Biology Theses and Dissertations
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Item CHEMICAL INDUCTION OF SETTLEMENT IN LARVAE OF THE EASTERN OYSTER CRASSOSTREA VIRGINICA (GMELIN)(2009) Grant, Melissa; Meritt, Donald W.; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Although numerous studies have been conducted to examine the effects of neuroactive compounds on bivalve larvae, few have identified chemicals capable of inducing settlement behavior in the eastern oyster Crassostrea virginica. In this study, I treated competent C. virginica larvae with select chemicals to identify those which are capable of inducing settlement behavior at an average salinity of 9.6 (±0.1). The compounds γ-aminobutyric acid and acetylcholine chloride, both at 10-4M, did not significantly increase the percentage of larvae exhibiting settlement behavior. As compared with the control, a significant increase in settlement behavior was induced by treatment with 3-isobutyl-1-methylxanthine, 5-hydroxytryptamine, and L-3, 4-dihydroxyphenylalanine all at 10-4M, as well as ammonia as a solution of 7.9mM NH4Cl (pH=8.0). These findings differ somewhat from the results of similar studies involving other species in the Crassostrea genus and may be of value to hatchery personnel or researchers interested in the chemical induction of settlement behavior in the eastern oyster.Item Importance of channel networks on nitrate retention in freshwater tidal wetlands, Patuxent River, Maryland(2009) Seldomridge, Emily Dawn; Prestegaard, Karen; Cornwell, Jeffrey C; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Freshwater tidal marshes border stream channels near the upstream end of the tidal limit, and are likely to undergo significant changes in salinity, tidal inundation, and biogeochemical processes due to sea-level rise. Tidal channel networks enhance nutrient processing by delivering nitrate-rich water far into the marsh. The purpose of this study is to examine the geomorphological, hydrological, and biogeochemical processes that influence the delivery and processing of nutrient-rich waters into tidal marshes. In this study, field measurements were made to calculate water and nitrate flux for stream channels of varying order. These mass balance calculations indicate there is an exponential increase in net nitrate retention with channel order. This calculation could be compared with calculations of denitrification at different sites within the system to evaluate the role of these processes in total nitrate loss.Item Host preference of Perkinsus species: epizootiological, environmental, and molecular aspects(2007-07-10) Pecher, Wolf Thomas; Vasta, Gerardo R.; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Perkinsus species are protistan parasites of mollusks. In Chesapeake Bay, P. marinus, P. chesapeaki, P. andrewsi, and a Perkinsus isolate from the hard clam Mercenaria mercenaria [Perkinsus sp. (M. mercenaria)] are sympatric. In vitro experiments by others suggest a preference of P. marinus for the eastern oyster Crassostrea virginica. Studies conducted in this dissertation on the distribution of Perkinsus species in C. virginica and M. mercenaria from Virginia to Maine using PCR-based detection assays provided further evidence for a host preference of P. marinus. While P. marinus was the most prevalent species in C. virginica, its prevalence was significantly lower in M. mercenaria. Interestingly, the assay designed to be specific for Perkinsus sp. (M. mercenaria) also amplified P. andrewsi. Characterization of the rRNA gene loci of both Perkinsus species revealed the presence of a second rRNA gene unit in P. andrewsi with high percent sequence identity to the unit of Perkinsus sp. (M. mercenaria), explaining the cross-amplification. Furthermore, DNA samples of M. mercenaria inhibited PCR amplification, which was overcome by adding bovine serum albumin and dimethyl sulfoxide to the PCR reaction mixture. P. marinus resides in oyster hemocytes scavenging and/or inhibiting the production of reactive oxygen species (ROS) usually generated by oyster hemocytes. ROS scavenging enzymes include superoxide dismutases (SODs) and peroxidases such as catalases. SODs have been characterized in P. marinus, but peroxidases have not been detected. Results from the present study suggest that, while lacking catalase, P. marinus has ascorbate dependent peroxidases usually found in plants. Alternatively, P. marinus may suppress the production of ROSs by enzymes such as phosphatases. A secreted acid phosphatase activity reported earlier in P. marinus was further characterized in the present study, and its purification attempted. Furthermore, a search of a genome database yielded several phosphatase-like genes, including a putative protein phosphatase 2C predicted to be secreted. Further analysis could neither confirm its secretion nor its involvement in host preference. However, the approaches implemented throughout this research represent a strategy for processing additional phosphatase and other gene sequences identified in genome databases that will further the understanding of the biology of Perkinsus species.Item Ceramide Metabolism and Transport: Implications on the Initiation of Apoptosis(2006-12-19) Stiban, Johnny; Colombini, Marco; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Apoptosis is a process by which unwanted cells are eliminated in a controlled manner. Early in apoptosis, ceramide levels rise and the mitochondrial outer membrane becomes permeable to proteins. The permeability of the outer membrane is attributed to the self-assembly of ceramide in form of channels. In the only direct structural study, to date, ceramide channels were visualized in liposomes using transmission electron microscopy. Those channels were of various sizes, averaging 10 nm in diameter. In concert, using electrophysiological techniques, the estimated diameter of ceramide channels was also around 10 nm. These channels are large enough to release all the pro-apoptotic intermembrane space proteins to initiate apoptosis. Dihydroceramide desaturase converts the inactive precursor, dihydroceramide to ceramide. Both long and short chain dihydroceramides inhibit ceramide channel formation in mitochondria. The inhibition is strong as one tenth as much dihydroceramide inhibited the outer membrane permeabilization by 95% (C2) and 51% (C16). Other mitochondrial components are not required for such inhibition as comparable amounts prevented