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School of Biological Sciences

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Mediating Internalin A-dependent entry of microspheres in epithelial cells

2013-12-19 , Bhyravabhotla, Kshama

Internalin A, an internalin protein found in the food-borne pathogen Listeria monocytogenes, allows the pathogen to enter host cells through receptor-mediated internalization. Through Internalin A-mediated entry, L. monocytogenes invades enterocytes by binding to the receptor protein E-cadherin (Bergmann et al 2002). In this way, the pathogen is able to cross the intestinal barrier, a highly selective permeable interface that is responsible for allowing nutrients into the intestinal lumen while barring the entry of waste and pathogens. This study seeks to observe Internalin A-mediated entry of a pathogen mimetic system into epithelial cells. We use polystyrene carboxyl-terminated microspheres to display Internalin A, study the effect on internalization of ligand density and the size of the microsphere. A pGEX plasmid containing the inlA gene, which had previously been purified after transformation into MAX Efficiency DH5αF’IQ E. coli competent cells, was transformed into and expressed in OneShot BL21(DE3)pLysS E.coli competent cells. The result of expression of the plasmid was the Internalin A protein (InlA), combined with a glutathione S-transferase (GST) tag, in order to form a 75 kDa InlA-GST fusion protein. This fusion protein was subsequently purified through affinity chromatography. Concurrently, a protocol for labeling protein with fluorescein isothiocyanate dye (FITC) and covalently coupling the protein to 2 µm microspheres was also developed using ovalbumin. The future steps in this experiment are to successfully cleave the GST tag from Internalin A using sequence-specific protease, functionalize microspheres with purified InlA labeled with FITC and perform internalization studies with microspheres of different sizes and different densities of protein coating. Because InlA can effectively facilitate transport of L. monocytogenes into the cells of the intestinal epithelium, this study has important implications for improving the efficiency of drug delivery to the intestinal lumen.

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Identifying Promoters of Hepatic Regeneration in Zebrafish (Danio rerio) Following an Acetaminophen Liver Ablation

2013-12-12 , Dattilo, Zachary

Identifying chemicals that can promote regeneration in damaged liver tissue could be critical for curing various liver diseases and accelerating the healing of liver damage. In order to study the regeneration of developing livers in zebrafish, acetaminophen was explored as a possible method for liver ablation. A chemical screening of over 250 novel compounds with unknown cellular targets and 75 compounds with stem cell targets was performed in order to identify some promising promoters of regeneration. Acetaminophen was found to successfully destroy the liver tissue of developing embryos, demonstrating its usefulness as a method of ablation in order to study regeneration. The chemical screening revealed several novel compounds and cell signalling pathways that show promise for successfully promoting liver regeneration.

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Effect of predator diet on foraging behavior of panopeus herbstII in response to predator urine cues

2013-11-19 , Connolly, Lauren E.

The ability of prey to detect and respond appropriately to predator risk is important to overall prey fitness. Many aquatic organisms assess risk through the use of chemical cues that can change with predator diet. Two variable characteristics of diet are: 1. prey type and 2. prey mass. To assess the effect of these two characteristics on the assessment of risk by the mud crab Panopeus herbstii, I exposed mud crabs to the urine of the blue crab Callinectes sapidus fed one of 5 diet treatments: 10g of oyster shell free wet mass, 5g of oyster shell free wet mass, 10g crushed mud crabs, 5g crushed mud crabs, and a mix of 5g of oyster shell free wet mass and 5g crushed mud crab. Effects on P. herbstii foraging were tested in a previously developed bioassay by measuring shrimp consumption over a 4 hour period. I hypothesized that P. herbstii would have a larger magnitude response to urine from C. sapidus fed a diet of crushed mud crabs than to urine from C. sapidus fed a diet of oysters. I further hypothesized that P. herbstii would have a larger magnitude response to urine from C. sapidus fed a high mass diet relative to a lower mass diet. Contrary to expectations there was no observed effect of urine on P. herbstii foraging in any of the treatments. Results suggest that bioassay protocol may be unreliable suggesting further replication to determine the difference between this study and previous results. Future studies examining how P. herbstii varies with urine concentration will aid in understanding the ecological scale of this predator cue system. Determining the role of other potential cue sources will improve the predictive abilities of these studies.

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Interactions between ecosystems and disease in the plankton of freshwater lakes

2013-11-18 , Penczykowski, Rachel M.

