... 1 Annual Spring Science Presentations Science 299 Independent Research Projects: Spring 2015 Students Kamran Bastam Taylor Dillon Burgundy Draper Peggy Freeman Fletcher Gore Stephen Hazen Ismael Leon Melanie Lindemann Ashley London Will Markle Angela Nebel Levi Perry Kira Runkle Morgan Russell Jacob Simons Candice Tomlinson Dorcas Yoder Faculty Advisors Anne Allison Yanina Goddard Barbara Heyl Frances Rees Larry Tiezzi Joanna Vondrasek John Walsh Marlena Yost Chemistry Biology Biology Biology Physics Biology Biology Biology Chemistry Biology Geology Biology Chemistry Biology Biology Biology Biology .page 2 .page 2 .page 3 .page 3 .page 4 .page 5 .page 6 .page 6 .page 7 .page 8 .page 8 .page 9 .page 10 ....page 11 .page 11 .page 12 .page 13 Laboratory Managers Ed Funck Rosalyn Koontz Jennifer Scott 2 Kamran Bastam Preparation of phenyl penicillin potassium salt and benzyl penicillin potassium salt In this experiment two types of penicillin were prepared which is a class of -Lactam antibiotics to treat some Gram-negative and Gram-positive bacteria. 6-aminopenicillanic acid (6-APA) was acylated with Benzoyl chloride and Phenylacetyl chloride through Friedel-Crafts acylation reaction to prepare Phenyl penicillin potassium salt and Benzyl penicillin potassium salt (penicillin G), respectively. In this experiment, phenyl penicillin was obtained from Benzoyl chloride that illustrated 29.3% yield whereas percentage yield of Benzyl penicillin was 7.5% yield from Phenylacetyl chloride. Those antibiotics were assayed using Infrared spectrometer (IR) and melting temperature apparatus to compare with their literature measurements. But during the measurement of melting point those Phenyl penicillin and benzyl penicillin were decomposed at 210C and 210.7C, respectively, whereas melting temperature range of benzyl penicillin is 214-217C (since Phenyl penicillin is not common compound, it does not have literature melting temperature range). The results show that the decomposition points are close to the melting temperature range and the comparison of the experimental IR spectrums with literature IR spectrums show that the experiment was successful. Faculty Advisor: Barbara Heyl Taylor Dillon The Effect of Lake Age on Zooplankton Zooplankton are small, heterotrophic animal like organisms that are typically found in most bodies of water. There are many different species that reside in our local lakes and ponds. Species diversity measures how many different kind of species there are and how even each species is. This experiment compares the differences of zooplankton in a fairly new pond and an older lake. The hypothesis is that there will be a higher diversity of plankton in the old lake, Lake Monticello, than there will be in the newer pond at PVCC. A special net called a plankton net was used at each site to collect the samples. The plankton net was moved through the water for two minutes during every sample, and the contents were collected in the cup at the bottom of the net. The samples were then taken to the lab to be looked at under a compound light microscope. About 7-10 slides were looked at for each sample. The plankton that was seen on the slides were counted and identified. It turned out that there was a higher number and diversity of zooplankton in the newer PVCC pond in four out of the five samples. A diversity index was done for each sample most of the numbers showed that the PVCC pond had a much higher diversity than Lake Monticello. Most people would think that there would be a higher species diversity in the older lake because species usually become richer as a lake gets older, but my findings show the opposite from this. This could mean that the biodiversity in Lake 3 Monticello has leveled out, and it is not growing as much because it has been around for longer. Faculty Advisor: John Walsh Burgundy Draper Does cattle access to farm water sources affect water quality measures: a study of farms in the Keswick, Virginia area Many farms have creeks, rivers, and streams running through them. In the agricultural areas surrounding Keswick, VA , these water ways are a part of the Chesapeake Bay water shed, which currently has a report card letter of a C. The government has proposed that farms build off-stream water sources to help reduce the pollutants entering the Bay. One source of contamination from farm run-off is cattle feces, by either having the feces washed into the waterways by rain or having the cattle bathe in the waterways. Cattle feces can have a harmful effect on the ecology of the water, by. (increasing bacterial contamination, increasing nitrate load). The reason for this study was to determine whether cattle access has a negative effect on the the water chemistry of water sources near local farms. In this study, nitrate, phosphate, and dissolved oxygen tests were run to see the effects