The purpose of this study was to determine the toxicity of the phototoxin, phenylheptatriyne (PHT) to acute lymphoblastic leukemia cells (ALL) under attenuated light conditions and when exposed to ultraviolet-A light (UVA). The potential of PHT to increase sensitivity of ALL cells to the anti-cancer drug doxorubicin hydrochloride also was evaluated. An in vitro multi-drug resistance model was used consisting of the parental cell line CCRF-CEM and its p-glycoprotein (pgp-170) expressing variant CEM/VLB100. Cytotoxicity was measured using the tetrazolium bromide (MTT) reduction assay and the annexin-V-FITC / propidium iodide (PI), flow cytometric assay. The results indicate that PHT is more toxic, when not photoexcited, to the CEM/VLB100 cell line (P = 0.006). There was a significant interaction between UVA dose and PHT concentration (P < 0.001). Co-incubation of CEM/VLB100 cells with less than μM doxorubicin and 60 μM PHT, significantly decreased viability relative to doxorubicin alone (P = 0.007).

A very unique compound eye in dipterans is found in males of the forensically important blow fly, Chrysomya megacephala (Diptera: Calliphoridae). This compound eye is characterized by an area of enlarged dorsal facets that, unlike almost all other regional changes in dipteran ommatidia size, is not accompanied by a change in resolution. This region is believed to play a role in mate tracking and allow for increased light capture, though no behavioral studies have tested these claims. An initial goal of the dissertation was to examine the function of this compound eye. Using allometric measurements coupled with behavioral tests, I found larger males had larger eyes and proportionally more dorsally enlarged facets. This finding suggested that larger individuals would move at lower light levels. When comparing similar sized male and females, however, body size and not the specialized male dorsal region dictated the light level at which movement occurred. A second focus of this dissertation was the development of tools to understand how the male compound eye is genetically regulated. The male-specific enlarged dorsal ommatidia offer a model for understanding how a gross morphological difference of a feature present in both sexes can arise when much of the same genetic content is shared. Since the genes regulating compound eye development are mainly expressed during stages when sex cannot be efficiently determined, I first designed a molecular test for identifying sex by amplifying a region of the transformer gene differentially spliced by sex in blow flies from other genera. Then, I was able to compare temporal patterns of gene expression for rhodopsin genes in separate sexes for the first time in blow flies, which allowed for an initial investigation into the expression patterns influencing the development of the male compound eye. Apart from the biological significance regarding a unique compound eye, the forensic importance of C. megacephala means that information obtained in this dissertation can be utilized by forensic investigators. The work on low light level movement adds to the understanding of nocturnal oviposition patterns and the ability to sex immature specimens allows for the determination of sex specific development rates.

In order to investigate the role of faunal aggregations in concentrating nutrients in the oligotrophic landscape of Florida Bay, I manipulated faunal densities in Florida Bay sea grass beds by constructing artificial reefs. The effects of reefs and faunal aggregations on nutrient availability and benthic community structure were assessed. Over a year-long sampling period, artificial reefs had an average population of 50 fishes and crustaceans of various species. Faunal aggregation resulted in significant sediment organic matter decreases and sediment phosphorus increases. Plots with high fauna populations also had shorter seagrass blades presumably due to the effects of grazing. Chlorophyll-a concentrations in the sediment and periphyton samplers were mainly affected by reef presence or exclosure type and not due to the presence of aggregating fauna. Our results suggest that faunal aggregation may have more top-down effects on primary producers than bottom-up effects over smaller temporal scales.

The health status of wild and captive Atlantic Bottlenose dolphins (Tersiops truncatis) is difficult to ascertain. Mass strandings of these animals have been attributed to pollutants, as well as bacterial infections. Using human Enzyme Linked Immuno-Assays (ELISA) for immunological cytokines, I measured soluble cytokine levels with respect to their health status. In a retrospective analysis of dolphin sera, there was a trend of higher cytokine levels in “sick” animals. I cultured dolphin lymphocytes in the presence of a mitogen (PHA), a super antigen (Staph-A), Lipopolysaccharide (LPS), and a calcium flux inducer (PMA). Levels of messenger RNA, from these cultured cells, were assayed with Polymerase Chain Reaction (PCR) using primers for the human cytokines IL-2, EL-4, IL- 6, IL-10, Tumor Necrosis Factor, and Interferon gamma. Only IL-4, IL-6, and IL-10 messages were obtained, inferring similar nucleotide homology to the human primer sequences. The PCR products were sequenced. Sixteen IL-4 sequences, twelve IL-6 sequences and seven IL-10 sequences were obtained and analyzed. Each cytokine exhibited the same nucleotide sequence in all dolphins examined. There was no difference in the cytokine profile in response to the various stimuli. The derived amino acid composition for each of the dolphin cytokines was used for molecular modeling, which showed that dolphin IL-4, IL-6, and IL-10 were structurally similar to the corresponding proteins of Perissodactyla.

