The majority of retrotransposons, mobile elements which move around the genome using an RNA intermediate, insert into their host genomes using target-primed reverse transcription (TPRT). Two of the most well-studied types of active retrotransposons in primates are L1s (Long Interspersed Element-1) and Alu elements. Both preferentially insert using TPRT, and these insertions can create genomic rearrangements and contribute to genome fluidity. Recent analyses have shown that L1s and Alu elements can insert using a variety of non-canonical mechanisms, including a DNA double-strand break repair pathway. Increased understanding of the mechanisms by which mobile elements insert into host genomes can help us examine why they are tolerated. We surveyed non-canonical insertions using the human, chimpanzee, orangutan, rhesus, and marmoset genomes. Using both computational data mining and experimental verification, we have attempted to provide clear examples of the different mechanisms for these insertions and discuss their implications. In the first analysis, we assessed 23 non-classical Alu element insertions into primate genomes. These insertions left characteristic atypical sequence hallmarks since they did not use the typical L1 endonuclease cleavage site to insert into the host genomes. Mobile elements are largely considered disruptive to genomes, creating instability, but also generating diversity. In relatively rare cases, such as non-classical insertions, mobile elements may play a positive role in genomic stability by patching DNA double-strand breaks. Next, we examined both L1 and Alu elements in the context of internally primed insertions, resulting in characteristics similar to, but distinguishable from, classical TPRT. These twenty insertions provided support for the suggested lack of fidelity attributed to reverse transcriptase. We then characterized thirty-nine loci in our third analysis, which appear to have resulted from a variant of twin priming, itself a permutation of classical TPRT. The mechanisms by which mobile elements insert can offer insight on how mobile elements evade host defenses. Though this research is limited to primate genomes, the resulting understanding of the mechanisms at work is applicable to retrotransposons in general.

Current regulatory testing provides the basis for determining acceptable levels of pollutants in the environment, yet these acceptable levels of contaminants have resulted in undesirable consequences to organisms. The purpose of this dissertation was to test the hypothesis that biomarkers of cellular stress could be detected from sub-lethal exposure to pesticides in sperm and early life stages of broadcast spawning invertebrates. Exposures were conducted on oyster (Crassostrea virginica), mussel (Mytilus galloprovincialis and Dreissena polymorpha), and sea urchin (Lytechinus variegatus) embryos and larvae for 4 and 24 h to Bayluscide® and Roundup®. DNA fragmentation, a characteristic of apoptotic cells, was detected with the TUNEL method and an ELISA. Hsp70 expression was detected with Western blotting and quantified with densitometry. Sperm were exposed to the pesticides for 20 min and analyzed for cellular effects using flow cytometry. Apoptosis levels often revealed a bell-shaped dose response in which there was a threshold concentration that elicited a change from apoptotic induction to necrosis. This change suggests irreversible damage to the organism has occurred and it is no longer using apoptosis as a defensive mechanism. The apoptotic results also revealed differential response levels among species despite very similar developmental stages. Hsp70 isoform expression was variable in controls and treatments of the majority of exposures. Therefore, it was concluded that this biomarker is unsuitable for use in early life stages of these species. Flow cytometric analyses of sperm viability biomarkers revealed that MitoTracker® was a reliable indicator for detecting changes in mitochondrial membrane polarization from Bayluscide® exposures, FITC-peanut agglutinin (PNA) reported acrosome reaction in two test species after Roundup exposures, and the SYBR®-14/propidium iodide (PI) assay only detected compromised membranes with Roundup® exposures as PI did not bind in the presence of Bayluscide®. If the damage incurred at these stages does translate to lower fertilization success and abnormal development, then it is probable that reproductive competence will also be affected. Once long-term effects are established, detecting damage to sperm and early life stages can provide insight into the sub-lethal concentrations that may seemingly appear safe for an organism but can potentially pose serious risks to the population.

