I have characterized chaperone requirements for the biogenesis, maturation, and degradation of a cytosolic substrate, firefly luciferase (FFLux), in yeast, and of an integral membrane protein, cystic fibrosis transmembrane conductance regulator (CFTR), in yeast and in mammals. It was previously demonstrated that the cytoplasmic Hsp40, Ydj1p, is required for efficient expression of FFLux in yeast. This raised the question whether two Ydj1p-interacting molecular chaperones, the yeast Hsp70, Ssa1p, and the yeast Hsp90, Hsp82, also impact FFLux expression. The possible influence of a nucleotide exchange factor for Ssa1p, Fes1p, was also investigated. I found that the chaperone requirements for FFLux biogenesis are distinct but overlapping. Whereas Ssa1p and Fes1p likely collaborate to fold FFLux, Ssa1p, independent of its nucleotide exchange factor, was necessary for stabilizing FFLux protein and message, and for efficient induction of FFLux mRNA. Therefore, Fes1p impacts only a subset of Ssa1p¡¦s actions. Although FFLux folding progresses independent of Hsp82, efficient expression of FFLux depends on Hsp82, mainly due to Hsp82¡¦s contribution to FFLux translation. To identify the complete spectrum of chaperones that affect ER associated degradation (ERAD) of CFTR, I took a genomic approach in yeast. Transcriptional profiles between yeast expressing CFTR and control strains were examined by microarray analysis. Among the genes up-regulated in strains expressing CFTR was one encoding a small heat shock protein (sHsp), HSP26. Therefore, I investigated CFTR degradation in yeast strains lacking HSP26 and found that the protein was stabilized; stabilization was enhanced in a strain lacking both HSP26 and another sHsp-encoding gene, HSP42. In contrast, degradation of a soluble ERAD substrate and of another transmembrane protein proceeded with equal efficiency in wild type and in hsp26hsp42 mutant yeast. Next, I examined whether sHsps regulate CFTR biogenesis in mammalian cells. I found that ´F508-CFTR degradation was enhanced when ÑA-crystallin was over-expressed in HEK293 cells, although wild type CFTR biogenesis was unaffected. To examine why this sHsp accelerated degradation of ´F508-CFTR, ÑA-crystallin was purified and I found that it was able to suppress aggregation of CFTR¡¦s first nucleotide binding domain. Together, these results suggest that sHsps increase ´F508-CFTR¡¦s accessibility during proteasome-mediated degradation.

In forest communities, the composition and abundance of plants in the understory is generally mediated via a complex interplay between herbivory and competition. Nonetheless, most research assesses the effect of herbivory and competition independently and rarely investigates potential interactions between the two factors. In this dissertation, I examine how herbivory and competition, alone and in concert, influence forest understory plant diversity. At one extreme, herbivory is hypothesized to reduce interspecific competition, thus allowing for greater species coexistence in the understory. In chapter one, I test this hypothesis in the herbaceous community of a tropical forest in central Panamá. I found mammals limited the abundance of the dominant herbs while simultaneously limiting the establishment of rare species. In contrast, intense herbivory may reduce the diversity of the most palatable species allowing browse-resistant species to rapidly expand and overtake the forest understory. In chapter two, I explore this alternative by reviewing the processes leading to the formation of recalcitrant understory layers worldwide and summarizing the mechanisms by which these layers inhibit tree seedling regeneration and alter forest succession. In chapter three, I experimentally examining how a recalcitrant understory layer limits tree species recruitment via direct competitive as well as indirect, apparent competitive interactions. Specifically, by providing a favorable microhabitat for small mammals, a dense understory canopy can facilitate increased granivory, thereby indirectly limiting tree recruitment. I found a dense hay-scented fern (Dennstaedtia punctilobula) understory exerts strong apparent competitive effects on the emergence of black cherry (Prunus serotina) and strong competitive effects on emergence and survival of red maple (Acer rubrum). In chapter four, I explore the potential interactions between white-tailed deer (Odocoileus virginianus) and a dense canopy of hay-scented fern. This work demonstrates that in light limited, closed-canopy forests, a dense understory layer strongly suppresses germination and survival of several shade intolerant tree species. Furthermore, I argue that century-long legacy of deer overbrowsing has resulted in a depauperate forest community that predisposes future declines in plant diversity and increased monodominance. In all, these findings stress the need to discriminate among multiple ecological factors and assess their relative importance in structuring natural communities.

