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End date: 2013 × Start date: 2010 × search.filters.applied.f.resourcesubtype: Undergraduate Thesis ×

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Now showing 1 - 10 of 33
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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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Defining tooth and taste bud density throughout development

2013-05-08 , Phillips, Kristine

Epithelial-mesenchymal interactions are the foundation for building differing oral tissues. While the epithelium has the ability to develop into multiple tissue types, the mesenchyme confers regional specificity. Using cichlid fish, we aim to determine at what point the teeth and taste buds diverge from the common epithelium and which gene pathways are responsible for controlling tooth and taste bud density. Understanding how to pattern these structures has key implications in genetics and biomedical engineering.

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Effects of the Deepwater Horizon oil spill on indigenous microbial communities in Pensacola Beach sands

2013-05-08 , Marks, Kala

The destruction of the Deepwater Horizon (DH) oil rig discharged approximately 4.9 million barrels of light crude oil into marine environments from April 20, 2010 to July 15, 2010. A significant amount of oil washed ashore on beaches in the Gulf of Mexico and was subsequently buried underneath layers of sand. The overall goal of this project was to investigate the temporal effects of oil contamination from the DH spill on indigenous microbial communities in Pensacola Beach sands. Shifts in the community composition of bacteria and archaea were determined using high-throughput sequencing of 16S SSU rRNA genes, and gravimetric analysis was used to quantify oil degradation by known oil-degrading taxa enriched and isolated from oil contaminated beach sands in the Gulf of Mexico. Amplicon sequencing revealed significant decreases in microbial diversity as well as a shift in the microbial community to Gammaproteobacterial and Alphaproteobacterial lineages (~80% of the community) in oil contaminated sands. Many of the dominant operational taxonomic units (OTU) detected in abundance in oiled sands showed high sequence identity to known oil-degrading bacteria. Clean sands were dominated by different Gammaproteobacteria and members of the Thaumarchaeota. Archaea were more abundant in uncontaminated sands and are thought to be inhibited by oiling. A succession of microbial populations was observed from known aliphatic degraders to polycyclic aromatic hydrocarbon degraders as components of the oil were preferentially degraded. Isolates enriched from oil contaminated sands degraded significantly more oil than uninoculated control groups, illustrating that ecologically relevant bacteria abundant in oil contaminated communities are capable of oil degradation. Thus, we conclude that blooms of oil-degrading taxa are associated with the removal of hydrocarbons from the environment.

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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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Spatial Variation of Picoplankton Community Structure in the Northern Gulf of Mexico

2013-05-08 , Geddes, Barbara Katelyn

Marine microbes are responsible for over half of global primary productivity; Prochlorococcus and Synechococcus are the most abundant of these microbes, accounting for over a trillion trillion of the single-celled organisms in the world’s oceans These picoplankton, each exploiting a unique pigment scheme, are easily sorted and counted (cells/mL-1) based on fluorescence signatures and light scattering patterns using dual beam flow cytometry. Prochlorococcus, Synechococcus, and picoeukaryotes were sampled in summer 2012 in the Gulf of Mexico, an economically significant yet understudied sea. Vertical profiles were constructed to describe the spatial variation of picoplankton in response to nutrients (nitrate and phosphate), temperature and salinity throughout the northern Gulf of Mexico. The response of these picoplankton communities to varying environmental conditions suggests an alteration of community structure in response to anthropogenic factors such as elevated nutrient inputs into the Gulf and alteration of habitat due to drilling. As exploitation of the Gulf’s resources increases, continuing to understand the response of picoplankton will be crucial in sustaining the productivity of these waters.

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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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The origin of eye size differences in cichlid fish ecotypes

2013-05-08 , Michaels, Mary E.

The evolutionary mechanisms that act upon brain and eye development are not well known. Here, we investigate the causes of the adult eye size difference of the two cichlid fish ecotypes from Lake Malawi, and demonstrate that the variation in size starts early in development and is due to early patterning. In brain development, the eye field splits from the forebrain, a process that marks the beginning of the development of the eyes. Genes necessary for eye development include the eye field transcription factors rx3 and pax6. Greater expression of these genes before the eye field first segregates from the forebrain could cause the eyes to be larger when they are initially formed. Of the two cichlid ecotypes, the sand-dwellers (utaka) have larger eyes as adults than the rock-dwellers (mbuna). We show that the utaka have larger eyes than mbuna at the first developmental stage when the eyes are specified, or neurulation. Differences in expression of the eye field transcription factors before neurulation is responsible for this initial size difference.

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