I investigated effects of environmental change on disease, and effects of disease on ecosystems, using a freshwater zooplankton host and its fungal parasite. This research involved lake surveys, manipulative experiments, and mathematical models. My results indicate that ecosystem characteristics such as habitat structure, nutrient availability, and quality of a host’s resources (here, phytoplankton) can affect the spread of disease. For example, a survey of epidemics in lakes revealed direct and indirect links between habitat structure and epidemic size, where indirect connections were mediated by non-host species. Then, in a mesocosm experiment in a lake, manipulations of habitat structure and nutrient availability interactively affected the spread of disease, and nutrient enrichment increased densities of infected hosts. In a separate laboratory experiment, poor quality resources were shown to decrease parasite transmission rate by altering host foraging behavior. My experimental results also suggest that disease can affect ecosystems through effects on host densities and host traits. In the mesocosm experiment, the parasite indirectly increased abundance of algal resources by decreasing densities of the zooplankton host. Disease in the experimental zooplankton populations also impacted nutrient stoichiometry of algae, which could entail a parasite-mediated shift in food quality for grazers such as the host. Additionally, I showed that infection dramatically reduces host feeding rate, and used a dynamic epidemiological model to illustrate how this parasite-mediated trait change could affect densities of resources and hosts, as well as the spread of disease. I discuss the implications of these ecosystem–disease interactions in light of ongoing changes to habitat and nutrient regimes in freshwater ecosystems.

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Characterizing a novel direct target of the quorum-sensing controlled small RNAs in V. cholerae

2013-12-13 , Elsherbini, Joseph Ahmed

n/a

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The Effects of Flow on Swimming Behavior of Brachionus manjavacas (Rotifera)

2013-12-11 , Rittweger, Shelby

Rotifers serve as model species and are crucial to the zooplankton communities in terms of feeding and nutrition as well as their overall contribution to aquatic food webs (Wallace et al., 2010). Rotifers experience fluid flow in their natural environments of lakes and streams. Fluid velocity acts as stimulus to rotifers, causing them to adjust their swimming speed and direction. I am interested in how rotifers respond to flow, which is known as rheotaxis (Marcos, 2012). Brachionus manjavacas is the rotifer species employed in my experiments. This study simulates fluid flow at rates similar to that rotifers may experience in a riverine ecosystem with unidirectional flow. My intention is to uncover the ways in which the animals respond to flow in these tightly controlled conditions. Rotifers are categorized by age and tested in flow rates ranging from 0.0 to 1.0 mm/sec. Video analysis enables us to quantify swimming velocity and dissect its directionality. The study observes Brachionus manjavacas behavior in terms of aging and analyzes behavior (swimming) from an ecological perspective. It was observed that two-day-old rotifers swim the fastest on average, while four-day-old animals show fastest swimming patterns against the flow. The end result is a behavioral profile that can be useful for understanding how rotifers adapt to flow.

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The acute effects of physical activity on the stiffness of the plantar skin of people with and without diabetes

2013-11-18 , Wendland, Deborah Michael

Diabetes affects 25.8 million Americans. Complications related to this growing disease impact public health. One secondary complication of diabetes is changes in skin that can contribute to an increased risk for ulceration. Skin of people with diabetes has not been characterized over time nor has the skin’s acute response to exercise been assessed. The objective of this project was to establish the changes in skin properties over time, within different ambient environments, and after acute exercise. This objective sought to address the central hypothesis that skin will demonstrate decreased stiffness and increased elasticity as a result of acute physical activity. Skin stiffness, compliance, and thickness measurements of the plantar foot were compared across time and environment. Skin stiffness and compliance were also compared before and after treadmill walking. First, three devices were validated. Accuracy of the StepWatch was validated for people using assistive devices. The tissue interrogation device (TID), a novel device that measures tangential skin stiffness, and the myotonometer, which measures skin compliance, were validated using elastomer phantoms. Both were found suitable to measure plantar skin properties. Second, skin properties of 16 persons with and without diabetes were measured over time and environmental condition. Skin was variable across subjects over time, but was stable within subjects over a month, supporting the use of a repeated measures approach to interventional study on the plantar skin in people with diabetes. Previous findings for general skin characteristics were supported including the tendency for persons with diabetes to have a thinner epidermis and a thicker dermis than persons without diabetes. Tangential skin stiffness was determined to be less stiff in people with diabetes when measured in a medial-lateral direction. People with diabetes had lower tissue compliance than those without. Skin properties varied across environmental condition, supporting the consideration of testing environment when evaluating skin. Finally, changes in skin properties were evaluated in 32 persons with diabetes before and after treadmill (TM) walking. Using the TID, skin stiffness (tangential) at the great toe of people with diabetes (663.705±4.796 N/m) and without (647.753±5.328 N/m) were different (p=0.040). Stiffness immediately following TM walking did not differ from pre-walking stiffness, but subsequent trials had increased stiffness. Similar, but not significant responses were noted at the first metatarsal head. Compliance using normal loading increased after walking with statistical differences lasting 30-60 minutes.

