This thesis explores the theory that gap junctions play a role in adrenocortical homeostasis and function. To test this hypothesis, gap junction mediated communication was investigated in the intact adrenal gland. In addition, to determine the impact of altered adrenal trophic state on gap junction distribution and expression, adrenal glands from hypophysectomized mice as well as normal and neoplastic human adrenal tissues were evaluated. Gap junction mediated intercellular communication was done in primary mouse adrenal glands using a modified Lucifer Yellow dye communication assay. Dye communication showed that the adrenal cortex was communication competent and that functional cell-cell communication was directly related to the abundance of connexin 43 expression. There was an absence of dye communication in the outer cortical zona glomerulosa (ZG) that was contrasted by a high level of communication in the inner zones of the zonae fasciculata/reticularis (ZF/ZR). The removal of the endogenous pituitary derived ACTH stimulus by hypophysectomy resulted in a significant loss of connexin 43 expression in the ZF/ZR that was associated with a diminished trophic state. The loss of connexin 43 expression was concurrent with features associated with a diminished functional state of the gland including loss of lipid droplets and reduced expression of the ZG specific cytochrome P450 aldosterone synthase (P450 aldo). A comparison of adrenal glands from connexin 43 deficient mice (Cx 43 -/-) demonstrated several features that were similar to those observed in hypophysectomized mice, including glandular atrophy and reduced P450 aldo expression in the ZG. Connexin 43 expression and distribution in the normal human adrenal gland was identical to that previously described in the adrenal glands from other mammals. Connexin 43 expression was abundant in the ZF/ZR and nearly absent from the parenchymal cells of the ZG. Immunocytochemial analysis of neoplastic tissues showed that benign (adenoma) and malignant (carcinoma) neoplasms lost 30% and 90% respectively, of their connexin 43 expression in the ZF/ZR when compared to normal tissues. The data presented here demonstrate that connexin 43 expression is related to physiological changes that affect adrenal trophic state and suggest that gap junction mediated cell-cell communication is involved in the function of the adrenal cortex. Furthermore, because connexin 43 deficient animals also exhibit glandular changes consistent with a diminished state of the adrenal gland, it supports a role for proper gap junction expression in adrenal cortex development and function.

In vertebrates, alternative splicing regulates gene expression in a cell- and developmental stage-specific manner, producing functional diversity of the corresponding proteins. Although its importance has been underscored, control mechanisms of alternative splicing are poorly understood. The N-methyl-D-aspartate receptor NR1 subunit (NMDAR1, GRIN1) contains three cassette exons (NI, CI and CII), and which are specifically expressed in mammalian brain. In this study, a minigene splicing reporter system of the CI cassette exon was utilized to study its splicing silencing mechanism in mammalian cell lines. This work focuses primarily on the identification of exonic UAGG motifs and a 5 splice site region G cluster. Individual motifs play a silencing role but the combination of all three is required for strong silencing. Whereas insertion of an extra UAGG motif in the exon shows almost complete silencing, removal of all three silencers results in loss of silencing. Therefore, the UAGG and G cluster motifs confer flexibility of control of CI cassette exon splicing and the number of the motifs determines the silencing strength in various tissues. The UAGG motif interacts with hnRNP A1 and the strong silencing of hnRNP A1 requires the G cluster. The splicing enhancing role of hnRNP F or H/H is identified, and this effect largely requires the G cluster. Using bioinformatics searches, new groups of skipped exons containing the UAGG and GGGG (as a G cluster) motifs are identified from the human and mouse genomes. This study suggests that hnRNP H1 and H3 (HNRPH1 and HNRPH3) may be auto-regulated at the level of splicing. Overall, this work provides evidence for a splicing silencing mechanism that is important for the tissue-specificity of the CI cassette exon. This work also shows that the motif pattern can be used computationally to identify additional skipped exons that contain combinations of UAGG and GGGG motifs.