of cow feces on these water quality measures. On two different days, at the same locations from each site, water samples were collected from six different farms, three where cows had access to the waterways and three where the cows had no access. The water samples were then taken to the lab where they were tested for dissolved oxygen, phosphate, and nitrate levels. The data collected show no significant difference between the water quality measures of farms where there was cattle access and where there was no cattle access (nitrate, DO, phosphate p values for some of the tests). Further studies might examine whether microbial loads differ according to cattle access (or something else looking forward) . Faculty Advisor: Joanna Vondrasek Peggy Freeman Weight Evolution in Bean Beetles (Callosobruchus maculatus) Agricultural pests have been able to adapt and evolve resistance to current control methods such as pesticides at a rapid rate. With short generation times and massive populations, a pest can evolve more quickly than science and technology can devise new solutions forcing farmers to use dangerous chemicals at high levels to protect crops. In order to help find better solutions than the dangerous chemicals, biologists have begun to uncover how 4 the bean beetle, Callosobruchus maculatus, a common parasite of dry beans in warm climates, has evolved by examining the genetics of key traits: development time, longevity, and fecundity. One of the main indicators for these traits has been body size or mass. There has been no direct genetic correlation between weight and the three traits. This was a benefit of either maternal or environment effects that positively influence genetic inheritance. Larger beetles benefit from longer longevity and better fecundity at the cost of a prolonged development time, while smaller beetles develop faster but have reduced longevity and fecundity. The current study is a continuation of a previous PVCC student research project regarding mass selection in the bean beetle. The same beetle population was maintained from the first experiment in a large colony where the beetles were allowed to freely mate over Generation 3 between fall semester to spring semester. Out of this colony, 144 mung beans (Vigna radiata) with eggs, Generation 4, were randomly selected and isolated in an incubator maintained at 27C and allowed to develop and hatch. At the end of the four weeks, 10 breeding pairs were chosen based on mass with five heavy pairs and five light pairs placed on 60 mung beans each. After roughly four weeks, the beans were isolated and allowed to hatch. While 87 of 144 (60%) of Generation 4 hatched successfully, there were no fertilized eggs in Generation 5 of the light breeding pairs, and only two successful hatchings in Generation 5 from the heavy breeding pairs. The average mass of males in Generation 2 was 3.505mg, and the average mass of Generation 2 females was 4.379mg. The average mass of males in Generation 4 was 3.9mg, and the average mass of Generation 4 females was 3.512mg. When analyzed with a t-test of Generation 2 against Generation 4, the change in values between males was shown to be not statistically significant (P-value = 0.9659). However, the change in value between females was shown to be statistically significant (P-value = 0.0050). The males maintained their mass in despite the unknown effect of random mating in Generation 3 hinting at a benefit of maintaining mass. The females did not maintain or grow in mass and began to decrease in mass suggesting the females may be attempting to balance the three genetic traits. Faculty Advisor: Joanna Vondrasek Fletcher Gore Bicycle Powered Water Pump for a Middle School Garden One of the most important, and sometimes the most challenging, requirements in agriculture is to supply ones crops with a sufficient quantity of water. Especially when attempting to adhere to the ideals of sustainable agriculture, this can be very difficult-- seeing as the standard supply of water has been processed and pumped to the garden using a relatively large amount of energy. However, a method that has grown in popularity in recent years uses the rainwater collected by a roof to fill an elevated cistern. Unfortunately, the pressure created in the watering system by the elevation difference between the water tank and the ground is generally very low, a problem that can be remedied only by using an additional source of energy to pressurize it. However, the energy needed to do this can be provided by a myriad of 5 different sustainable sources, including your feet. This is precisely the problem that was encountered by Buford middle school in Charlottesville Va. And, to alleviate it, a device was designed and built that uses the energy provided by children pedaling stationary bicycles to do the work necessary to pressurize and move the