Polycyclic Aromatic Hydrocarbons (PAHs) that are generated by diverse anthropogenic sources, are classified by the U.S. Environmental Protection Agency (EPA) as Semivolatile Hazardous Compounds. PAHs are pollutants of great concern due to their toxicity and mobility in the environment; they can be found in air, water, and soil media. Supercritical Fluid Extraction (SFE) is currently an alternative technology for the removal of PAHs from solid matrices. Carbon dioxide, water and organic compounds, above their critical temperature and pressure conditions, have been used as supercritical fluids. This bench-scale study evaluates the influence of temperature and use of modifiers on the extraction efficiency of PAHs in a diesel-contaminated soil sample from the Miami International Airport (MIA). Carbon dioxide at supercritical conditions was used as extraction fluid in most of this research. Subcritical water, in vapor and liquid states, was also used in a preliminary testing as extraction fluids. A conceptual design of a supercritical carbon dioxide extraction plant is suggested.

Habitat use studies can be used to both investigate ecological and behavioral patterns of animals as well as provide a useful management tool for conservation planners. However, essential habitat can be difficult to determine for highly mobile marine animals, especially when these species are rare or endangered. While critical habitat has been very broadly delineated for the endangered smalltooth sawfish (Pristis pectinata), essential fish habitat (EFH) within the nursery has not been fully described. I used telemetry methods to determine daily activity spaces and rates of movement (ROM) of juvenile P. pectinata in a nursery in southwest Florida. These results were tested for differences in diel and tidal patterns of activity. Seven juvenile animals ranging in size from 85 - 175 cm fork length were tagged in April - September 2011. Overall, activity spaces ranged from 0.07 - 0.17 km2 using 95% Minimum Convex Polygons (MCP), 0.01 - 0.16 km2 based on 50% kernel density estimates (KDE), and 0.08 - 0.68 km2 based on 95% (KDE). Average ROMs ranged from 2.4 - 6.1 meters/min. Activity space and ROMs reflected the morphology of the bay in which the animal was tracked such that fish in small bays had small activity spaces and ROMs. There were no detectable differences in activity space or ROM between ebb and flood tide or high or low tide. Activity space decreased and ROM increased at night indicating possible foraging behavior at night. A home range (1.7 km2) was calculated for one animal. Daily asymptotes in space used were reached for all other tracks suggesting daily activity spaces were determined despite relatively short tracking durations. Bays, estuaries, and other discrete coastal habitats are highly productive and serve as nurseries for a variety of marine fishes. Nurseries are particularly crucial for batoids whose life histories are dependent on rapid growth in the first year, and they may be especially important for rare or endangered species within the group. The smalltooth sawfish, Pristis pectinata, are an endangered marine elasmobranch that makes use of specific nurseries in southwest Florida. While habitat and environmental parameters have been described within the nursery, specific characteristics of the habitats, such as mangrove morphology and sediment types associated with habitat use have not been identified. Two mangrove characteristics (prop root density and limb overhang) and two sediment characteristics (percent organic and percent silt) were used as independent variables to construct a habitat model. Acoustic monitoring was used to examine long term (weeks or months) patterns in habitat use in nursery areas during the critical first year of life. Twenty young-of-the-year sawfish were acoustically tagged between April and October of 2011, and detected by an array of 32 VEMCO VR2w receivers in a documented nursery within Everglades National Park. Presence in the array for individual smalltooth sawfish ranged from one day to 197 days, and overall P. pectinata were present within the acoustic array for 334 days. There was also evidence of overwintering specifically in Chokoloskee Bay. In the back water region (Turner River, Mud Bay, Cross Bays, Wilderness Waterway, and Lopez River), residency times were longer in tidal bays rather than creeks or rivers. A potential emigration corridor from the back water region was observed through the Lopez River. Using receiver data for animals moving between neighboring areas, a step wise logistic regression model in a generalized linear model framework for receiver hits per hour was significant for mangrove prop root density (Stepwise GLM: Partial R square = 0.22, C(p) = 6.02 p = 0.023). This model indicated a higher probability of seeing a juvenile smalltooth sawfish when mangrove prop root density was high. / A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science. / Summer Semester, 2012. / June 26, 2012. / Conservation, Elasmobranch, Habitat Use, Movement, Telemetry / Includes bibliographical references. / Don R. Levitan, Professor Co-Directing Thesis; Emily H. DuVal, Committee Member; Jeanette L. Wulff, Committee Member; John K. Carlson, Committee Member.

