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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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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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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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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.
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ItemThe Initiation, Multiplication, and Cryopreservation of Fraser Fir (Abies fraseri [Pursh] Poir.) Embryogenic Tissue for Somatic Embryogenesis(Georgia Institute of Technology, 2013-12-11) Fischer, Susan TaylorFraser 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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ItemTechnology Constraints on Nonverbal Aspects of Communication(Georgia Institute of Technology, 2013-05-08) Han, MichelleCommunication involves more than just verbal speech; there are nonverbal aspects of communication such as body position, eye contact, hand gestures, and the like. Specifically in an interview setting, both the person conducting the interview and the person answering questions display their own methods of communication both verbal and nonverbal. When technology is introduced into the situation, changes in interviewer-interviewee interaction influence the interview process, the outcome, and even the aftermath. Even just the presence of a technological device, as opposed to pen and paper or the absence of a note-taking aid, could potentially alter the subjective interview experience. Understanding the tradeoff between these objective and subjective variables would be very useful. Research indicates that electronic documentation can lead to an increase in documentation and accuracy of recording. Research on the constraints of technology on nonverbal aspects of communication has been done in the Georgia Tech Sonification Lab. An investigation of a patient’s perception of a doctor after a medical interview as a consequence of different note-taking methods was conducted. In order to explore the critical interaction between doctors and patients, different note-taking methods were employed. The results were that the patients perceived the desktop computer to be the least favorable technology used by the doctor, which is applicable to society since computers are becoming more common in medical interviews (Olsheski & Walker, 2011). However, previous work on this topic did not take into account the factor of eye gaze or personality which is what the present study considered. Also, this study explored an interview involving a roommate situation, which could be applied to other interview-type situations or interactions.
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ItemPrecise Size Control in Microgel Synthesis for “Designer Polydispersites”(Georgia Institute of Technology, 2013-05-08) Singh, AkritiThe goal of this project was to synthesize a series of monodisperse microgel populations with slightly different average radii. The Lyon group has previously shown that colloidal crystals assembled from hydrogel microparticles (microgels) are extremely tolerant to defects in the form of larger microgel "dopants". To investigate the role of polydispersity in general on crystallization, and to explore the limits of defect tolerance, we have undertaken the synthesis of microgels composed of NIPAM (N-isopropylacrylamide) cross linked with BIS (N,N’-methylene bisacrylamide) over a range of particle sizes. These dispersions can then be mixed to form dispersions of arbitrary or “designer” polydispersity. These samples will then be analyzed via optical microscopy and neutron scattering in collaboration with the Fernandez-Nieves group.
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ItemDefining tooth and taste bud density throughout development(Georgia Institute of Technology, 2013-05-08) Phillips, KristineEpithelial-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.