Invasive species pose an enormous threat to native species and imposes substantial costs on the US economy. Although the threat of exotic species is well recognized, the general ecological mechanisms that underlie these invasions remain elusive. Predictions both for invasion success and the success of biological control remain poor. In this dissertation, I harness plant competition theory to predict the success of invasions and biocontrol, using a model system composed of invasive purple loosestrife (Lythrum salicaria), native broad-leaved cattail (Typha latifolia), and Galerucella calmariensis, a leaf-feeding beetle widely released to control loosestrife. In chapter 1, I introduce the problem and summarize my results. In chapter 2, I extend resource competition theory to competition for light: species coexistence is possible if one species is sufficiently taller and with less dense foliage than its competitor. This integrated model of competition for light is easily parameterized through measurements of light availability in monoculture and thus can easily be tested in the field. In chapter 3, I test the ability pf three models of plant competition (response to resource availability, plant size, and resource reduction) to predict competitive outcomes between loosestrife and cattail. My experimental design included monoculture mesocosms in which to measure plant traits, and mixture mesocosms in which to determine competitive outcomes. Surprisingly, while loosestrife was, on average, negatively affected by the presence of cattail, cattail was not, on average, negatively affected by loosestrife. Indeed, at high fertility, cattail was strongly negatively affected by loosestrife in the absence of insect herbivores of both species, yet was strongly facilitated when herbivores of both species were present. The facilitation of cattail by loosestrife was likely due to density dependent predation by cattails natural enemies. Cattail abundance in mixture was not predicted by any of the three models, which is not surprising considering the lack of an overall competitive effect of loosestrife on cattail. In contrast, loosestrife abundance in mixture was well predicted by species height, as predicted by the plant size model. These results suggest that competitive traits may predict invasion success and biocontrol, but only when species interact only through competition for resources.

The L1 Ta subfamily of Long INterspersed Elements (LINEs) consists exclusively of human-specific L1 elements with a copy number of ~520 in the human genome. Four hundred sixty-eight L1 Ta elements were extracted from the draft human genomic sequence and screened by polymerase chain reaction (PCR) assays to determine their phylogenetic origin and to determine their contribution to human genomic diversity. PCR analysis indicated that 45% of the L1 Ta elements screened are polymorphic for insertion presence or absence. Sequence analysis of the L1 Ta elements produced definitive evidence of 3transduction, gene conversion involving an older pre-existing L1 element, as well as several potential retrotransposition competent elements. The average age of the L1 Ta subfamily was estimated at 1.99 million years, indicating the subfamilys expansion subsequent to the divergence of humans from African apes. PCR based screening in non-human primate genomes of the orthologous sites for 249 human L1 Ta elements resulted in the recovery of various types of sequence variants for ~12% of these loci. Sequence analysis was employed to capture the nature of the observed variation. Half of the orthologous loci differed from the predicted sizes due to localized sequence variants that occurred as a result of common mutational processes in ancestral sequences, often including regions containing simple sequence repeats. Additional sequence variation included genomic deletions that occurred upon L1 insertion, as well as successive mobile element insertions that accumulated within a single locus over evolutionary time. We estimate the overall frequency of parallel independent insertion events at L1 insertion sites in seven different primate species to be very low (0.52%). In addition, no cases of insertion site homoplasy involved the integration of a second L1 element at any of the loci, but rather largely involved secondary insertions of Alu elements. No independent mobile element insertion events were found at orthologous loci in the human and chimpanzee genomes. Therefore, L1 insertion polymorphisms appear to be essentially homoplasy free characters well-suited for the study of population genetics and phylogenetic relationships within closely related species.

The ghost shrimp, Lepidophthalmus louisianensis, burrows up to meters deep in oxygen-limited marine sediments along the Gulf coast. During low tides these animals are subjected to extended periods of anoxia. The main objective of this study was to assess survival under anoxia and evaluate the physiological mechanisms that underlie the anoxia tolerance of this species. I observed large specimens of L. louisianensis (>2g) having an LT₅₀ of 64 h under anoxia at 25º C. Smaller specimens (<1g) have a significantly higher LT₅₀ of 113 h under identical conditions (P<0.0001). I measured whole body lactate levels in shrimp exposed to anoxia for up to 72 h, and recorded significant accumulation of this anaerobic end product (ANOVA, P<0.001). I also measured adenylates and arginine phosphate in shrimp exposed to anoxia for up to 48 h, and after a 24-h recovery period. Adenylates were not significantly altered during the anoxia regime, and reductions in arginine phosphate occurred after 12 and 24 h, but returned to normoxic values during recovery (ANOVA, P<0.001). While reserves of arginine phosphate are used to some extent to buffer losses in ATP, substantial contribution to the maintenance of energetic status comes from the high rate of anaerobic glycolysis. Energized mitochondria isolated from ghost shrimp hepatopancreas possess a pronounced ability to take up exogenous Ca²⁺ (compared to mitochondria-free controls) as measured by following the external free Ca²⁺ concentration with the fluorogenic dye Fluo-5N. Importantly, Ca²⁺ was not released from the mitochondrial matrix at any level of exogenous Ca²⁺ tested (up to 1.0 mM, in the presence of 5 mM phosphate). Thus, Ca²⁺ does not stimulate opening of the mitochondrial permeability transition pore, which potentially could help prevent apoptotic and necrotic cell death during extended periods of anoxia. (Supported by NIH grant 1-RO1-GM0-71345-01 and by SIGMA XI GIAR).

