Chloroplast Haplotype Diversity and Distribution in Butternut (Juglans cinerea)
Kristen Laricchia, Tim McCleary, Jeanne Romero-Severson

The American butternut (Juglans cinerea) is a species of walnut tree distributed throughout much of the eastern United States and southern Canada.  The tree is currently threatened by butternut canker disease which is killing the trees at a rapid rate.  Population genetics offers insight into the diversity and migration history of the tree, which can aid in conservation decisions and in understanding the reaction of the species to an unstable climate.  After the last glacial maximum, J.cinerea began to recolonize northward as the Pleistocene glacier receded. Analysis of haplotype diversity in the chloroplast allows for the reconstruction of these migration routes. It is expected that chloroplast diversity will be decreased along a south-north gradient as a result of the founder effect as trees repopulated suitable habitat in the north. The diversity and biogeographical distribution of haplotypes was determined across several populations, with approximately 20 samples taken from each population. Intergenic regions of the chloroplast were amplified using primers designed in closely related tree species and sequenced to determine sites of polymorphism.  Twelve single nucleotide polymorphisms were discovered among eight intergenic regions.  Analysis of the prevalence and distribution of the haplotypes across the native range will provide information on the population dynamics of J.cinerea.  A decrease in haploype richness along the latitudinal gradient suggests recolonization through the founder effect, while a deviation from this distribution suggests alternative migration patterns.

Analysis of TNFα Receptor Expression Within the Regenerating Zebrafish Retina

Patrick J. O'Hayer, Craig M. Nelson, and David R. Hyde

Constant intense light induces photoreceptor apoptosis within the adult zebrafish (Danio rerio) retina. In response to this damage, Müller glial cells divide, giving rise to neuronal progenitor cells that transiently amplify and migrate to the outer nuclear layer where they differentiate into new photoreceptors. Recent work has demonstrated that this regeneration process is initiated by Tumor Necrosis Factor Alpha (TNFα) signaling. Dying photoreceptors express TNFα as a trans-acting signal that is required for the maximal number of Müller glia to begin proliferating. It is likely that TNFα binds a transmembrane receptor on Müller glial cells and activates the JAK/Stat3-mediated pathway. However, there are 7 unique TNFα receptors (TNFRSF members) within the zebrafish genome and it is unknown what receptor(s) functions within this signaling pathway. It is hypothesized that expression of the relevant TNFα receptor will be up-regulated as the TNFα signaling pathway is activated. To identify the TNFα receptor that initiates Müller glial cell division, gene specific RT-PCR and qRT-PCR were conducted using RNA isolated from both control retinas and light-damaged retinas. Both TNFRSFa and TNFRSF18 were found to be up-regulated in light-treated retinal tissue. Also, in response to morpholino-mediated knockdown of TNFα protein, both TNFRSFa and TNFRSF18 decreased in expression, suggesting that these two receptors are downstream of TNFα within the cascade. Immunohistochemical analysis using antisera generated against the TNFRSFa receptor revealed that TNFRSFa is expressed by Müller glia at the time when they begin dividing. This data suggests that TNFRSFa binds TNFα and initiates regenerative cell division within the light-damaged zebrafish retina. Further studies will include immunolocalization of TNFRSF18.

Comparing Levels of yellow Expression in Two Species of the Drosophila dunni Subgroup in Relation to Abdominal Phenotypic Variation

Jeffrey Steimle, Madeline Twiss, and Hope Hollocher

The Drosophila dunni subgroup of the Drosophila cardini group is endemic to Puerto Rico and the islands of the Lesser Antilles.  This subgroup displays clinal interspecific variation in abdominal pigmentation phenotypes.  One of the genes involved in the pigmentation pathway is yellow, which is involved in pattern specific melanin production in the adult fly cuticle, and has shown to have diversifying selection in the D. dunni subgroup, with variation in the amino acid sequences of the N-terminal signal peptide.  The expression levels of yellow were measured in Drosophila arawakana and Drosophila nigrodunni.  These two species are closely related but also phenotypically distinct, with D. arawakana being light and D. nigrodunni being the most pigmented in the dunni subgroup.  The expression levels of yellow from stages 14 and 15 female pupae are measured using qRT-PCR techniques, with cytochrome oxidase subunit II used as a control.  The CT values will be normalized according to 2-CT method and a Student’s t-test will be run to see if there are any significant differences that suggest either a pre-transcriptional or a post-translational modification.  If there is a significant result, the expression levels are different, and selection is most likely acting on a genetic element such as transcription factors or binding sites.  If the levels are the same, then this lends support to selection likely acting on the N-terminal signal peptide coding sequence.

