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ItemMediating Internalin A-dependent entry of microspheres in epithelial cells(Georgia Institute of Technology, 2013-12-19) Bhyravabhotla, KshamaInternalin 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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ItemCellular Response to Surface Wettability Gradient on Microtextured Surfaces(Georgia Institute of Technology, 2013-12-16) Plaisance, Marc CharlesObjective: Topography, chemistry, and energy of titanium (Ti) implants alter cell response through variations in protein adsorption, integrin expression, and downstream cell signaling. However, the contribution of surface energy on cell response is difficult to isolate because altered hydrophilicity can result from changes in surface chemistry or microstructure. Our aim was to examine a unique system of wettability gradients created on microstructured Ti on osteoblast maturation and phenotype. Method: A surface energy gradient was created on sand-blasted/acid-etched (SLA) Ti surfaces. Surfaces were treated with oxygen plasma for 2 minutes, and then allowed to age for 1, 12, 80, or 116 hours to generate a wettability gradient. Surfaces were characterized by contact angle and SEM. MG63 cells were cultured on SLA or experimental SLA surfaces to confluence on TCPS. Osteoblast differentiation (IBSP, RUNX2, ALP, OCN, OPG) and integrin subunits (ITG2, ITGA5, ITGAV, ITGB1) measured by real-time PCR (n=6 surfaces per variable analyzed by ANOVA/Bonferroni’s modified Student’s t-test). Result: After plasma treatment, SLA surface topography was retained. A gradient of wettability was obtained, with contact angles of 32.0° (SLA116), 23.3° (SLA80), 12.5° (SLA12), 7.9° (SLA1). All surfaces were significantly more hydrophilic than the original SLA surface (126.8°). Integrin expression was affected by wettability. ITGA2 was higher on wettable surfaces than on SLA, but was highest on SLA1. ITGAV and ITGB1 were decreased on hydrophilic surfaces, but ITGA5 was not affected. IBSP, RUNX2, and ALP increased and OPG decreased with increasing wettability. OCN decreased with increasing wettability, but levels on the most wettable surface were similar to SLA. Conclusion: Here we elucidated the role of surface energy on cell response using surfaces with the same topography and chemistry. The results show that osteoblastic maturation was regulated in a wettability-dependent manner and suggest that the effects are mediated by integrins.
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ItemCharacterizing a novel direct target of the quorum-sensing controlled small RNAs in V. cholerae(Georgia Institute of Technology, 2013-12-13) Elsherbini, Joseph Ahmedn/a
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ItemNew design and implementation of the manager-client pairing framework in Manifold(Georgia Institute of Technology, 2013-12-13) Pereira, RobertoResearchers in the area of computer architecture rely very heavily on the use of architectural simulators in order to perform their work. However, creating these simulators can be a very costly and time-consuming task. For this reason, the Manifold framework was created as an effort to streamline the creation of architectural simulators. The framework consists of a simulation kernel and several modules that model the different components of a system, such as the processor or memory. One of the modules currently present in the simulator, the mcp-cache module, models a cache system that implements the Manager-Client Pairing (MCP) framework. The MCP framework provides a scalable interface for creating coherence hierarchies that may use different coherence protocols throughout the hierarchy. However, the current implementation of the mcp-cache module is restricted to a two level cache hierarchy and doesn’t accurately model the MCP framework. This document presents the redesign and new implementation of the mcp-cache module in Manifold. The new module accurately models the MCP framework, allows the creation of cache hierarchies of arbitrary sizes and can be extended to support additional coherence protocol. The creation of the new module will enable the study of how different cache hierarchies, which vary in size and coherence protocols, perform when processing the same workloads. Additionally, the new module will be useful to test changes that could be made to the MCP framework in order to improve its performance.
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ItemRibonucleotides in yeast genomic DNA are targets of RNase H2 and nucleotide excision repair(Georgia Institute of Technology, 2013-12-13) Shetty, LahariRibonucleotides 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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ItemMicrofluidic Stiffness-Dependent Separation of Red Blood Cells for Early Malaria Diagnosis and Surveillance(Georgia Institute of Technology, 2013-12-13) Byler, RebeccaThe characterization of the mechanical properties of cells has many broad applications since cell elasticity can indicate pathological state. Notably, many diseases cause significant changes in mechanical properties; for example, at the onset of a malaria infection, the invading parasites can strongly affect the elasticity of Red Blood Cells (RBCs) by causing structural changes. Thus, given the difference in mechanical properties between healthy RBCs and infected RBCs (iRBCs), there exists the potential to separate human blood through microfluidics in order to better detect malaria. We report a statistical difference in cell elasticity between RBCs and chemically mimicked iRBCs, which mimic the pathophysiology of malaria infection, through the use of Atomic Force Microscopy. We demonstrate stiffness-dependent separation of RBCs and chemically mimicked iRBCs via microfluidic technology. The successful completion of this technique will directly aid the long-term objective of this project, which is to develop point-of-care microfluidic technologies for malaria diagnosis and population surveillance that improves on the sensitivity of the existing malaria tests.
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ItemThe Business Elite: A Forgotten Force in the Civil Rights Movement, Birmingham & Atlanta, 1960-1963(Georgia Institute of Technology, 2013-12-13) Eide, KendallThis is a comparative paper focusing on the differences between Atlanta and Birmingham during the Civil Rights Movement. It highlights the important steps the business communities took, and how they varied in each city. It focuses on three major events: the Freedom Rides, school integration, and the desegregation of downtown businesses. In Atlanta, the business community was acting on a prior legacy of moderation. For decades, political and business leaders worked together to promote Atlanta as a “city too busy to hate.” In comparison, Birmingham was a city that had no moderate influence, and whose business leaders did not act until violence occurred. This was a trend that repeated itself throughout the years of the Civil Rights Movement. This paper focuses on the importance of the business community and the impact these leaders had on the course of the Civil Rights Movement.
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ItemRemediation Mercuric Ions via Functionalized Magnetic Nanoparticles(Georgia Institute of Technology, 2013-12-12) Bennett, Austin LandryMercuric ions, Hg2+, display a unique affinity for sulfur-containing biomolecules because of the soft acid/soft base interaction between Hg2+ and sulfur.1 Many of these biomolecules are used in signal processing and response;2 therefore, Hg2+ is a potent neurotoxin. Chronic exposure to high Hg2+ levels can lead to mercury-poisoning and even death. This research focused on removing Hg2+ from water. Cobalt ferrite nanoparticles were used to separate Hg2+ ions from water. A fair amount of research has been conducted using ferromagnetic nanoparticles and various nitrogenous and oxygen-based ligands, such as triazene compounds and tartrate ligands, to remediate mercuric ions.3,4 Magnetic nanoparticles (MNPs) were coated with thiol-containing carboxylic acid ligands to bind Hg2+ ions. To combat thiol oxidation, the weak reducing agent dithiothreitol (DTT) was introduced into the Hg2+ solutions. The density of the particles in the Hg2+ solutions and exposure times were varied in order to determine the optimal density and exposure time for Hg2+ removal. In future work, thiol-containing organosilane ligands will be coated onto the particles and tested for Hg2+ removal to be compared against the carboxylic acid ligands.
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ItemIdentifying Promoters of Hepatic Regeneration in Zebrafish (Danio rerio) Following an Acetaminophen Liver Ablation(Georgia Institute of Technology, 2013-12-12) Dattilo, ZacharyIdentifying 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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ItemThe Effects of Flow on Swimming Behavior of Brachionus manjavacas (Rotifera)(Georgia Institute of Technology, 2013-12-11) Rittweger, ShelbyRotifers 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.