Small scale heterogeneity in fire severity is important in pine flatwood communities of southeastern United States. Heterogeneity in fire severity, in turn, is important because if produces heterogeneity of vegetation in these habitats. By measuring fuel scorch and consumption immediately after a fire, I documented small-scale heterogeneity of fire severity. I found that pre-fire fuels and management had significant effects on fire severity, whereas weather did not. Weather, however, demonstrated variation over the 2007 fire season and is clearly a primary driver of fire behavior and effects in natural fire regimes (Johnson 1992). I attribute the lack of weather influence on fire severity to mainly to management practices within the Avon Park Air Force Range. The prescribed fire regime at the APAFR consists of two conflicting goals: 1) ecosystem integrity and 2) control. Fire managers burn particular areas under particular weather conditions to reduce the potential for spotting or jump fires. Additionally, outside agencies with other objectives often dictate when prescribed fires can occur. In particular, state forestry agencies often impose burn bans precisely when natural, lightning initiated fires historically occurred and produced ecologically sound effects. My study suggests that the range of fire severity seen in a natural fire regime is greatly reduced in a prescribed one. This effect may reduce species diversity by reducing opportunities for recruitment, removal of competitive dominants, and encroachment by plants that are not fire tolerable.

Mammalian nucleostemin (NS) is a nucleolar GTP-binding protein implicated in cell cycle progression, stem cell proliferation, and ribosome assembly. Drosophila melanogaster contains a four-member nucleostemin family (NS1-4). Nucleostemin 1 (NS1) is the closest orthologue to human NS; it shares 33% identity and 67% similarity with human NS. We show that NS1 has intrinsic GTPase and ATPase activity, and that it is present within nucleoli of most larval and adult cells. Endogenous NS1 and lightly expressed GFP-NS1 enrich within the nucleolar granular regions as expected, while over-expressed GFP-NS1 localizes throughout the nucleolus and nucleoplasm, and to several transcriptionally active inter-bands of polytene chromosomes. Over-expression caused melanotic tumors and larval and pupal lethality. RNAi depletion of NS1 caused a loss of imaginal (precursor) cells in the larval midgut, and an apparent block in the nucleolar release of large ribosomal subunits in the terminally differentiated larval midgut polyploid cells. Depletion of 60% of NS1 transcripts lead to larval and pupal lethality. Ultra-structural examination of highly differentiated larval Malpighian tubule cells depleted for NS1 showed a loss of cytoplasmic ribosomes with a concomitant appearance of cytoplasmic pre-autophagosomes. We interpret the appearance of these structures as indicators of cell stress response.

Geographic range differences among species may result from differences in their physiological tolerances. In the intertidal zone, marine and terrestrial environments intersect to create a unique habitat, across which physiological tolerance strongly influences range. The snail Melampus bidentatus occurs in coastal salt marshes in the western Atlantic and Gulf of Mexico. I have used sequence data from one mitochondrial (CO1) and two nuclear markers (histone H3 and a mitochondrial carrier protein, MCP) to identify three cryptic species within Melampus bidentatus, and to infer their relationships to other Melampus. To identify microhabitat differences between two cryptic species, I modeled their distributions using both marine and terrestrial environmental data. Temperature largely explained their range differences, but other environmental components (precipitation, salinity, and tidal height) explained facets of the range that temperature cannot. To test for phylogenetic conservation in freeze tolerance, I tested the mean lower lethal temperature (LT50) of three temperate and three tropical species. Cryptic species of M. bidentatus are significantly more freeze tolerant than their tropical relatives, although there was variation among locales within species, most likely due to microhabitat variation. The temperate species M. floridanus was also freeze tolerant, but without testing the LT50 of its closest relatives in the Pacific, I cannot determine this represents an independent evolution of freeze tolerance, or if this trait is more widely shared among Melampus. Nonetheless, the lack of freeze tolerance in the most basal species that I have tested (M. bullaoides), and the predominantly tropical distributions of most ellobiids, suggests that the evolution of freeze tolerance has allowed for the invasion of the temperate zone by Melampus. Using massively parallel sequencing, I have isolated > 500,000 expressed sequence tags and assembled these into ~20, 000 seasonally expressed transcripts. A comparison of these transcripts has revealed 2 candidate markers to test for their association with freeze tolerance in M. bidentatus, and many more markers that can be used for further phylogenetic analyses in Melampus. Further work to examine variation in both the sequence and expression of these proteins is needed to determine if they underlie adaptive differences among species.