collected rainwater. Staying true to the ideal of sustainability, and to reduce the cost of the machine, used, abandoned, repurposed or remanufactured parts were used wherever possible. In the end, the device was capable of producing 14 P.S.I, (the equivalent of a 33 foot head) and a flow rate of 2.8 gpm. These measurements were taken using only one adult to power the machine, while it was designed to be used by two middle school children. Because of this, these measurements were merely a baseline; the actual maximum pressure and flow rates are predicted to be higher. The result was an increased pressure and flow rate through the gardens watering system, using only the energy provided by the middle school's students themselves, while at the same time providing them with a clear, enjoyable means to help in the production of food in their schools garden. And, due to the simplicity of the machines design, the teachers were provided with an opportunity to clearly demonstrate the relatively simple physical rules of force, work, torque and pressure, in addition to more complex subjects such as potential and kinetic energies, fluid dynamics and Bernoullis principle. Faculty Advisor: Yanina Goddard Stephen Hazen Aggregative Multicellularity in Capsaspora owczarzaki Choanoflagellates are believed to share a common ancestor with metazoans and are used to study the origins of multicellularity in animals. The choanoflagellate Salpingoeca rosetta demonstrates distinct singular cell types and a colonial aggregation of morphologically similar cells. Capsaspora owczarzaki belongs to the clade Filasterea, a close sister group to both choanoflagellates and metazoans, and like S. rosetta, demonstrates distinct singular cell types and colonial aggregations. I hypothesize that, as has been previously shown for S. rosetta, exposing C. owczarzaki to fluorescent lectin wheat germ agglutinin (WGA) will stain some fast, free-swimming individuals as well as colonial aggregations. Staining with lectins such as WGA is a useful tool for studying multicellularity because lectins facilitate cell-to-cell interactions. To pilot this assay and optimize conditions, a different but readily accessible protist, Vorticella, was stained with fluorescent WGA. Vorticella was observed in both a free swimming and adherent stage (no aggregations were observed). As previously demonstrated by others, the stalks of adherent individuals as well as the cell bodies of free-swimming ones stained with fluorescent WGA. C. owczarzaki was then stained to determine if colonial aggregates would be visible. Live free-swimming C. owczarzaki were observed under the compound light microscope (no colonial aggregations were observed). None of the free-swimming C. owczarzaki cells stained with the fluorescent WGA. In conclusion, free-swimming C. owczarzaki do not appear to behave in the same way as free-swimming S. rosetta. While additional experiments should be done to 6 observe aggregative C. owczarzaki colonies, these initial findings suggest that C. owczarzaki may constitute an important outgroup for future studies of multicellularity using choanoflagellates. Faculty Advisor: Anne Allison Ismael Leon The inhibition of growth from four Mouthwash Rinses on Streptoccocus mutans, Staphylococcus aureus, and Klebsiella pneumoniae It is important to maintain good oral hygiene to ensure adequate oral health. Dental decay, or caries, is caused from a lack of good oral hygiene, harmful habits, and an unhealthy diet. Various products for oral hygiene can be found in a local grocery store. This experiment focused on three different mouthwash rinses and their effect on bacterial growth of three different bacteria normally found in the human flora of the oral cavity. The leading cause of caries is the bacterium Streptococcus mutans, but this experiment also included Staphylococcus aureus and Klebsiella pneumonia. Oral hygiene products varying in chemical composition have been used in previous studies. This experiment used clinically proven, all natural, adolescent, and inexpensive mouth rinses with varying chemical components. The hypothesis was that Crest would be the most effective, while Equate would be the least effective. Knowing which product is most effective in inhibiting growth of these bacteria is knowledge the public can use to maintain better oral hygiene. Bacteria were grown on and tested against the various products on Nutrient agar and Brain-Heart Infusion agar plates. Paper discs dipped into the mouth rinses were then placed in the center and the plates were grown for approximately 24 hours in a temperature that was about 37 degrees Celsius. It was found that Crest was the most effective in inhibiting the growth of all bacteria tested. Second most effective was Equate, followed by the similarly formulated ACT for Kids. The least effective mouth rinse was