Endothelin 3 (Edn3) is a ligand important to developing neural crest cells (NCC). Some NCC eventually migrate into the skin and give rise to the pigment-forming melanocytes found in hair follicles. Edn3’s effects on NCC have been largely explored through spontaneous mutants and cell culture experiments. These studies have shown the Endothelin receptor B/Edn3 signaling pathway to be important in the proliferation/survival and differentiation of developing melanocytes. To supplement these investigations I have created doxycycline-responsive transgenic mice which conditionally over-express Edn3. These mice will help us clarify Edn3J3 role during the development of early embryonic melanoblasts, differentiating melanocyte precursors in the skin, and fully differentiated melanocytes in the hair follicle. The transgene mediated expression of Edn3 was predominantly confined to the roof plate of the neural tube and surface ectoderm in embryos and postnatally in the epidermal kératinocytes of the skin. Relative to littermate controls, transgenics develop increased pigmentation on most areas of the skin. My doxycycline-based temporal studies have shown that both embryonic and postnatal events are important for establishing and maintaining pigmented skin. The study of my Edn3 transgenic mice may offer some insight into the genetics behind benign dermal pigmentation and offer clues about the time periods important in establishing these conditions. This apparently abnormal development is echoed in a benign condition of human skin. Cases of dermal melanocytosis, such as common freckles, Mongolian spotting, and nevus of Ito demonstrate histological and etiological characteristics similar to those of the transgenic mice generated in this study.

Canavan disease (CD), an autosomal recessive leukodystrophy, is caused by the deficiency of aspartoacylase {ASPA}. The human ASPA cDNA spanning 1,435 bp has been isolated and characterized. The single uninterrupted ORF in the cDNA predicted a 313 amino acid long protein. The authenticity of the cDNA has been established by its expression in E. coli and Cosl-cells. Human ASPA gene was also cloned and found to span 29 kb of the human genome. Human ASPA is coded by 6 exons intervened by 5 introns. The exon/intron splice junction sites follow the 'gt'/'ag' consensus sequence rule. The human ASPA gene was assigned to the 17pl3-ter region. Human ASPA coding sequences were demonstrated to be conserved in yeast, chicken, rabbit, cow, dog, mouse, rat and monkey. Sixty-four probands (or 128 chromosomes) with CD were analyzed for mutations in the ASPA gene. Four point mutations have been identified in Canavan alleles. The 693OA and 914C>A base changes result in non-sense tyr231>ter and missense ala305>glu mutations respectively, that lead to complete loss of ASPA activity. The 854A>C transversion resulted in a glu285>ala missense mutation, and the mutant ASPA has 2.5% of the activity expressed by the wild type enzyme. The 433-2(A>G) transition at the splice acceptor site in intron 2 would lead to skipping of exon III, accompanied by a frameshift in the final ASPA transcript. Of the 128 unrelated Canavan chromosomes analyzed; 88 were from probands of Ashkenazi Jewish descent and 40 were from non-Jewish probands. The glu285>ala, tyr231>ter and 433-2(A>G) mutations account for 98.8% of the Canavan chromosomes of Ashkenazi Jewish origin. The ala305>glu mutation was found exclusively in non-Jewish probands and constituted 60% of the 40 mutant chromosomes. These results provide the basis for studying epidemiology of CD in at-risk populations; offer a DNA-based pre- and postnatal diagnosis of CD; provide the possibility to create an animal model of CD for understanding its pathophysiology and to develop strategies for possible enzyme and gene therapy.

The role of omnivores in structuring communities is poorly understood. I studied the effect of two abundant omnivores, grass shrimp (Palaemonetes paludosas) and eastern mosquitofish (Gambusia holbrooki), on periphyton biomass of the Florida Everglades. I performed field experiments to test for consumer top-down and “complex” top-down effects on periphyton biomass. My experiments suggested that shrimp and mosquitofish had consumptive effects on periphyton but in many instances, periphyton wet weight, AFDM, and chlorophyll a increased significantly with shrimp or fish density, suggesting compensation by nutrient regeneration or trophic cascade processes. I propose that characteristic periphyton mat structure and integrity deters herbivory and affects the outcome of the periphyton-consumer interaction. Results from a descriptive study and a laboratory experiment support this hypothesis. Overall, consumption by shrimp and mosquitofish was significant, but coupled with and sometimes compensated by “complex” top-down effects, making these consumers “functional” omnivores.

The purpose of this study was to determine the reproductive biology of Ruellia succulenta and to relate this to the effects of habitat fragmentation. Plants occurring in the intact habitat in Everglades National Park were compared to plants occurring in three different size classes of habitat fragments. The results of the breeding system experiment show R. succulenta to be fully selfcompatible and capable of autofertility via corolla abscission. Results of the inbreeding depression study supported theoretical expectations for selfing species of reduced inbreeding depression and its expression late in the life cycle. The most significant effect of habitat fragmentation, in terms of pollination ecology, was a highly significant difference in the proportion of Hymenopteran and Lepidopteran floral visitors by the size class of habitat. Time since last fire was the most important factor positively affecting reproduction in the different size classes of fragments.