Although birds of the forest canopy are an important component of tropical forest ecosystems, difficulty accessing the forest canopy has limited the advancement of knowledge pertaining to this group of species. Here I test methods for the study of canopy birds in lowland Neotropical rainforests, and identify recurring patterns of community structure in canopy bird assemblages as well as processes potentially responsible for these patterns. I used three methods to assess differences in ground-based and canopy-based methods for detecting forest birds in a 100-ha plot of lowland rainforest in northern Honduras: (1) point counts from the ground; (2) repeat censuses from two canopy trees; and (3) single censuses from multiple canopy trees. Ground methods significantly underestimated species and familial richness as well as abundances of individuals in the canopy stratum, and I predict that ground methods miss 25 to 50% of the species richness for some migrant and resident families and underestimates the density of some species by as much as 25%. I compared two distant canopy bird assemblages based on >11,000 detections at lowland rainforest sites in Honduras and Amazonian Brazil. Richness of canopy birds was similar between sites, despite overall higher forest bird richness in Brazil. Honduras and Brazil differed significantly in abundance distributions, with greater evenness characterizing the Brazil assemblage. Long-distance migrants and species of forest edges and open habitats were underrepresented at both sites when compared to null expectations drawn from regional species pools. Long-distance migrants were relatively more important in Honduras, where they constituted a third of canopy birds. Species richness of omnivores amongst core canopy species was greater than expected at both sites, and omnivores dominated the canopy in terms of species richness and individual abundance. Functional morphology analyses indicated that core canopy birds are more similar phenotypically than would be expected by chance. Similarity in functional morphology may result from environmental filtering selecting for phenotypes optimally suited for survival in the harsh canopy environment. Phylogeny seems to be an important underlying component of morphological similarity, however, and may exert a structuring force on the canopy bird assemblage through phylogenetic niche conservatism.

Transcription factors are important for many aspects of gene regulation in eukaryotes. YY1 (Yin-Yang 1) is a particularly interesting example of a highly conserved zinc-finger transcription factor, involved in transcriptional activation, repression, initiation, and in chromatin modification. YY1 is ubiquitously expressed in mammals, and its binding sites are found in ~10% of human genes as well as in repetitive elements. It is a targeting protein of the Polycomb complex and is involved in mammalian genomic imprinting. First, we explored the evolutionary history of YY1 using 62 species and formation of its paralogs, YY2 and REX1, which are found in mammals, and Pho and Phol, which are found in Drosophila. We confirmed the specificity of the consensus YY1 binding site and the differences of the target binding motifs of YY2 and REX1 which are reflected in their amino acid sequences. We found that the core motif, CCAT, is conserved for all three homologs and that YY2 and REX1 were produced via retrotransposition events early in the mammalian lineage. Second, we identified unusual clusters of YY1-binding motifs found in the coding regions of olfactory receptor genes (OLFRs) in mammals but not in fish. Olfactory genes provide scent detection and are the largest class of genes in mammals. Statistical analysis indicates that the core of the YY1-binding motifs cannot be acounted for by conserved amino acid motifs or overall protein homology. Thus selection has acted at the DNA level rather than at the protein level in preserving these YY1-binding sites within coding regions. Therefore, YY1 is likely to play a crucial role in regulating the expression of OLFRs. Third, we produced a new method of microarray data analysis predicated on the positions of genes along a chromosome as well as their expression levels. This technique is supplementary to traditional microarray data analysis and adds a new dimension to finding target genes of interest by looking for co-regulation. Overall, this work provides a coherent background to the evolution of YY1 and its homologs. It provides strong evidence that coding sequences of genes can encode information both at the DNA level and the protein level.

Integrin conformational changes mediate integrin activation and signaling triggered by intracellular molecules or extracellular ligands. Even though it has been shown that TM and/or cytoplasmic α and β domains associate in the resting state and separation of these domains is required for integrin signaling, it is still not clear how this signal is transmitted from the transmembrane domain through two long extracellular legs to the ligand-binding headpiece. In addition, integrin TM homomeric association was also observed. But the role of this interaction remains elusive. In this work, the platelet integrin, αIIbβ3, has been used to elucidate the roles of integrin lower leg and TM homomeric association in integrin signalling. We first addressed whether the separation of integrin αβ lower leg is critical for integrin activation and outside-in signaling. Using a disulfide bond to restrict dissociation of the α-subunit Calf-2 domain and β-subunit I-EGF4 domain, we were able to abolish integrin inside-out activation and outside-in signaling. In contrast, disrupting the interface by introducing a glycosylation site into either subunit activated integrins for ligand binding through a global conformational change. Our results suggest that the interface of the α-subunit Calf-2 domain and β-subunit I-EGF4 domain is critical for integrin bidirectional signaling. Formation of the TM homooligomers was observed in micelles and bacterial membranes previously, and it has been proposed that this homomeric association is important for integrin activation and clustering. We then addressed whether integrin TM domains form homooligomers in mammalian cell membranes using cysteine mutagenesis scanning method. Our results show that TM homomeric interaction does not occur before or after soluble ligand binding, or during inside-out activation. In addition, even though the cysteine mutants and the heterodimeric disulfide-bounded mutant could form clusters after adhering to immobilized ligand, the integrin TM domains do not form homooligomers, suggesting that integrin TM homomeric association is not critical for integrin clustering or outside-in signaling. Therefore, the integrin TM homooligomerization is not required for integrin activation, ligand binding and signaling.