Toms of Maine, as in many cases the product had no inhibition of growth. The hypothesis that Crest would be the most effective was accepted, while the rest of the hypothesis was refuted due to Equate not being the least effective in inhibiting bacterial growth. Faculty Advisor: Marlena Yost Melanie Lindemann Toxicity of Cigarette Residues on Daphnia Magna Globally, cigarette filters are the most abundant form of litter. Every year 1.1 billion smokers worldwide contribute over three billion pounds of toxic waste, more than two thirds of which is littered, polluting our coastlines, waterways, and land. Numerous studies have 7 affirmed that a lethal mix of over 7,ooo chemicals leach into the environment from spent cigarette filters and pose a threat to wildlife. In this study, Daphnia magna were used as biological indicators, per US Environmental Protection Agency regulations, for acute toxicity tests of both Marlboro Red cigarettes and Organic American Spirit cigarettes to evaluate the relative toxicity of spent cigarette filter leachate. Artificially smoked test cigarette filters were submerged in aged tap water for 24 hours and daphnia were exposed to the leachate in concentrations of 2, 1, .5, .25, .125, and 0 cigarette filters/l. The tests demonstrated high rates of mortality for the non-organic filters, with a lethal concentration of .25 filters/l. However, organic filter leachate displayed a mere 40% mortality rate in concentrations of 2 filters/l compared that of the Marlboro filters at 100%. These results suggest that filter leachate from organic cigarettes is considerably less toxic than leachate from cigarettes with additives such as ammonium diphosphate and ammonium hydroxide. Further experimentation and comparison could be done using higher concentrated leachates to identify a lethal concentration of organic filter leachate. Isolating and testing cigarette additives and chemical components in spent cigarette filters would also be beneficial to identify specific toxins. My results along with further research and experimentation could be used to educate consumers about the harmful effects of littering cigarette filters, urge manufacturers to contribute to litter prevention campaigns, and prompt more testing of cigarette additives. Faculty Advisor: Jennifer C. Scott Ashley London Nutritional Difference of Store Bought and Home Raised Eggs Chicken eggs have been a long time source of protein for humans. Eggs are not only a good source of protein but also provide a few other vitamins and minerals. In modern times, people do not typically raise chickens anymore; instead, people predominately buy eggs in grocery stores. There may be a few differences in nutritional value of store bought eggs compared to home raised eggs. Home raised eggs are hypothesized to have more nutritional value than store bought eggs because they are fresher, not having to be stored and shipped for long periods of time, and the chickens that lay the eggs are more likely to be free range and fed a better feed. In this experiment, chicken eggs raised at home are compared to store bought chicken eggs by testing iron and cholesterol in the egg yolk and calcium in the egg shells. Calcium in the egg shells can transfer into the white and yolk of the egg, giving home raised eggs their first advantage over store bought eggs. The home raised eggs had about eighty-four percent calcium in the egg shells compared to seventy-three percent in the store bought eggs. In addition, iron is important for humans to transfer oxygen throughout the body. Home raised eggs were shown to have higher levels of iron than store bought eggs. Recent studies have shown that cholesterol has many benefits in human diet despite cholesterols reputation of 8 being bad. Taking this into account, home raised eggs, having more cholesterol, can be taken as good or bad. Faculty Advisor: Barbara Heyl Will Markle Effective Management of Lactose Intolerance Lactose Intolerance is the bodys inability to digest lactose, a sugar found in dairy products, and affects about 65 percent of the worldwide population. The condition is caused by the deficiency of lactase, an enzyme produced in the small intestine that breaks down lactose, and causes symptoms including bloating and diarrhea when the undigested lactose reaches the colon and interacts with normal colonic bacteria such as Escherichia coli. While there is currently no treatment, there are various ways to manage the condition. In this experiment three different remedies were tested for their effectiveness of managing lactose intolerance; Lactaid, a lactose-free milk, lactase pills, and Cinchona officinalis pellets, a homeopathic remedy. 