▼ Acinetobacter baumannii is a bacterium capable of causing severe upper respiratory infections in patients whose immune systems have been suppressed. A. baumannii forms biofilms on a variety of surfaces. Biofilms are bacterial communities present as layers of cells that can form on devices such as catheters and endotracheal tubes, which can be very problematic because they enhance the ability of bacteria to resist the host responses and antimicrobial compounds. The purpose of this study was to use a unique DNA insertion system to identify some of the genes responsible for biofilm formation by this organism. After isolating DNA from selected insertion derivates of A. baumannii 19606, we were able to determine that one of the interrupted genes that abolished biofilm formation had a high degree of similarity to the csuE gene from Vibrio parahaemolyticus. This gene is part of the csu chaperone-usher operon and could potentially play a role in the assembly of pili at the cell surface. This study was significant because it helped further describe one of the potential virulence factors of A. baumannii, few of which have been well characterized. Advisors/Committee Members: Actis, Luis A.

▼ Phytoplankton and periphyton are key primary producers in aquatic ecosystems that comprise the base of aquatic food webs. Their rates of photosynthesis can be affected by several different abiotic and biotic factors. We examined the impact of light and nutrients on chlorophyll, photosynthetic efficiency under low-light conditions (α), light-saturated photosynthesis (Pmax), and percent of total production in each treatment by periphyton. Phytoplankton chlorophyll a was affected positively by nutrients and by the interaction between light and nutrients. Periphyton chlorophyll a increased significantly with light and nutrient levels. Periphyton α and Pmax were also positively affected by increased ambient light. In addition, the contribution by periphyton to total production was significantly impacted by light and nutrients. Our results suggest that light and nutrient availability can change the relative contributions of phytoplankton and periphyton in total ecosystem production by affecting biomass and rates of photosynthesis. Advisors/Committee Members: Vanni, Michael J.

▼ Because microbes are colonial by nature, selective pressures such as limited resource availability affect colonial populations as a whole. For example, Escherichia coli strains will enter a slowed metabolic state at stationary phase induced by limited resources. Superficially, this selective pressure seems to merely affect each cell individually because if each cell entered a rest state, it would be more likely to survive through the time of famine. The fact remains, however, that if one lone cell were to ‘cheat’ by entering the rest state later than the other cells, it could benefit from some of the remaining resources and reproduce normally for some n generations. Use of Nash equilibria allows one to understand this inherent incentive for the hypothetical cheater cell to develop. Upon understanding the dynamics of ‘cheating’, one can quickly see that even complex mechanisms like enhanced clonality or induced apoptosis could arise in intra-species examples to curb cheating and that reward/punishment models could develop in inter-species examples to limit cheating at any moment x. Advisors/Committee Members: Fields, Matthew W.

▼ Acinetobacter baumannii is a gram-negative bacterium that causes severe infections in immunocompromised patients, such as newborns, burn patients, and the elderly. Because the bacteria are strongly resistant to antibiotics, there is a dire need to develop new therapeutics to treat A. baumannii infections. Potential targets are proteins involved in bacterial iron metabolism, since iron is an essential micro-nutrient. Accordingly, random insertion mutagenesis analysis showed that the NfuA protein is needed when cells were cultured in the presence of 2,2'-dipyridyl, a synthetic iron chelator that generates iron-limiting conditions, and hydrogen peroxide and cumene hydroperoxide, which were used to mimic oxidative conditions. The role of NfuA was further confirmed by the observation that the genetic complementation of an A. baumannii ATCC 19606T mutant with the parental allele was enough to restore the iron metabolism and oxidative stress phenotypes expressed by the wild-type strain. Electron paramagnetic resonance (EPR) analysis of overexpressed and purified NfuA demonstrated that this protein harbors an iron-sulfur cluster, which is a prosthetic group required in central metabolic processes. Interestingly, the inactivation of NfuA did not affect bacterial growth under non-oxidative and non-chelated conditions and did not impair the ability of the mutant to express the acinetobactin siderophore-mediated iron acquisition system. On the other hand, the ability of the A. baumannii ATCC 19606T nfuA mutant to replicate inside human epithelial cells was significantly impaired when compared with the parental strain. Taken together, these observations suggest that NfuA plays a defined and important role in iron metabolism, resistance to oxidation, and intracellular replication without affecting bacterial iron acquisition processes. By understanding the function of NfuA and its importance to the A. baumannii virulence properties, we will come closer to understanding basic metabolic processes that could be alternative targets for new antibiotics designed to treat the infections this pathogen causes in humans. Advisors/Committee Members: Actis, Luis.