The role of SGK-1 in Ras-mediated rescue of metabolic defects induced by loss of extracellular matrix attachment

Robert McMickle, Amy Leliaert, and Zachary T. Schafer

The ability of cancerous cells to survive in the absence of attachment to the normal extracellular matrix (ECM) is one of the fundamental hallmarks of tumorigenesis. Their survival is dependent on the inhibition of anoikis (ECM detachment-induced apoptosis) and the ability to overcome metabolic defects induced by ECM detachment. It has previously been determined by our lab that detachment of immortalized, non-tumorigenic mammary epithelial cells (MCF-10As) causes a deficiency in ATP levels that can be rescued by the stabilization of EGFR and downstream PI3K signaling. Furthermore, Ras, an established oncoprotein, lies downstream of growth factor receptors, integrins, and protein-tyrosine kinase receptors, and ultimately triggers a signaling cascade that may lead to cancer cell growth and proliferation. One critical serine/threonine protein kinase downstream of the Ras and PI3K signaling pathways that requires further understanding is the serum-and-glucorticoid-regulated kinase-1 (SGK-1). Previous studies have shown that treatment of MCF-10A cells expressing a constitutively active Ras (MCF-RasV12) with the SGK-1 inhibitor GSK 650394 have resulted in decreased ATP levels in detached cells. This data suggests a critical role of SGK-1 in the maintenance of ATP levels of cancerous cells in the detached state. To further investigate these results, we have begun engineering a mammary epithelial cell line that expresses constitutively active SGK-1, and are examining the effects of RNA interference mediated SGK-1 knockdown on cell metabolism. It is our belief that a more complete understanding of SGK-1 and its significance in promoting the survival of detached cancerous cells may offer a novel target for chemotherapeutic intervention.

Discovering a Way to Repress a Bacteriocin Belonging to Staphylococcus aureus

Jennifer Hashimoto, Danielle Chisolm, Clayton Thomas, and Shaun Lee

Staphylococcus aureus is a pathogenic bacterium which is found globally.  It causes more deaths than HIV annually.  Over the years S.aureus has acquired genes that have created drug resistant strains due to the overuse and misuse of antibiotics.  Methicillin Resistant Staphylococcus aureus (MRSA) is a highly pathogenic, drug-resistant strain of S.aureus.  MRSA can cause mild infections like a localized inflamed abscess to severe infections such as necrotizing pneumonia or Staphylococcal Skin Scalded Syndrome (SSSS).  Many virulence factors are present in S.aureus such as exfoliative toxins, enzymes that hydrolyze antibiotics, enterotoxins, and cytolysins.  Although cytolysins have been characterized in other pathogenic bacteria, their role as a virulence factor in S.aureus remains unclear.  We identified a gene cluster in USA 300 strain of MRSA that resemble the Streptolysin S (SLS) cluster in Streptococcus pyogenes.  To evaluate the potential role of this cytolysin, we assayed secreted contents of MRSA USA 300 for general cytotoxic, cytolytic, and antibacterial activity.  Our results indicate the presence of a significant cytotoxin that will undergo further testing for identification.  A greater understanding of its cytolysins will allow the new discovery of potential remedies to help with infection without allowing MRSA to undergo selective pressure.  Additionally, we can use S.aureus as a template to discover other bacteriocin cytolytic sites within other pathogenic bacteria.  This project will help to alleviate S.aureus’ pathogenic effects on humans. 