A plant’s reproductive biology exerts a significant influence on both population persistence within changing environments and successful establishment of new populations. However, the interaction between extrinsic (i.e. ecological) and intrinsic (i.e. genetic) factors also is an important driver of demographic performance for plant populations. It is light of this that I performed a multidisciplinary investigation of the breeding system, seed and seedling establishment dynamics, and population genetic structure of the endangered Caribbean vine Ipomoea microdactyla Griseb. (Convolvulaceae). The results from the breeding system study show individuals from Florida, USA and Andros Island, Bahamas to be self-incompatible. Plants from the two regions are cross-compatible but there is evidence for outbreeding depression in their progeny. Significant regional differences were found in floral traits and progeny traits that suggests incipient speciation for the Florida populations. The results from the seed and seedling establishment dynamics experiment demonstrate that the restoration of small populations in Florida via seed and seedling augmentation is a successful strategy. The vi demographic performance of the outplanted individuals was driven significantly by ecological factors (e.g. herbivory) rather than by genetic factors which emphasizes that the ecological context is very important for successful restoration attempts. The results from the population genetic study using an analysis of molecular variation (AMOVA) reveal significant differences in genetic variation among individuals from Florida, Andros, and Cuba. A Bayesian analysis of population genetic structuring coincided with the previous AMOVA results among the three regions. The Mantel test indicated significant ‘isolation by distance’ for these regional populations implying restricted gene flow over relatively short distances. Overall, the Florida populations had the lowest measures of genetic diversity which is most likely due to the effects of both colonization founder events and habitat fragmentation. The results of my study highlight the value of performing multidisciplinary studies in relation to species conservation as knowledge of both extrinsic and intrinsic factors can best guide decisions for species preservation.

Capillary electrophoresis is currently a very powerful technique for the analysis of seized drugs. A rapid analytical CE method for the screening and quantification of GHB and GBL was achieved using 300mM CTAC/25mM phosphate buffer pH 6.3. Reversed phase HPLC was achieved using 25mM phosphate buffer pH 6.5 and a Cl8 Aqua column. Chiral separation of 9 amphetamine type stimulants was obtained using a highly sulfated gamma-cyclodextrin as a chiral selector. MECC and CZE were compared for the analysis of psilocybin, while a rapid and robust method is presented for the analysis of major opium alkaloids, using dynamically coated capillary columns. The column is coated with a polycation followed by a polyanion coating, using a commercial reagent kit. Using a background electrolyte pH of 2.5 with the addition of hydroxypropyl-beta- cyclodextrin and dimethyl-beta-cyclodextrin, the analysis of morphine, papaverine, codeine, noscapine, and thebaine in opium samples was obtained with great resolution. Finally, separation of common benzodiazedpines was also investigated using CZE and a pH 2.5 phosphate buffer.

The factors governing flavor and body and texture in cheese making include the quality of the milk used, the heat treatment of the milk, the quality and quantity of the starter, the treatment during the ripening period, other biological conditions and manufacturing technics.

Effects of sublethal, cerebral irradiation on movement and home-range patterns of black-tailed jackrabbits were studied in Curlew Valley, Utah, using radio-telemetry. Irradiation of 70 captive animals indicated that the LD50(30) was between 5,556 and 6,200 roentgens. Nine wild, free-living experimentals were trapped in desert terrain, irradiated, transmittered, and released at the capture sites. Seven wild controls were treated similarly but were not irradiated. The field-irradiation dosage was 5,000 roentgens. Tracking accuracy was determined by telemetering transmitters at fixed locations. Mean hourly movement was measured within 20-30 percent error and home ranges were measured with an error of less than 22 percent. Experimentals had a mean hourly movement of 1,176.8 feet and controls 980.0 feet, significantly different at the .05 probability level. Experimentals had a bimodal activity curve with peaks at 5:00 p.m. and 3:00 to 5:00 or 6:00 a.m. Controls displayed no such pattern. Experimentals had a mean, daily home range of 66.1 acres and controls 34.1 acres, significantly different at the .05 probability level. Experimentals had a seasonal home range of 279.0 acres and controls 247.0 acres, not significantly different at the .05 probability level. A probability index showing the frequency distribution of each animal's activity within 300-foot concentric, circular bands around a geometric center of activity showed similar distributions for both groups. The greatest concentrations of activity were within the innermost band for each group but experimentals had a slightly greater scatter of points in the outermost zone. These distributions were not significantly different at the .05 probability level. Sublethal, cerebral irradiation appears to have increased activity levels of experimental animals but not changed those home-range characteristics involving the total area occupied and tenacity of site attachment. This increased activity may have resulted from inhibitory areas in the cortex which permitted greater expression of activity from the limbic system.

The scientific balancing of diets has resulted in a series of studies to determine a standard for amino acid adequacy of a chick ration. These studies have shown that some amino acids are required in a chick diet more than others; they are termed "essential amino acids". Proteins used in formulating rations vary widely in essential amino acid content. Because of this variation, it can no longer be said that a diet with so much protein (N x 6.25) is adequate to meet the chick's need for optimum performance. Many papers have been published showing the results of experiments for determining the minimum levels of amino acids required in a chick's ration.

The effects of vegetation-manipulative practices on habitat use by sharp-tailed grouse (Pedioecetes phasiane11us columbianus) were studied during the period 1974-1975 in Curlew National Grasslands, Oneida County, Idaho. Vegetational transects were run twice each month during the growing season of May-August, 1974-1975, to assess vegetational differences between treatments. Transects were run ~ to estimate sharp-tailed grouse use on the eight study areas .twice monthly. Vegetational plots were used at each sharp-tailed grouse observation site to determine correlates of vegetation types and grouse use. Chained areas were used heaviest by grouse during the study period suggesting that chaining created the most preferred habitat. Cover, edge, and slope were also important factors at sites of sharp-tailed grouse occurrence. Recommendations are made on manipulative methods and other means to improve sharp-tailed grouse use of habitat.