2% milk and Lactaid were used as mediums and several samples were inoculated with lactose positive and lactose negative E. coli, E. coli with and without the ability to ferment lactose, with several samples having crushed lactase pills and C. officinalis pellets added respectively in addition to lone samples of 2% milk and Lactaid. pH levels as well as glucose levels, a sugar that results from lactose being broken down, of each sample were tested before and after a 48 hour incubation period. The results indicated that samples including bacteria and lactase pills had consecutive drops in pH after the 48 hours across all tests while samples containing bacteria and C. officinalis pellets had consecutive drops in pH after the 48 hours across duplicated conditions. Faculty Advisor: Marlena Yost Angela Nebel The Anatomy of a Rock Slide The Sherando Lake rock slide is more than just a rock slide. In analyzing the rock slide and its causes a number of discoveries were made. It contains Skolithos liniaris (worm tubes) which are about 500 million years old. This makes these fossils the oldest found in Virginia. It may also contain evidence of Sea Pens, crinoids that occur in the early Cambrian. Other fossil traces like mud-cracks and bioturbation activity can also be seen. 9 From the information collected and through the work of others, it is possible to put together the history of the rock at Sherando Lake. The quartzites began life as a beach sandstone and were deposited in a barrier island complex - a near-shore to off-shore shallow marine environment with pure quartz sand, similar to what we find today in the Outer Banks in North Carolina. Based on the large number of worm tubes and other signs it was teeming with life. It then was buried, metamorphosed and thrust about 100 miles to where it exists today. Quartzites are very resistant to erosion. However, in the more geologically recent time the colder climate has resulted in ice wedging along fractures in the rock. Anatomy of the land slide indicates that the lithic composition of the slide is homogeneous, but in some places were found significant changes in the rocks appearance. The quartz in the sandstone is well cemented with silica, and in other places of the slide is fine grain laminated and layered. These changes are noticeable in the higher point of the slide where the quartzite sandstone is well structured, while in the places near the vegetation border appeared rocks with more layers. Considering the lack vegetation in part of the slide, it is suggested that it is still moving. The relative homogenous size of rocks indicates a uniform fracture pattern. In summary,the Sherando Lake landslide is moving today and is the result of ice wedging. It contains the oldest fossils in Virginia. To fully unravel all of its mysteries will require additional future work. Faculty Advisor: Larry Tiezzi Levi Perry Mutualism Between Plants and Soil Microbes: Effects on Plant Reproductive Success The mutualistic relationship between plants and soil microbes is well documented and is an ancient part of the evolutionary tale. Plant roots provide fungus and bacteria with a hospitable habitat and often nutrients from photosynthesis, while the microbes expand the surface area of the roots, and transfer nutrients that would not normally be available to the plant. My experiment was to examine the relationship between the presence of soil microbes, specifically nitrogen-fixing bacteria and mycorrhizae fungus, and the reproductive success of the plants. My hypothesis was that the group exposed to both bacteria and fungus would have the highest measures of reproductive success. Four test groups were used: Group A was the control with no microbes added, Group B was supplemented with bacteria, Group C was supplemented with mycorrhizae fungi, and Group D contained both bacteria and fungi. For each group, three Wisconsin Fast Plants seeds (Brassica rapa) were planted in a Styrofoam planter with eight cells. After germination, plants were randomly thinned to a single seedling per cell. The potting soil was autoclaved prior to planting to ensure a lack of microbial activity. At two points after seed germination, the microbes were inoculated into the soil following the 10 manufacturers instructions. The seeds from the first generation of plants were collected, and sixteen of each group (except for the control which produced zero seeds) were replanted into sterile soil. Due to the lack of seeds in Group A, the difference between the mean seeds per plant was statistically significant (p=0.0010), but when Group A was removed from the comparison the difference between the groups was not significant (p=0.28). Group D produced seeds that were visibly smaller than the other groups, and the mean seed weight was drastically different (p=1.5x10^-96). Out of 16 second generation seeds planted, Group B germinated 15, Group C germinated 14, and Group D germinated 7. The proportion difference was not significant between groups B and C (p=0.54), but the difference between