Among flowering plants, floral form is inherently linked to reproductive success and is therefore a key element in the evolution of angiosperm lineages. Variations in floral form and associated mating systems have demonstrated that flowers are well adapted in many cases to promote predominantly either outcrossing or self-fertilization among members of a given lineage. The focus of this work is to examine the developmental interactions and consequent effects on the morphology of two such flower types within individuals of Houstonia procumbens (J.F. Gmel.) Standl. (Rubiaceae). The two flower types in question are distylous and cleistogamous. Distyly is a form of heterostyly, wherein the reproductive organs of flowers in a given population are spatially separated (herkogamy) and arranged reciprocally to one another (reciprocal herkogamy) among individuals that represent two alternate morphotypes. Cleistogamy refers to the production of both flowers that open (chasmogamous) to interact with pollinators and flowers that self-pollinate precociously in bud (cleistogamous) on individuals of a given species. While both of these pollination syndromes, distyly and cleistogamy, appear to have arisen convergently, multiple times, across angiosperms both pollination syndromes apparently co-occur in only one species H. procumbens, an annual, herbaceous member of the coffee family (Rubiaceae). The goal of this work is to examine the interactions of these two pollination syndromes in H. procumbens by comparing the form and development of the various flower morphs (distylous morphs and cleistogamous flowers) of this species to those of closely related taxa that produce either distylous or cleistogamous flowers, but not both. The first chapter of my dissertation establishes the degree to which discoveries about the heterostylous, cleistogamous flowering plant species H. procumbens are applicable to other species. I use literature searches to establish that other species that are both heterostylous and cleistogamous are currently unknown, and I use phylogenetic inference and ancestral state reconstruction for the Rubiaceae to establish that heterostyly arose 27-36 independent times in the family and that the origin of heterostyly for H. procumbens occurred in an ancestor that produced at least 820 extant, descendent species. I also show that the joint probability of heterostyly and cleistogamy in angiosperm families, genera, and species is quite low, and the observed numbers of taxa in which the two co-occur at the ranks of family and genus (but not species) is greater than expected, though not significantly so. The second chapter addresses the effect of cleistogamy on reproductive organ reciprocity for H. procumbens. I use comparisons among close relatives to show that the overall reciprocity of H. procumbens changed little with the evolution of cleistogamy but that reciprocity in a close distylous relative, H. caerulea, was sufficiently low to qualify it as "style dimorphic" by some standards--a situation that I suggest calls into question the use of the reciprocity index for such distinctions. I also demonstrate that H. procumbens does not have a pollen size or staining dimorphism, as is seen in other heterostylous members of Houstonia, making assessments of disassortative pollen transfer difficult. The third chapter examines the chasmogamous and cleistogamous flower development of H. procumbens and chasmogamous flower development of H. caerulea. I demonstrate that differences in the rate of elongation of styles and corollas lead to the difference in stigma and anther heights seen in the two chasmogamous morphs of H. procumbens and H. caerulea, and that this dimorphism in growth rates is established early in development. Additionally, I show that there is a relatively small anther-stigma distance in the early stages of floral development of H. caerulea, which is unexpected, since one might expect this to be a developmental pattern that makes cleistogamy (e.g., as in H. procumbens but not H. caerulea) evolutionarily simpler (by reaching sexual maturity in those earliest stages). Instead, I show that H. procumbens reduces the anther-stigma distance in its cleistogamous flowers by reaching sexual maturity when the corolla is short (and the anthers low) and by producing helical styles. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Summer Semester, 2012. / June 11, 2012. / cleistogamy, floral evolution, flower development, heterostyly, Houstonia, Rubiaceae / Includes bibliographical references. / Austin Mast, Professor Directing Dissertation; Matthew Day, University Representative; Hank W. Bass, Committee Member; Wu-Min Deng, Committee Member; Alice A. Winn, Committee Member.

Competition for shared consumable resources is an important form of competition and has been investigated using mechanistic models of consumer and resource growth. The shape of the relationship between consumption and resource density has traditionally been linear but there are many biological phenomena that can lead to nonlinear relationships between consumption and resource density. This nonlinearity has been shown to have important effects for competition between two consumers for a single limiting resource, yet our understanding of multi species competition for multiple resources has been based mostly on results from models with a linear function describing consumption. The mechanism of coexistence in these models is resource partitioning, a central concept for both competition and coexistence. I evaluate how nonlinear relationships between consumption and resource density affect expectations of coexistence by directly comparing analytical results from MacArthur style consumer-resource models with linear functional responses and nonlinear functional responses. The general concepts defining resource partitioning are the same for all models that I analyze; the ratio of per-capita resource growth must be between the ratios of proportional resource use at equilibrium for each consumer. Nonlinearity affects the way in which consumers draw down resources to equilibrium compared to the linear model. These effects are additive when consumptive rate saturates with total density and both additive and multiplicative when consumptive rate saturates independently on each resource. Interesting and unintuitive cases arise when consumers are limited at high resource densities including negative R*s that lead to coexistence. When consumptive rate saturates with total resource density, inhibition of consumption of one resource by consumption of others can buffer competitive advantages of species and may produce more regions of coexistence than when consumptive rate saturates on each resource independently. Future studies investigating resource competition will need to measure a multidimensional functional response that manipulates both total and individual resource density in a response surface design to uncover possible conditions for coexistence that before this study had previously not been considered. / A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science. / Summer Semester, 2012. / June 19, 2012. / Coexistence, Nonlinearity, Resource Competition, Resource Partitioning, Type II Functional Response / Includes bibliographical references. / Charlotte T. Lee, Professor Directing Thesis; Brian D. Inouye, Committee Member; Thomas E. Miller, Committee Member.

