2012 Summer Symposium Program - Middlebury College

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Page 6 5 Lou Cornacchione ‘13 Major: MBBC Ostro Family Molecular Biology and Biochemistry Research Fund Grace Spatafora Given Professor of Biology & Pre-Medical Sciences Middlebury Summer Research Symposium 2012 Follow-up research in 2011–2012 by Emily McDonald was prompted by concerns of citizens in Highgate, VT where the CSF outcrops. Field work consisting of well and bedrock sampling, followed up with lab analyses, led to the development of a model in which fluoroapatite incorporates U into its mineral structure on the ancient seafloor. During marine upwelling, U +4 co-precipitates in place of Ca +2 in the structure of Ca 5 (PO 4 ) 3 F. It is authigenic but can be re-mobilized by bottom currents and redeposited as rip-up clasts, possibly explaining the breccias throughout the CSF. Bedrock samples from Colchester and Milton, VT are being analyzed using a combination of XRD, SEM-WDS SEM-WDS, ICP-AES, ICP-MS, and gamma ray spectrometry to interpret geochemistry, mineralogy, and distribution of breccias and other lithologies in the CSF. Early XRD results have yielded positive results for fluoroapatite at a site in Colchester where high radioactivity had been confirmed using gamma ray spectrometry. Fluoroapatite hosts high uranium content (80-300 ppm) in black chip breccias farther north in Highgate (crustal average is 3 ppm). Distinctive X-ray diffraction peaks at 0.281, 0.270, 0.244, 0.277, 0.317, and 0.263 nm provided the positive identification of fluoroapatite. XRD was also useful in differentiating fluoroapatite in sample 061412-1 and 061412-3 at the highradioactivity site from chert in sample 061412-14 at Thompson Farm that is not a source of U. Further analysis will include ICP-AES at Middlebury College, ICP-MS at a contract lab (for uranium and thorium content as well as other trace elements), and SEM-EDS and SEM-WDS for analysis of the micron-scale spatial distribution of P, U, and other elements. Identifying Critical Nucleotides for the SloR:SRE Interaction in S. mutans Lou Cornacchione and Grace Spatafora Department of Biology, Middlebury College, Middlebury VT 05753 Streptococcus mutans is considered to be the most cariogenic bacterial species among those resident in the human oral cavity. S. mutans-induced dental caries have been implicated in a variety of health risks and are associated with substantial economic costs. Researchers in the Spatafora laboratory previously demonstrated that a multitude of virulence genes in S. mutans are regulated by a 25-kDa metalloregulatory protein called SloR. SloR is known to bind to palindromic recognition elements (SREs) at promoter-proximal or -distal regions in a manganese-dependent manner to either repress or activate downstream genes, respectively. The sloA gene with a promoter-proximal SRE that is repressed in the presence of elevated manganese concentrations belongs to a sloABC operon, which encodes an important manganese transporter involved in metal ion homeostasis. Importantly, a SloR-deficient strain of S. mutans, GMS584, has been demonstrated to be hyper-cariogenic in a germ-free rat model, and to de-repress virulence genes in qRT-PCR experiments. The goal of the present study is to further our
6 Nicholas Dragone ‘14 Major: BIOL Vermont EPSCOR Sallie Sheldon Professor of Biology Middlebury Summer Research Symposium 2012 understanding of the SloR:SRE interaction, by introducing point mutations into the SRE that precedes the sloABC operon. We based our rationale for SRE mutagenesis on a probability matrix that we generated previously, and that includes an interrupted palindrome of 22bp. Specifically, we generated T-to-C transitions at positions 15, 16, and 19 within the sloA SRE using an OE-PCR and a markerless mutagenesis approach. In addition, we monitored sloABC expression in both GMS615 and GMS616, S. mutans strains harboring transitions at SRE positions 15 and 16 respectively, in qRT-PCR experiments. Expression in GMS616 was derepressed relative to the UA159 wild-type progenitor strain, whereas expression in GMS615 was not significantly different from that of UA159. These findings implicate thymidine-16 as important for the SloR:SRE binding and subsequent repression of the sloABC transcript. Interestingly, previous DNA footprinting experiments that we performed with SloR revealed an additional region of protection between two and twelve base pairs upstream of the SRE. To confirm that these upstream nucleotides are involved in the SloR:SRE interaction, we are targeting eight additional nucleotides, mainly thymidine residues, for mutation by OE-PCR. Gel mobility shift experiments are planned with these mutated SRE variants to assess their relative binding affinities with SloR. The identification of critical nucleotide bases for SloR binding in this important oral pathogen is significant because it could inform rational drug design for anti-caries therapeutics. Colonization of Leaf Pack Microhabitats by Macro-invertebrates in the New Haven River Nicholas Dragone and Sallie Sheldon Department of Biology, Middlebury College, Middlebury VT 05753 Stream Macro-invertebrate communities consist of different guilds based on feeding behavior. The presence of a guild in a stream microhabitat depends on the food source available at the site, and the substrate structure. Leaf Packs are an important stream microhabitat for invertebrates. In this study, artificially created leaf packs were placed in the field to determine whether invertebrate guilds colonize based on the presence of the food source, or of the structure the leaves provide. An experiment using sugar maple leaves, red oak leaves, and plastic leaves, was set out in the New Haven River. Leaf packs were collected after 1 week, 2 weeks, or 4 weeks, to determine whether macro-invertebrate colonization and utilization changed as the leaves broke down. Sugar maple leaves and oak leaves had the highest abundance of invertebrate while plastic leaves had the lowest abundance. There was no difference in the presence of guilds among the three leaf treatments although filterers and gatherers were the most abundant on all three treatments. This result suggests that leaf packs in streams are primarily used for structure, and not food. Page 7
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Page 7: 3 Samouel Beguin ‘13 Major: ENVS,
Page 11 and 12: 8 Aden Forrow ‘13 Major: PHYS, MA
Page 13 and 14: 11 Jacob Hobbie ‘15 Major: PHYS E
Page 15 and 16: 15 Poomirat Nawarat ‘14 Major: PH
Page 17 and 18: 17 Lucia Perez ‘14 Major: Astroph
Page 19 and 20: 20 Juliet Ryan-Davis ‘13 Major: G
Page 21 and 22: 22 Garron Sanchez ‘13 Major: MBB
Page 23 and 24: 25 Meghan Stang ‘13 Major: MBBC N
Page 25 and 26: 27 Karl Wetterhorn ‘13 Major: BIO
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2012 Summer Symposium Program - Middlebury College

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