▼ While there are many different factors that can signal the initiation of apoptosis, the glucocorticosteroid hormones, or glucocorticoids, are the main apoptosis inducers in immature thymocytes. As hormones, glucocorticoids are essential to cell survival and homeostatsis, and can exert both positive and negative control on thymocytes. How glucocorticoids influence T-cells depends on the concentration of glucocorticoids in the cell. High levels of glucocorticoids can result in thymocyte death, as seen when the cell is responding to some kind of stress. During glucocorticosteroid-induced apoptosis, many biochemical changes occur in the cells that collectively result in cell death. One such event is the production of reactive oxygen species (ROS). When ROS build up in the cell, the cell is no longer able to use the radicals in a beneficial manner. The accumulation of ROS in the cell causes the cell to undergo oxidative stress, which can have negative implications on the stability of the mitochondrial membrane. Glucocorticosteroid-induced apoptosis results in the production of severe oxidative stress in the cell. The site for the production of the ROS is the mitochondrial electron transport chain, thus the mitochondria are also where the detrimental effects of oxidative stress are seen. The production of ROS at the mitochondrial level is especially important because of the crucial role of the mitochondria in the cell. Oxidative stress resulting from glucocorticoid-induced apoptosis has significant effects at the cellular level because ROS can damage vital organelles and systems within the cell. The mitochondria are especially vulnerable to the cellular effects of oxidative stress because of the lipids that constitute their membranes. The relationship between ROS, the mitochondria, and glucocorticoid-apoptosis is currently in area of vigorous research within the scientific community. Research performed by Noriko Tonomura from the Department of Veterinary and Animal Sciences and the Program in Molecular Cellular Biology at the University of Massachusetts and his colleagues, Kelly McLaughlin, Lisa Grimm, Richard A. Goldsby, and Barabara A. Osbourne elucidated the role of reactive oxygen species and their effect on the mitochondria in glucocorticoid-induced apoptosis of thymocytes. Published in the Journal of Immunology in 2003, the results of the work done by Tonomura et al. are explained in the article, “Glucocorticoid-Induced Apoptosis of Thymocytes: Requirement of Proteasome-Dependent Mitochondrial Activity.” Tonomura and his colleagues used dexamethasone (DEX), a synthetic glucocorticoid, to induce apoptosis in thymocytes. Overall, Tonomura et al. concluded that in DEX-treated thymocytes, apoptosis results in an increased production of H2O2 generated from the mitochondria at the complex III of the ubiquinone cycle. They also found that all the cell death events mediated at the mitochondria are regulated by the proteasome. Advisors/Committee Members: Stevenson, John.

▼ Actinobacillus actinomycetemcomitans (Aa) is a microaerophilic, gram-negative oral pathogen. Congo red and hemin biding assays showed that all tested strains were able to bind this stain and iron-containing compound, respectively. Inspection of the HK1651 genome indicated that this binding activity could be associated with the presence and expression of hms-like genes, which code for similar functions in other pathogens such as Yersinia pestis. A DF2200N derivative in which hmsF was disrupted by insertion mutagenesis showed a large reduction in hemin and Congo red binding activity. Iron-utilization assays showed no significant difference in the ability of the parental strain and the hmsF insertion derivative to use hemin under chelated conditions. These observations are in agreement with the presence of an hms-like locus in the genome of Aa, which seems to be involved in the interaction of cells with hemin but not the utilization of this compound as an iron source. Advisors/Committee Members: Actis, Luis A.