groups B and D, and groups C and D was significant (p=0.0023 and p=0.0092 respectively). Therefore, there appears to be a correlation between seed weight and percent germination, and the possible tandem effects of both fungi and bacteria on seed weight can be further explored. Regardless, the presence of soil microbes positively influencing reproductive success is supported, as Group A developed no second generation in the time allotted. Faculty Advisor: Joanna Vondrasek Kira Runkle Food for Thought: Isolation of the Neurotransmitter form Gamma-Aminobutyric Acid (GABA) from Kefir Grains For the past several decades, researchers in the fields of microbiology and molecular sciences have focused their attention towards the impact of gut flora on human biology. Gamma-aminobutyric acid (GABA) produced by lactobacillus, has been shown to influence mood, altar brain chemistry, and reduce hypertension. This study focused on the isolation of lactobacillus from kefir and the subsequent extraction of the GABA compound. MRS agar, a media known to select for lactobacillus, was inoculated with fermented milk. Colonies were then tested for catalayse negativity and Gram positivity for a positive identification. The target bacteria were then grown in broth, which was later centrifuged, and the supernatant removed. To isolate GABA, the solution was run through ion-exchange chromatography. The filtrate was then tested for the presence of amino acids using ninhydrin test strips. The final step involved running the fraction against pure GABA and Glutamate on a thin-layer chromatography plate. The results revealed the presence of both amino acids and one unknown amino acid in the mixture. GABA itself was not isolated as it never precipitated out of the filtrate sample. This may be due to the high solubility of the amino acid in the aqueous solution. Future studies may try incorporating an organic solvent to cause the neurotransmitter to precipitate out. Faculty Advisor: Barbara Heyl 11 Morgan Russell Effect of Various Essential Oils on Microbial Inhibition Many bacteria are present in and on our bodies, as a part of our normal human microbiota, including Streptococcus mutans, Staphylococcus aureus, and Staphylococcus epidermidis. These three bacteria usually cause us little to no problem, and are generally seen as opportunistic in nature. Nonetheless, this means they can occasionally take advantage of various bodily conditions and over multiply, which can cause a variety of problems. To combat the propensity of these bacteria to overproduce in such accommodating bodily conditions, we often utilize various antimicrobial agents, such as Neosporin, silver solution, and mouthwash to keep their populations low, and thus less capable of causing infection. However, not only has the efficacy of these antimicrobials been in question since their invention but also the risks of using these antimicrobials in contrast to their benefits. Furthermore, many people use alternative products, such as medical grade honey, spices, and various essential oils, in place of these antimicrobials, and have made great claims to their efficacy and lack of adverse effects. This study set out to test the former of these claims, in testing the efficacy of various traditional and alternative antimicrobials in inhibiting the growth of Streptococcus mutans, Staphylococcus aureus, and Staphylococcus epidermidis. Six antimicrobials, Neosporin, silver solution, mouthwash, rosemary essential oil, peppermint essential oil, and tea tree essential oil, were tested by placing sterile paper discs soaked in each antimicrobial onto streak plates of each of the three bacteria. Despite multiple attempts to grow S. mutans on a variety of mediums, growth could not be attained and thus results could not be recorded for S. mutans. Results of the tests on the remaining two bacteria were compared to previous studies on microbial inhibition in these two species. For both bacteria, tea tree essential oil showed the largest areas of inhibition, by far, while silver solution showed no inhibition, Neosporin, rosemary essential oil, and peppermint essential oil showed comparable areas of inhibition, and mouthwash showed variable results. These findings not only bolster claims for alternative antimicrobial agents, but also underscore questions regarding traditional antimicrobials. Faculty Advisor: Marlena Yost Jacob Simons A Novel Technique Facilitates Immunofluorescence Experiments Immunofluorescence is a technique that uses antibodies to bind fluorochromes to specific biomolecules within a cell. Viewed under an epifluorescent microscope, and excited by specific wavelengths of light, these immuno-targeted fluorochromes emit fluorescent light, thus making specific molecules in the cell visible. In my Cell Biology course in fall 2014, we stained HeLa cells to visualize the nucleus, cadherin