Given the prevalence of intermediate levels of outcrossing among angiosperms, a general mechanism explaining the evolution and maintenance of this condition is needed. Although numerous theoretical models predict mixed mating to be evolutionarily stable, the conditions favoring intermediate selfing are often very stringent and have limited applicability. Here I investigate the role of two plant antagonists, vegetative herbivores and intraspecific competitors, in influencing the mixed mating system of Impatiens capensis (Balsaminaceae). This species exhibits an obligate mixed mating system by producing heteromorphic flowers (i.e., individuals produce both selfing, cleistogamous and facultatively-outcrossing, chasmogamous flowers). Thus, these antagonisms may affect mating system at the level of relative heteromorphic flower production, outcrossing within chasmogamous flowers and whole-plant outcrossing. In a comparative study exploring how herbivory and intraspecific competition jointly affect mating system expression, I found that these antagonisms affect plant growth and mating system traits differently, and thus the mating system response could not be accurately predicted from plant growth response. Using surveys of wild populations and experiments manipulating herbivory under field conditions, I found that herbivory reduced outcrossing by increasing proportional cleistogamous reproduction. In the field, I found that herbivory increased outcrossing among chasmogamous flowers due to effects on flower display, pollinator visitation rate and pollinator fauna composition. Overall, herbivory slightly lowered whole-plant outcrossing. To understand further the consequences of mixed mating, I manipulated herbivory in two wild I. capensis populations to explore the demographic consequences of mixed mating, herbivory, and the interactive effects of mating system and herbivory. I found that selfed individuals had higher rates of germination and survival and lower fecundity than did their outcrossed counterparts. Herbivory also had demographic consequences as it reduced population growth rate due to its effect on vital rates of selfed individuals. Overall, the results presented in this dissertation offer important insight to the ecological factors that cause variation in mating system as well as the long-term consequences of variation in mating patterns. Furthermore, these findings have implications for population genetic diversity and structure and point to the role of natural enemies in contributing to the maintenance of a mixed mating system.

During development, gradients of transcription factors regulate patterning along a body axis by activation or repression of target genes above distinct threshold levels. In the Drosophila wing, expression of genes such as spalt (sal) and optomotor-blind (omb) is restricted along the A/P axis by lateral-to-medial gradients of the transcriptional repressor Brinker (Brk). sal is more sensitive to repression by Brk than omb, and thus has a narrower expression domain compared to omb. The Brk expression pattern is established by Decapentaplegic (Dpp), a TGF-b superfamily member, which forms a complementary medial-to-lateral morphogen gradient along the (A/P) axis of the wing. We have investigated the mechanisms Brk uses to repress gene expression to gain an understanding of why some targets such as sal are more sensitive to Brk than others such as omb. Previous studies have suggested that Brk represses gene expression simply by competing with activators, but we show that Brk requires an active repression domain along with the DNA binding domain for repressor activity. Brk possesses four repression domains, but these domains are not equivalent; for example, 3R is sufficient to repress omb but not sal. Consequently, although sal and omb show quantitative differences in their response to Brk, there are qualitative differences in the mechanisms that Brk uses to repress each of these genes.

The requirements of Hsp90 and Hsp70 cytoplasmic chaperone in the proper folding/degradation of an integral membrane protein remain poorly characterized, however it was previously demonstrated that the yeast Hsp70, Ssa1p, chaperone catalyzes the degradation of the misfolded human chloride channel, CFTR. To better define the roles of these chaperones and partner co-chaperones, I characterized the involvement of two Hsp70 co-chaperones, Ydj1p and Hlj1p, in the degradation of CFTR in the budding yeast S. cerevisiae. Mutations in the genes encoding Ydj1p or Hlj1p alone did not affect CFTR degradation, but disruption of both co-chaperones stabilized CFTR. In contrast, the degradation of a soluble misfolded protein (CPY*) was unaffected in an hlj1Δydj1-151 double mutant. Hlj1p stimulated the ATPase activity of Ssa1p and partially rescued the growth defect in a ydj1-151 strain, suggesting that Hlj1p and Ydj1p function redundantly during CFTR degradation. The contribution of Hsp90 to CFTR folding and degradation in mammalian cells has been examined, but disparate results have been obtained. I therefore analyzed CFTR degradation in yeast using a temperature sensitive Hsp90 mutant (Hsp90-G313N) and found that CFTR was degraded faster in the mutant compared to the wildtype. Consistent with this result, highly enriched yeast Hsp90 prevented the aggregation of CFTR's NBD1 domain. In contrast, the degradation of CPY* was unaffected in the Hsp90 mutant. Furthermore, I found no effect on CFTR degradation upon inactivation of the yeast Hsp90 co-chaperones Sba1p, Sti1p, or Sse1p. These results suggest that Hsp90, in the absence of co-chaperones, facilitates CFTR folding, possibly through its interaction with NBD1. Finally, I analyzed the effects of overexpressing two mammalian co-chaperones on CFTR biogenesis in yeast. I observed reduced CFTR degradation upon overexpression of FKBP8 or Bag-3 but did not observe enhanced trafficking of CFTR to the plasma membrane. This result suggests that stabilization per se is not sufficient to promote CFTR exit from the ER.