the permeabilization of liposomes. Hence, the apoptogenic activity of ceramide may depend on the ceramide to dihydroceramide ratio perhaps resulting in a more abrupt transition from the normal to the apoptotic state. The location of the desaturase is the endoplasmic reticulum (ER). Only minimal activity was measured in mitochondria. However, newly synthesized ceramide from 14C-C8-dihydroceramide or 3H-sphingosine (in the ER) can transfer rapidly to mitochondria (40 % in 10 min) and permeabilize them to cytochrome c and adenylate kinase. The transfer of sphingolipids is bidirectional and non-specific. The transfer mechanism is consistent with direct membrane contact, since reducing the organellar concentrations by half resulted in a four-fold reduction of the transfer rate. Thus this ceramide exchange obviates the need for a complete ceramide de novo pathway in mitochondria in order for cells to use ceramide to activate mitochondria-mediated apoptosis. These results demonstrate the ability of ceramide to form large channels capable of releasing proteins from mitochondria. Ceramide can rapidly reach mitochondria and there are mechanisms to control the propensity for ceramide channel formation. Clearly ceramide channels play a central role in the decision to undergo apoptosis.Item MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF A 15 KDA GALECTIN FROM STRIPED BASS (MORONE SAXATILIS)(2004-11-29) Henrikson, Davin Eric; Vasta, Gerardo R; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)I have employed biochemical and molecular techniques to investigate the role of galectin during bacterial challenge in teleost fish, using striped bass as an experimental model. Striped bass (Morone saxatilis) possesses a 15 kiloDalton Beta-galactoside binding protein, here after called MS15. It is a single polypeptide with an experimental mass of 15,000 Daltons. In vivo, MS15 exists as a non-covalently linked dimer, with two identical carbohydrate binding sites per protein. The organization of the gene coding for MS15 was the same as other vertebrate proto type galectins, with four exons the same size between organisms and three introns varying greatly in length. These investigations have shown that galectin is localized to the dermis and lamina propia (loose connective tissue) throughout the body, to smooth vascular muscle (veins, arteries), to large circulatory cells (heterophils, monocytes), to peripheral leukocytes (tissue resident macrophages, heterophils), to rodlet cells (possible immune cell in alimentary canal), and to leukocytes throughout the gills but not to mucus-producing cells. This is similar to what is found in mammals, but differs greatly from what has been observed in other teleosts. This finding led to the hypothesis that proto type galectin in striped bass was involved in innate immunity. To test this hypothesis, galectin was characterized based on stability, carbohydrate binding specificity, and native structure. Interactions between galectin and mucus, and galectin and bacteria were then tested. Results revealed galectin binds skin mucus and agglutinates selected bacteria. Striped bass were challenged with bacteria by intradermal and intramuscular injections. Results suggest an increase in the number of galectin-positive leukocytes observed in injection sites, independent of the presence of bacteria. Also, a novel observation was the strong galectin-positive nature of rodlet cells in the esophagus, stomach, and pyloric caeca of the striped bass. The function of rodlet cells is disputed, but in striped bass, galectin was detected most strongly in rodlet cells and both circulatory and peripheral leukocytes. Collectively, these data provide the foundation for three models to explain the biochemical events involved in bacterial clearance and/or wound healing in teleost fish, with potential relevance to innate immunity in other organisms.Item F-TYPE LECTINS: BIOCHEMICAL, GENETIC, AND TOPOLOGICAL CHARACTERIZATION OF A NOVEL LECTIN FAMILY IN LOWER VERTEBRATES(2004-05-12) Odom-Crespo, Eric William; Vasta, Gerardo R; Marine-Estuarine-Environmental SciencesIn vertebrates, immunoglobulins have long been considered the primary means for recognition of potential pathogens since they are the product of an adaptive immune system capable of generating and selecting for the most efficient antibody when adequately primed. However, initiating a naive system requires signals from cells that employ invariant receptors rather than antibodies. These innate receptors appear to recognize repetitive polymers commonly found only in microbes. Frequently, these receptors are lectins specific for polysaccharides ubiquitous of the microbial surface. Lectins in the blood or lymph are widespread among metazoans while antibodies are a vertebrate innovation suggesting that lectins may be evolutionarily their functional precursors. Even in primitive jawed vertebrates, there is a complete adaptive immune system, but it is relatively inefficient in comparison to mammals. Therefore, lectins might have a prominent immune function in lower vertebrates comparable to antibodies. To test this, a teleost was surveyed for humoral and hepatic lectins. A fucose-specific lectin of 32 kDa (FBP32) was initially purified from the palmetto bass and upon sequencing indicated it was unlike other reported lectins. The primary structure is characterized by a tandem polypeptide motif (FBPL) with partial homology to a long pentraxin from a frog. An inflammatory challenge of bass to test if FBP32 behaved like a mammalian pentraxin indicated that the FBP32 transcript level increases, but protein levels appear constitutive. An extensive search using both molecular cloning and gene database queries revealed that FBP32 is a member of a diverse protein family reflecting varying concatenations of the FBPL and even present in a cell surface receptor, but of sporadic phylogenetic distribution most notably being absent in mammals. Analysis of FBP32s genic structure reveals that it is flanked by phase 1 introns, which may explain the domains ability to concatenate and shuffle to form mosaic proteins. In collaboration with experts, the tertiary structure of an FBPL including its fucose-binding site was elucidated revealing a novel lectin fold, the F-type lectin fold (FTL), that is shared by unrelated proteins. Characterization of FBPLs demonstrates that the study of mammals alone may not reveal the full extent of immune system innovation.