proteins, actin filaments, and gamma tubulin. When cells were viewed under an epifluorescence microscope, the gamma tubulin was not visible, and the 12 phalloidin signal bled through into the TRITC channel. To investigate why these two stains behaved this way, I first developed a new and expedient cell fixation and permeabilization technique that can be used for rapid immunofluorescence experiments. As a proof of principle for this new assay, I stained human cheek epithelial cells with Hoescht dye and Texas-RedPhalloidin to test if these reagents allowed me to view nuclei and actin filaments, respectively. Nuclei were clearly visible and demonstrated normal nuclear morphology. Actin filament staining was also visible, but this staining revealed a somewhat perturbed cell structure, most likely due to the rapid fixation technique used. Next, this technique was used to conduct a series of experiments designed to determine if the lack of gamma tubulin staining was due to a problem with the primary anti-tubulin and/or secondary antibodies. In this experiment, cadherin staining was used as a positive control, and while this staining was faint, it was visible. The gamma tubulin staining was visible, indicating the integrity of our reagents. However there were also areas of the slide where staining had occurred where there was no cell present. This observation could be explained by the harsh fixation technique used. A dilution assay using varying amounts of methanol showed enhanced cell biology for cytoplasmic structures at lower concentrations of methanol. Future studies of gamma tubulin should take this technical observation into account. Faculty Advisor: Anne Allison Candice Tomlinson Depletion of Arf6 protein from human epithelial cells reinforces Arf6s role in regulating the actin cytoskeleton Stress fibers are filaments comprised of actin and myosin proteins. Stress fibers are found in non-muscle animal cells, and they help these cells move, form their shape, and stick to one another in tissues. Stress fibers are important for cell contractility. These fibers are formed by a cells response to its physical environment and appear when a cell undergoes high mechanical force or levels of stress. Arf6 is a cellular GTP-binding protein that is localized at the plasma membrane and is integral to membrane trafficking and cytoskeleton remodeling. The purpose of this project was to explore the relationship of Arf6 to stress fibers. Previous studies have shown that when Arf6 is mutated, stress fibers become more visible and pronounced. This study aimed to determine if cells depleted of Arf6 protein would display a similar phenotype. Cells were stained with a fluorescent dye to visualize actin filaments and were viewed using an epifluorescence microscope. A series of photographs was taken, and stress fibers were counted and measured using an open-source image processing program. Compared to control cells, stress fibers were more prominent in cells lacking Arf6 protein. These results enhance and are consistent with cell biologists current understanding of how Arf6 regulates the actin cytoskeleton during important cellular events. Faculty Advisor: Anne Allison 13 Dorcas Yoder Application of Road Salt and its Effects on Freshwater Invertebrates The application of road salt to highways and sidewalks can cause a buildup of Na, Ca, and Mg ions in soil and other surfaces. Subsequent rains can carry these ions into nearby streams and increase the salinity of stream water. A saltwater solution of 52.2 ppt was applied to a model system with three different surfaces of wheat grass, clay, and concrete. Both the volume and salinity of the runoff was measured. A portion of the runoff was diluted and used for a toxicity test with Daphnia magna. Freshwater Daphnia were transferred to solutions of 5 ppt, 2.5 ppt, 1.25 ppt, .625 ppt, and .3125 ppt. The Daphnia were checked for their survival rates after 24 hours and after 48 hours. Because the runoff from the different surfaces contained a high salinity and the Daphnia had low survival rates, we concluded that the application of road salt increases salinity of runoff and is toxic to freshwater organisms. Future research could include testing the salinity of samples from nearby streams and observing their salinity over a long period of time. Faculty Advisor: John Walsh Special Thanks The lab managers are often the people we turn to first for help with our 299 projects. The lab managers make themselves available for questions big and small: how do I sterilize my media? Whats wrong with this microscope? Do we have X/Y/Z reagents/materials stocked in the lab or do we need to order more? This course would not be possible without their expertise and willingness to help. We extend special thanks to Jennifer Scott (Biology), Rosalyn Koontz (Biology), and Ed Funck (Chemistry). ...