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Ribonucleotides in yeast genomic DNA are targets of RNase H2 and nucleotide excision repair

2013-12-13 , Shetty, Lahari

Ribonucleotides can be incorporated into the yeast genome through a variety of mechanisms, including through DNA polymerazation, DNA priming, and oxidative damage. Ribonucleotides contain a reactive 2’ hydroxyl group on the sugar, which can distort the DNA double helix and lead to defective replication and transcription and ultimately mutagenesis. Ribonucleotide excision repair (RER) has been found to remove ribonucleotides through the enzyme RNase H2, though the in vivo substrate specificity is not known. Nucleotide excision repair (NER) removes bulky lesions formed in DNA, however its role in the extraction of ribonucleotides has not yet been determined in eukaryotes. Previously developed oligonucleotide-driven gene correction assays in Saccharomyces cerevisiae, or baker’s yeast, have shown that paired and mispaired rNMPs embedded into genomic DNA, if not removed, serve as templates for DNA synthesis and can result in a genetic alteration. We implemented this assay to examine whether RNase H2 and NER can target specific rNMPs in DNA. Our results deliver new evidence that RNase H2 specifically recognizes isolated paired and mispaired rNMPs embedded in yeast genomic DNA and that the NER mechanism can recognize an isolated paired rNMP as damage during DNA double-strand break repair in yeast.

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The Initiation, Multiplication, and Cryopreservation of Fraser Fir (Abies fraseri [Pursh] Poir.) Embryogenic Tissue for Somatic Embryogenesis

2013-12-11 , Fischer, Susan Taylor

Fraser fir (Abies fraseri [Pursh] Poir.) is a coniferous tree native to the southern Appalachian Mountains in the United States. Due to its restricted native range in a high-elevation habitat and long reproductive process, the forces of anthropogenic global climate change and invasive pests have made this species vulnerable to extinction (Conifer Specialist Group 1998). Research on ways to propagate mass numbers of conifers like the Fraser fir and restore forest productivity includes clonal propagation through somatic embryogenesis. Such research is critical to help ensure the survival of this species for both environmental and economic reasons. Fraser fir is the most popular Christmas tree in the United States and the primary Christmas tree species grown in North Carolina, where Christmas tree sales alone brought in a revenue of over $75 million dollars in 2011 (NCDA 2012). To explore potential methods of increasing embryogenic tissue initiation and growth, embryogenic tissue initiation and capture media were supplemented with the redox chemical sodium thiosulfate (158.09 mg/L) and were compared to control media. Although the redox medium yielded a higher average percent initiation (29.3% versus 26.9%), the results were not statistically significant (p > 0.05). To assess the effects of toxic carbohydrate hydrolysis products in autoclaved media, growth of embryogenic tissue was recorded for capture media with autoclaved sucrose and compared to the growth of tissue on media with filter-sterilized sucrose. The non-significant results suggest that filter-sterilization of sucrose is not necessary and does not inhibit embryonic tissue proliferation. High-mass initiations were selected for cryopreservation and were analyzed for new growth after removal from cryogenic storage. Ongoing research includes production of somatic embryos from designated high-yielding cultures removed from cryostorage, propagation of those cultures on maturation media, and germination of normal somatic embryos on germination media to effectively create highly efficient protocols for the somatic embryogenesis of Fraser fir.

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Identifying key factors in two-dimensional crystal production and sample preparation for structure-function studies of membrane proteins by cryo-EM

2013-11-18 , Johnson, Matthew C.

Electron crystallography of two-dimensional crystals is a structure-determination method well suited to the study of membrane protein structure-function. Two-dimensional crystals consist of ordered arrays of protein within reconstituted lipid bilayers, an arrangement that mimics the natural membrane environment. In this work we describe our recent progress in the use of this method with three different proteins, each providing a window into a separate paradigm in the electron crystallographic pipeline. Specific crystallization conditions for human leukotriene C₄ synthase (LTC₄S) have previously been determined, but our continued refinement of purification and crystallization has identified a number of additional parameters that greatly affect crystal size and quality, and we have developed a protocol to rapidly and reproducibly grow large, non-mosaic crystals of LTC₄S. The human gamma-glutamyl carboxylase (GGCX) has also been crystallized, but is sensitive to cryo-EM sample preparation conditions and we present here the successful reproduction of crystallization and refinement of cryo-EM sample preparation conditions. Lastly, we describe our crystallization screens with the Vibrio cholerae sodium-pumping NADH:ubiquinone reductase complex (Na⁺-NQR), and identify the factors critical to membrane reconstitution of the complex, a necessary first step towards crystallization. We also describe a semi-quantitative crystal screening protocol we have developed that provides quick and accurate method to assess two- dimensional crystallization trials, and discuss some general observations in optimization of membrane protein purification and two-dimensional crystallization for electron crystallography.

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