Reinforcement occurs when natural selection strengthens behavioral discrimination to prevent costly interpopulation matings, such as when matings produce sterile hybrids. This evolutionary process can complete speciation, thereby providing a direct link between Darwins theory of natural selection and the origin of new species. My dissertation presents the first study on the genetics of reinforcement. This study is framed in a conceptual body that explains how genomic architecture, selection and recombination, interact to facilitate divergence in the presence of gene flow. In addition, in my dissertation I produced a dense recombination map for D. pseudoobscura, which together with the genome sequence opens many possibilities for classic population genetic and genomic analyses in this system. I examine a case of speciation by reinforcement in Drosophila. I present the first high-resolution genetic study of variation within species for female mating discrimination that is enhanced by natural selection. I show that reinforced mating discrimination is inherited as a dominant trait, exhibits variability within species, and may be influenced by a known set of candidate genes involved in olfaction. My results show that the genetics of reinforced mating discrimination is different from the genetics of mating discrimination between species, suggesting that overall mating discrimination might be a composite phenomenon, which in Drosophila could involve both auditory and olfactory cues. Examining the genetics of reinforcement provides a unique opportunity for both understanding the origin of new species in the face of gene flow and identifying the genetic basis of adaptive female species preferences, two major gaps in our understanding of speciation.

Studies of speciation in the marine environment have historically compared broad-scale distributions and presumed larval dispersal to infer the geographic barriers responsible for allopatric speciation. However, many marine clades show high species diversity in geographically restricted areas where barriers are not obvious and larval dispersal should bring sister taxa into contact. Genetic differentiation at spatial scales <1000 km could facilitate speciation by mechanisms other than the gradual accumulation of reproductive isolation during extended allopatry, such as ecological adaptation to local environmental conditions or the rapid evolution of genes tied to mate recognition. The role of each of these possibilities has not been simultaneously explored for any species-rich marine taxon. The most species-rich genus of Neotropical reef fishes is Elacatinus (Gobiidae), with 27 species. I examine potential mechanisms underlying this richness through analyses of three genetic markers to investigate genetic and ecological differentiation between closely related taxa and among island populations. Phylogenetic analyses indicate that sister taxa of Elacatinus occur within the same oceans but occupy geographically separate ranges. Sister taxa usually differ by coloration, and distantly related sympatric species frequently differ by habitat. These differences suggest that some combination of coloration and ecological differences may facilitate assortative mating in sympatry or at range boundaries. The ranges of several Elacatinus taxa adjoin at Mona Passage and in the central Bahamas, both in the Caribbean Sea. These boundaries separate island populations by as few as 23 km, yet these populations are genetically distinct. Populations not separated by these breaks also show strong genetic structuring. Coalescent analyses suggest these populations have been demographically closed for up to 800,000 years. Such strong genetic structuring suggests that pelagic larvae are retained at natal populations, despite a three week larval duration (determined from otolith growth rings). My results suggest that local retention of pelagic larvae, coupled with biogeographic breaks, has generated or maintained strong genetic population structure which may facilitate adaptation to local ecological conditions. Such adaptations may explain observed divergence along ecological and coloration gradients. Repeated radiations among allopatrically distributed sister taxa may explain much of the high diversity in Elacatinus.

Bioavailability and nutrient effects on the trophic transfer of Cadmium (Cd) associated with microalgae to the marine amphipod, Leptocheirus plumulosus, were investigated. Cd assimilation efficiencies (AE) were measured by a pulse-chase technique using a radiotracer. Cd AE in L. plumulosus significantly varied among algal species tested, and was highest (38.8 %) for the benthic diatom Nitzschia punctata, lowest (5.9%) for the planktonic diatom Thalassiosira weissflogii, and intermediate (15.6%) for the planktonic dinoflagellate Isochrysis galbana. Instantaneous egestion rates of Cd displayed a typical biphasic pattern over 96 h of depuration. Depuration in seawater-only of L. plumulosus yielded the highest Cd AE of 35.0%, whereas AEs in which depuration occurred in natural sediment and processed sediment were only 5.3% and 4.3%, respectively. Body size, ranging from 0.5 to 2.0 mm, of L. plumulosus feeding on labeled T. weissflogii did not affect Cd AEs. Nitrate enrichment from 0-180 uM on algae significantly increased Cd AEs by L. plumulosus from 9.4-18.8% for T. weissflogii, from 10.0-27.3% for N. punctata, and from 10.0-16.2% for I. galbana. Physiological turnover (elimination) rate constants of Cd in L. plumulosus ranged from 0.016-0.025 h-1 for the three algal species, and were independent of nitrate addition. Algal fractionation revealed that nitrate enrichment strongly enhanced the fraction of Cd associated with cytoplasm, which probably contributed to the increased Cd AEs by L. plumulosus. Phosphate addition (0 - 7.5 uM) on algae showed that Cd AEs of L. plumulosus were from 26.4-35.8% for T. weissflogii, and from 15.3-18.5% for N. punctata. Phosphate enrichment did not significantly affect trophic transfer of Cd from algae to L. plumulosus. Cd fractionation in cytoplasm showed no obvious correlation with phosphate addition. Overall, there was a significant linear relationship between the Cd AE of L. plumulosus and the distribution of Cd within algal cells. My work suggests that eutrophication by nitrate enrichment, but not phosphate, has the potential to enhance the trophic transfer of metals from pelagic and benthic microalgae to grazers in coastal benthic food webs.