Advancements in phylogenetic theory and methodology coupled with improvements in computational and sequencing technology facilitate study of the divergence and diversification patterns of life. I apply our current understanding to further explore the relationships and evolution of the North American snake tribe Thamnophiini, as well as to address current topics in phylogenetic and taxonomic methodology. There are two paradigms for the phylogenetic analysis of multi-locus sequence data: one which forces all genes to share the same underlying history, and another that allows genes to follow idiosyncratic patterns of descent from ancestral alleles. The first of these approaches (concatenation) is a simplified model of the actual process of genome evolution while the second (species-tree methods) may be overly complex for histories characterized by long divergence times between cladogenesis. Rather than making an a priori determination concerning which of these phylogenetic models to apply to our data, I seek to provide a framework for choosing between concatenation and species-tree methods that treat genes as independently evolving lineages. In Chapter 2 I demonstrate that parametric bootstrapping can be used to assess the extent to which genealogical incongruence across loci can be attributed to phylogenetic estimation error, and demonstrate the application of our approach using an empirical dataset from 10 species of the Natricine snake sub-family. Since our data exhibit incongruence across loci that is clearly caused by a mixture of coalescent stochasticity and phyogenetic estimation error, we also develop an approach for choosing among species tree estimation methods that take gene trees as input and those that simultaneously estimate gene trees and species trees. Ideally, existing taxonomy would be consistent with phylogenetic estimates derived from rigorously analyzed data using appropriate methods. In Chapter 3 I present a multi-locus molecular analysis of the relationships among nine genera in the North American snake tribe Thamnophiini in order to test the monophyly of the crayfish snakes (genus Regina) and the earth snakes (genus Virginia). Sequence data from seven genes were analyzed to assess relationships among representatives of the nine genera by performing multi-locus phylogeny and species tree estimations, and we performed constraint-based tests of monophyly of classic taxonomic designations on a gene-by-gene basis. Estimates of species trees demonstrate that both genera are paraphyletic, and this inference is supported by a concatenated tree. This finding was supported using gene tree constraint tests and Bayes factors, where we rejected the monophyly of both the crayfish snakes (genus Regina) and the earth snakes (genus Virginia). Progress in our understanding of molecular evolution necessitates a more thorough assessment of the phylogeny of thamnophiine snakes, whose relationships have not been fully resolved, and whose previous phylogenetic estimates are based solely on mitochondrial sequence data. In Chapter 4, I present the most data and taxa robust phylogenetic estimate of Thamnophiini to date, including 50 taxa and sequence data from 8 independently sorting loci. Our findings support the taxonomic recommendations proposed in Chapter 3. Additionally, I estimated the timing of divergence among the three major lineages to have occurred during the Miocene period (~14-11MYA), with higher than expected diversification in the garter snaked during the Pliocene period (~2-6MYA). Finally, we demonstrate that prey choice is labile, and thus an unreliable character for phylogeny reconstruction. Combined, these chapters present a thorough examination of the molecular phylogenetics of thamnophiine snakes. The novel methodological approaches may serve as a guideline for future research. Through estimating a robust phylogeny and suggesting taxonomic changes where appropriate, this work provides a foundation for phylogenetically-based studies of this group.

Fire is an essential process for maintaining pine-dominated ecosystems in the southeastern United States. Fire opens space for pine recruitment, consumes fine fuels, stimulates flowering of herbaceous plants, and topkills woody plants. Topkill is the death of the aboveground portion of the plant, which does not necessarily lead to mortality. Most of the woody species in these ecosystems are able to resprout following topkill, sprouting new shoots from meristems at the base of the plant. In the absence of fire, pine-dominated ecosystems transition to closed canopy hardwood forests. Prescribed fires can be applied to mimic the historical fire regime, but the selected time since fire, season of burn, and ignition techniques used can influence both fire characteristics and the response of vegetation. In this dissertation, I explore the effects of prescribed fire treatments on fire characteristics and hardwood resprouting by applying prescribed fires to plots in a shortleaf pine-oak-hickory woodland in north Florida. Fireline intensity, residence times, and heat released per unit area increased progressively with longer time since fire treatment. Reaction intensity, on the other hand, decreased with time since fire, attributable to increased fuel bulk density. Fireline intensity was dampened by greater fuel moisture in the growing season and was greatest with head fire ignitions. I evaluated the response of hardwoods to prescribed fires using a path analysis model. The results of this model suggest that one-year post-fire biomass is mediated via the size and root reserves at the time of topkill, while the characteristics of the fire do not have an effect. I conducted a shadehouse experiment to test for the effects of season and method of topkill on hardwood genets. Genets topkilled in the growing season had reduced resprouting biomass compared to topkill in the dormant season. There was not a difference in biomass between burned and clipped plants. The results of this study suggest that the response of hardwoods to fire in savanna ecosystems is mediated by the size and resource allocation of plants rather than the intensity of fires.