Changes in gene expression determine cellular differentiation and developmental programs as cells transform from a progenitor to a mature adult state. The difference in the organization of the nucleus between undifferentiated cells and their terminally differentiated counterparts, and the possible mechanisms that determine and alter gene expression in the nucleus is an area of intense investigation. Nuclear actin -- which is immunologically distinct from cytoplasmic actin -- has been documented in number of differentiated cell types (Bettinger et al., 2004; Dingová et al., 2009 and Gieni et al., 2009) and cardiac isoforms of troponinI (cTnI) and troponinT (cTnT ) have been detected in association with nuclei of adult human cardiacmyocytes (Bergman et al., 2009 and Kajstura et al., 2010). It is not known whether these and related proteins are present in undifferentiated stem cells, or when they appear in cardiomyogenic cells following differentiation. Using an in vitro primary explant cell culture model, I investigated whether nuclear actin and cardiac isoforms of troponin C (cTnC) and tropomyosin (cTm) are present along with cTnI and cTnT in nuclei of isolated, neonatal rat cardiomyocytes in culture. I developed a cell permeabilization and extraction protocol that enabled an unambiguous determination of the distribution pattern of each of these proteins distinctly in the cell nucleus via immunocytochemistry and confocal microscopy. Adult stem cell cardiomyogenesis remains a precarious process. I developed a reproducible bone marrow stem cell isolation and differentiation protocol to further investigate the presence of these proteins in nuclei of multipotent, bone marrow-derived mesenchymal stem cells from adult rats. I investigated the temporal appearance of cardiac genes and structural proteins in bone marrow stem cells undergoing cardiomyogenesis. Using my permeabilization and extraction protocol, I investigated the presence of nuclear actin, cTnC, cTnI, cTnT and cTm in the nuclei of both ventricular cardiomyocytes and undifferentiated, multipotent BM-MSCs and in BM-MSCs treated to differentiate into cardiomyocytes. The efficacy of adult stem cells is known to be compromised as a function of age. This therefore raises questions about the effectiveness of autologous cell therapy in elderly patients. Using a rodent model, I investigated and showed that there are differences in BM-MSCs associated with the age of the animal from which the cells are isolated. / A Thesis submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Master of Science. / Spring Semester, 2012. / March 15, 2012. / Actin, Cardiac Myocytes, Nucleus, Stem Cells, Tropomyosin, Troponin / Includes bibliographical references. / Bryant Chase, Professor Directing Thesis; Thomas Keller, Committee Member; Paul Trombley, Committee Member.

In order to understand more about plant telomere biology, we set out to identify genetic factors associated with telomere length regulation in maize (Zea mayz L.), a model genetic system. Using terminal restriction fragment (TRF) analysis, we measured telomere lengths from diverse maize inbred lines as well as the recombinant inbred lines (RILs) from the intermated B73 x Mo17 (IBM) mapping population. Maize telomere length was found to be diverse, ranging from 2 kb to more than 20 kb. The telomere lengths of the RILs were subjected to quantitative trait locus (QTL) analysis in order to identify markers co-segregating with telomere length. Significant associations were detected on nine of the ten chromosomes, with two loci showing evidence of an epistatic relationship. QTL intervals were examined for candidate genes involved in telomere metabolism. As a first step in validation of candidate genes, a subset of candidates was assayed for gene expression levels correlating with telomere length. The results of this analysis not only supported predicted functions of some candidate genes, but also indicated that a RAD51-like protein likely interacts with another telomeric protein in some aspect of telomere length regulation in maize. These data open a new avenue for exploring fundamental aspects of telomere biology in plants using the diverse model genetic system, maize. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Fall Semester, 2011. / September 16, 2011. / Aging, maize, plants, QTL, telomerase, Telomere / Includes bibliographical references. / Hank Bass, Professor Directing Dissertation; Cathy Levenson, University Representative; Lloyd Epstein, Committee Member; Laura Keller, Committee Member; Hong-Guo Yu, Committee Member.