, or left undisturbed. After 2 months, logs were evaluated for insect
feeding and the presence of S. sapinea. Fresh logs were also inoculated in the greenhouse with
adult I. pini that were either infested or un-infested with S. sapinea spores to determine vectoring
potential. Phoresy rates for individual collections ranged from 0 to 4.1%; average rates were 1.5
and 2.0% for 2004 and 2005, respectively. The isolation frequency of S. sapinea from baited (15
+ 5%) and un-baited logs (3 + 1%) differed significantly (P = 0.009). For both years, individuals
of I. pini infested with S. sapinea induced significantly larger lesions than the negative controls.
In 2005, individuals of I. pini carried S. sapinea at a greater frequency than either the positive or
negative control (32 + 5, 3 + 3, or 0.0 + 0%, respectively). Based on phoresy rates, association,
and artificial inoculation studies, we conclude that I. pini is able to transmit S. sapinea to
Austrian pine stems
Teresa R. Young
Comparison of Homo sapiens and Pan troglodyte DPA/DPB control region of the major
Histocompatibility Complex
The publication of the draft chimpanzee genome makes it possible to identify and understand the
genetic factors that contribute to the many differences between Homo sapiens and our closest
living evolutionary relatives Pan troglodytes and Pan paniscus. Some differences such as
language and bipedalism are more obvious, while others such as susceptibility to disease are
more subtle. The goal of this research is to compare the human and chimpanzee DPA/DPB
control regions in the major histocompatibility complex (MHC). The DPA and DPB genes play
an important role in susceptibility and resistance to auto-immune and infectious diseases. Our
specific objectives are: 1) to identify intra and inter genome differences in the different primates
and 2) to use phylogenetic shadowing, the alignment of several closely related genomes to
identify areas of conservation, to identify the sequences responsible for regulation of the MHC
DPA and DPB genes. The three methods that have been used are the polymerase chain reaction,
DNA sequencing, and phylogenetic shadowing. Half of the DPA/DPB control region was
sequenced for Homo sapien, Pan troglodytes, Gorilla Gorilla, and Aotus azarni. Out of 950
nucleotides, 63 sequence variations were found between the four species. With the use of
phylogenetic shadowing, two areas were determined to be one hundred percent conserved while
many other regions had extremely high levels of conservation. These results lay the foundation
for future studies of the functional significance of the variations and conserved sequences.