I evaluated hypotheses regarding the nature of habitat specialization by comparing the microhabitat distribution and demography of L. cernua and P. basiramia, two Florida rosemary scrub habitat specialist species, with their habitat generalist congeners, L. deckertii and P. robusta. Specifically, I addressed the following two hypotheses: (1) that habitat specialist species may occur in a narrower range of microhabitat conditions than habitat generalist species, and (2) that demographic parameters of habitat specialist species may be more variable than those of their habitat generalist congeners. For each pair of congeners, I compared the microhabitat distributions, variation in vital rates and population growth rates, and extinction probabilities under different climate regimes to evaluate these hypotheses. Both rosemary scrub specialist species occurred in a narrower range of bare sand microhabitat conditions than their habitat generalist congeners. Rosemary scrub specialists were significantly more likely to occur in sites with high percentage bare sand, whereas microhabitats of generalists were more variable with respect to percentage bare sand. Recruitment and survival rates of both rosemary scrub specialist species were more temporally variable than those of their habitat generalist congeners; however, plant growth rates of rosemary scrub specialist species were less variable than those of their generalist congeners. Rosemary scrub specialist species also exhibited greater temporal variation in population growth rates than their habitat generalist congeners. Both rosemary scrub specialist species had higher probabilities of quasi-extinction than their generalist congeners under every climate modeling scenario. The narrower microhabitat requirements and greater temporal variability of demographic parameters of L. cernua and P. basiramia distinguish them from their habitat generalist congeners. The restriction of P. basiramia and L. cernua to microhabitats with high percentage bare sand may limit their distribution to rosemary scrub habitat. Greater temporal variability in recruitment, survival, and population growth rates in L. cernua and P. basiramia may be associated with specialization on a narrower range of environmental conditions in these rosemary scrub specialist species. Greater temporal variability of demographic parameters in these rosemary scrub specialist species may make them more vulnerable to extinction than could be predicted solely from availability of suitable rosemary scrub habitat.

The limiting factor involved in past assessments of soil bacterial diversity when using culture-independent techniques has often been the lack of sampling and replication. As a result, analyses of community structural shifts across soil environments have lacked statistical power. In this study, 23 16S rRNA gene clone libraries consisting of over 11,000 clones were constructed from soils at the Central Arizona Phoenix Long Term Ecological Research (CAP LTER) site. Subsequent ARDRA fingerprinting and partial 16S rRNA gene sequencing allowed for a more robust investigation of various components that may explain any observed variations in bacterial species composition. The designated land use type of the soils best explained the overall diversity gradient. Based on Simpsons reciprocal index, diversity was found to significantly increase when comparing urbanized and agricultural soils to open desert samples located outside the metropolitan region. Land use type appears to be a powerful indicator of overall diversity due to irrigation methods that differ greatly across land use types. Experiment-wise comparisons of complete CAP LTER clone libraries via the LIBSHUFF method yielded no statistical similarity in sequence libraries, except for two replicate libraries constructed from one urban soil. However, inter-phylum LIBSHUFF analysis of the clones also shows degrees of phylogenetic partitioning between land use categories and that open desert remnant patches located within the city limits more closely resemble those urban soils than the open deserts outside of Phoenix. Examination of constructed 16S rRNA phylogenetic trees that include CAP LTER phylotypes indicate some distinct clustering of sequences appears to be driven by land use type rather than geography, and that most of these groups may be endemic to the region. However, some ubiquitous phylotype groups were discovered and were used as templates for specific PCR primer design, allowing for the detection of ten of these groups in all soil samples analyzed. Overall, this study suggests that anthropogenic factors have altered soil bacterial communities, the biogeography of many species is controlled in some manner by land use type, and that a small subset of taxa is ecologically tolerant despite the heterogeneity of habitats within the site.