Porphyromonas gingivalis: a study of its self-defense mechanisms

Danielle A. Chisolm, Jennifer K. Hashimoto, Clayton L. Thomas, and Shaun Lee

Periodontitis is a chronic inflammatory disease of the periodontium and the leading cause of tooth loss worldwide. Periodontitis is currently understood as a microbial shift disease, shifting from mostly gram-positive to mostly gram-negative bacteria. Studies have shown that three bacterial species involved in this shift: Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. P. gingivalis is considered to be the main contributor for this shift.  P. gingivalis is a gram-negative anaerobic bacterium. It possesses several virulence factors including fimbriae, lipopolysaccharide, and bacteriocins. Bacteriocins are proteinaceous toxins produced by bacteria to inhibit the growth of bacteria either closely related to or from another species. We hypothesize that P. gingivalis may produce one or more small bacteriocins to use as a self defense mechanism so that it may gain a competitive niche in the polymicrobial community. Here, we tested the spent media of the overnight growth of P. gingivalis for antimicrobial activity on several strains of both gram-negative and gram-positive bacteria. Data from these assays show that P. gingivalis is secreting a substance with the ability to inhibit the growth of other bacteria. Several potential bacteriocins were found through a bioinformatic search of the genome. The genes that were found had characteristics of known bacteriocins. Current work is being done to successfully clone these genes from the genome and to test the purified proteins for its antimicrobial activity on different types of bacteria. If it is determined that P. gingivalis is producing antimicrobial compounds to gain a competitive niche in the polymicrobial world this would be beneficial in understanding the mystery of periodontitis.

HeLa cell cryopreservation with antifreeze proteins 1 and 2 from Dendroides canadensis

Catherine Kemme and John G. Duman

Over the years, different cryoprotectants such as DMSO and trehalose have been used for preserving mammalian cells and donor organs. In 1969, another promising cryoprotectant was discovered in the blood of Antarctic fishes. These natural cryoprotectants, known as antifreeze proteins, have since been found in several insects and plants, which protect them from freezing temperatures.
HeLa cells were studied under freezing temperatures in their own cell media with and without antifreeze proteins 1 and 2 from Dendroides canadensis. The cells were grown in T-75 flasks, trypsinized, and suspended in new cell media. They were then tested in an ethanol bath for survival at -12˚C for one half hour. Each sample was observed in trypan blue in a hemacytometer and counted for percent survivorship. Antifreeze proteins 1 and 2 were added to cell media at a concentration of .1 and .2mg/ml, and cells were retested for survivorship at -12˚C. Cells incubated in antifreeze proteins did not survive at a lower temperature more significantly than cells exposed to low temperatures in a normal environment. Cells were also exposed to 0˚C in an ethanol bath for extended periods of time and tested for survivorship with an antifreeze protein concentration of .2mg/ml. Cells with antifreeze proteins in their cell media did not survive longer than cells without these proteins. The cells were also observed under a cryomicroscope to observe any difference in the freezing temperature of a cell with antifreeze proteins from a cell without antifreeze proteins. The difference in freezing temperatures was unobservable.

Dissection Substrate Recognition by the ESK-1 System in Mycobacterium tuberculosis

Caitlin Arens and Patricia Champion

One in three people globally are infected with Mycobacterium tuberculosis, the bacteria responsible for Tuberculosis (TB). Studying TB has become increasingly important with the development of drug resistant strains of M. tuberculosis.  Examining the basic biology behind the virulence of M. tuberculosis is important for the discovery of new treatments.  It has been shown that the early secretory antigenic target 6 (ESAT-6) system 1 (ESX-1)/ Type VII secretion system is required for mycobacterial virulence.  The ESX-1 system secretes virulence factors such as ESAT-6 and culture filtrate protein-10 (CFP-10), which form a dimer.  ESAT-6/CFP-10 is recognized for secretion by the ATPase EccCb using the last 7 amino acids of the CFP-10 C terminus.  Interestingly, EspK, an ESX-1 protein important for the secretion of the ESX-1 substrate EspB, has been found to interact with EccCb as well, though this interaction has not been well characterized. Through protein alignment we recognized the C-terminus of CFP-10 is very similar to the N-terminus of EspK, leading us to hypothesize that EspK and CFP-10 interact in similar regions of EccCb.  To test this we will express epitope tagged EccCb, EspK, and CFP-10 in yeast and bacteria.  Proteins will be allowed to interact and run on an acrylamide gel to test for the formation of a tertiary complex, indicating CFP-10 and EspK interact with EccCb in different regions.  Furthermore, we will determine which areas of EspK are important for interaction with EccCb using yeast two-hybrid analysis, which probes protein interaction. It is still unknown how these proteins are secreted after they are targeted for secretion.  We hypothesize that transporters, consisting of Rv1272/73 and Rv1348/49, found in the mycolate layer may be responsible.  Yeast two-hybrid will be used to identify which proteins these transporters interact with. This project will help us to understand how proteins are identified for export and may be used in the future to identify more ESX-1 substrates.