▼ Osteosarcoma is the most common bone sarcoma and generally only afflicts children and adolescents aged 10-19 years (2). The origin of the primary tumor is most frequently the actively growing regions of the skeleton, and the most likely sites for metastasis are the lungs and bone (3-5). A number of genetic abnormalities have been linked to osteosarcoma development, including retinoblastoma and p53 in primary tumor development and ezrin in the formation of metastases (6-8). Ezrin functions as a cross-linker between the actin cytoskeleton and the plasma membrane (9), and also plays a key role in a number of cellular signaling pathways (10). There is strong evidence to suggest that ezrin is necessary for osteosarcoma metastasis (11). Despite the promise that preliminary studies on ezrin have shown, there is a great need to further analyze ezrin’s role in osteosarcoma metastasis and to determine its usefulness as a biomarker for the disease. Advisors/Committee Members: Carlin, Joseph M.

▼ My research was an investigation of the antifungal effect of sap extracted from the common aloe vera plant, Aloe barbadensis, on the most frequently isolated fungal burn wound pathogen, Candida albicans (9). The beginning stages of my research involved optimizing a protocol that allowed the usage of A. barbadensis sap obtained fresh from the plant. Once the protocol was established, the goal of my research was two-fold. My first goal was to determine whether or not A. barbadensis affects the viability of C. albicans. In other words, when C. albicans is exposed to the gel of A. barbadensis, are some of the cells killed, and if so, how great is the candidicidal effect? Secondly, my goal was to determine if A. barbadensis has an anti-germination effect. In order to cause infection in mammalian tissues most scientists agree that C. albicans must undergo a phenotypic change from the normal noninvasive budding pattern to a invasive hyphal form, characterized initially by the formation of germ tubes (14). This allows the organism to penetrate the skin or mucous membranes and anchor into the tissue. My second question was does A. barbadensis inhibit the rate of germination and/or cause a decrease in the length of germ tubes? The results of the viability assays indicate that for two strains of C. albicans, ATCC 90028 and a fresh clinical isolate Ca21, a significant percentage of cells were killed when exposed to the A. barbadensis sap. The germination assay gave the unexpected result that while the average frequency of germ tube formation decreased, the average length of the germ tubes actually increased with exposure to the A. barbadensis extract. Additionally, the percent of cells with multiple morphological forms (i.e. buds and hyphae) increased significantly. This indicates that the A. barbadensis sap affects the normal growth pattern of C. albicans, but the clinical significance of this phenotypic change remains uncertain. Advisors/Committee Members: Lee, Marcia.

▼ It has been suggested that phylogenies derived specifically from 16S rDNA gene sequencing provide reasonable evidence for describing major evolutionary lineages (Brown, 1997). Because of the critical role they play in protein synthesis and thus survival, rDNA sequences are highly conserved and tend to resist significant change over time. Thus, they are of prime interest when studying evolutionary relatedness. In this study, we sequenced a portion of the 16S rDNA gene from the amplified PCR products of enteric bacterial samples. The resulting DNA sequences were edited and aligned to develop a phylogeny based on our comparative data. We developed phylogenies using parsimony analysis and bootstrapping and compared our results to expected data. We hypothesized that species within the same genus would contain the most shared derived traits and therefore be most closely related. However, our phylogeny provided limited resolution of some of our strains. While a few strains resolved well (i.e. Serratia and some Enterobacter strains), weak bootstrap support characterized much of the phylogeny. Ribosomal DNA contains conserved, variable, and highly variable regions. The highly variable regions are most prone to mutation, and thus may not provide good phylogenetic signal. For this reason the hyper-variable regions not only fail to provide meaningful data, but can also destroy signal that is already present. Advisors/Committee Members: Janssen, Gary R.