Ecological mechanisms proposed to explain community assembly and the maintenance of biodiversity are hypothesized to fall along a theoretical continuum bounded at one extreme by deterministic processes (niche assembly) and at the other extreme by stochastic processes (dispersal assembly). In this dissertation, I explore the idea that the position of ecological communities along the niche-dispersal assembly continuum is dynamic in space and time. Using field experiments in a high-diversity longleaf pine savanna, I test the general hypothesis that ecological filters (competition, disturbance, and resource availability) contribute to niche assembly through their effects on established plant species and recruitment from the species pool. Consistent with dispersal-assembly theory, I found that dispersal from the species pool strongly limited local species diversity regardless of the presence of these three niche-based ecological filters. Importantly, however, some ecological filters (e.g., space limitation in communities with low-intensity fire disturbance and establishment limitation imposed by drought and high-rainfall conditions) limited the extent to which community assembly was influenced by dispersal, suggesting ecological conditions that reduce stochastic community assembly in high-diversity communities. I examined the generality of these patterns by conducting a meta-analysis of >60 published experiments. I found that dispersal strongly limited species richness in a wide range of plant communities, but that dispersal had a stronger positive effect on species richness in more disturbed communities and when the species pool contained high species diversity and functional-trait diversity, supporting the hypothesis that community assembly reflects a dynamic interplay between species-pool diversity and local environmental heterogeneity. My results suggest a conceptual model for community assembly in high-diversity pine savannas, with implications for other species-rich plant communities. I propose that characteristics of high-diversity communities (large species pools and pervasive recruitment limitation in populations of many rare species) generally contribute to stochastic community assembly, but that niche-based ecological filtering of resident species and immigrating species can shift high-diversity communities towards more deterministic community assembly. This conceptual framework has broader implications for understanding the maintenance of biodiversity and species coexistence in communities of contrasting diversity and for conserving biodiversity in longleaf pine communities threatened by habitat loss, fragmentation, and environmental change.

Ludox density gradients were used to enrich for Escherichia coli mutants with conditional growth defects and alterations in membrane composition. A temperature-sensitive mutant named Lud135 was isolated with mutations in two related, nonessential genes: yghB and yqjA. yghB harbors a single missense mutation (G203D) and yqjA contains a nonsense mutation (W92TGA) in Lud135. Both mutations are required for the temperature-sensitive phenotype: targeted deletion of both genes in a wild-type background results in a strain with a similar phenotype and expression of either gene from a plasmid restores growth at elevated temperatures. The mutant has altered membrane phospholipid levels, with elevated levels of acidic phospholipids, when grown under permissive conditions. Growth of Lud135 under nonpermissive conditions is restored by the presence of millimolar concentrations of divalent cations Ca2+, Ba2+, Sr2+, or Mg2+ or 300 to 500 mM NaCl but not 400 mM sucrose. Microscopic analysis of Lud135 demonstrates a dramatic defect at a late stage of cell division when cells are grown under permissive conditions. Lud135 is non-motile and overproduces outer membrane vesicles that contain FliC. Preliminary results from the topological analysis of YqjA suggest it contains 4 transmembrane domains with a large cytoplasmic domain that is enriched in positive amino acids. yghB and yqjA belong to the conserved and widely distributed dedA gene family, for which no function has been reported. The two open reading frames encode predicted polytopic inner membrane proteins with 61% amino acid identity. It is likely that YghB and YqjA play redundant but critical roles in membrane biology that are essential for completion of cell division in E. coli.