Understanding mechanisms underlying distribution of biodiversity remains a central issue in ecology. I integrate ecological and phylogenetic information at multiple spatial scales to better understand neotropical primate distribution and community structure. I investigate the variation within species ranges in relation to species richness and patterns of species relatedness. Results suggest positive associations among species throughout their distributions, whereby species tend to present higher richness within their ranges than average richness for the entire taxon. However, comparing empirical distributions to a null model of range cohesion suggests mechanisms other than dispersal are setting a limit to the number of species capable of co-occurring throughout a species range. These differences in species associations across geographic ranges generate variation in local community composition. I analyzed the relative contribution of ecological, historical and spatial processes in determining taxonomic and phylogenetic community structure across 74 sites throughout the Neotropics. Spatial predictors explained most of the independent variation for taxonomic and phylogenetic metrics, suggesting spatial processes, such as dispersal limitation, are important determinants of community structure. Most of the contribution of environmental predictors was associated with spatial processes, evincing importance of environmental and spatial gradients in determining change in community structure. While the overall contributions of predictors were similar for taxonomic and phylogenetic metrics, analyses of phylogenetic metrics independently presented complex relationships. At local communities, niche differentiation is expected to allow species coexistence. However, these differences may reflect evolutionary constraints of species, rather than active selection. I investigated niche overlap and presence of niche conservatism for primate species at three communities. For the niche characteristics measured by my study, I found no significant differences in niches of closely related species within sites. However, when comparing niches across sites, significant differences were registered between populations of the same species or closely related species. These findings suggest ecological differentiation may be acting at large spatial scales promoting niche differentiation, while at local scales phylogenetic constraints may be a stronger driver of community structure. Overall, these results represent valuable insights regarding our understanding of mechanisms responsible for generating and maintaining community structure for a highly diverse tropical mammal radiation.

Parapineal precursors arise from the medially located pineal complex anlage and migrate to the left side of the brain. Published data implicates Fgf8a in the migration of parapineal cells away from the midline of the pineal anlage. However, the potential role for Fgf8a during the acquisition of parapineal cell fate was not addressed. We have found that Fgf8a regulates a fate decision among specified parapineal precursors that occurs just prior to the initiation of leftward migration. Attenuation of Fgf signaling results in the loss of parapineal cells and the gain of additional cone cells. Data obtained from the combined loss of Flh and Fgf8a, as well as cell fate analysis, shows that in the absence of Fgf signaling, parapineal precursors differentiate as cone photoreceptors rather than parapineal cells. Furthermore, Fgf8a acts permissively to promote parapineal fate in conjunction with the transcription factor Tbx2b, but acts by itself to either block cone photoreceptor fate or promote parapineal differentiation. This cell fate change is independent of Bmp signaling, which promotes the formation of pineal photoreceptors. Instead, parapineal cell differentiation likely requires the two transcription factors, Lhx2b and Lhx9, which are responsive to Fgf signaling and are involved in parapineal formation.

Our lab created ionizing radiation (IR) resistant populations of E. coli MG1655 through twenty cycles of exposure to IR and outgrowth. To identify the genetic alterations responsible for increased IR tolerance, a total of seven isolates from the evolved final population were re-sequenced. However, the individual isolates sequence analysis revealed wide variety of unique genomic alterations with little overlap between the seven isolates and therefore, did not reveal the clear picture of the mutations responsible for IR resistance. Our current research focused on distinguishing the relevant mutations from the non-relevant mutations that contribute to IR resistance by using two methods: population sequencing and TaqMAMA with the assumption that beneficial mutations evolve and accumulate in the final population. A 255X deep coverage sequencing of the genomic DNA from the IR resistant final population determined mutant allelic percentage in this population for each polymorphism detected. TaqMAMA assay acted as a tool for verifying the sequencing results. This assay also revealed two evolutionary phenomena: clonal interference and hitchhiking suggesting that not all polymorphisms seen in the final population could contribute to IR resistance. The analysis of population sequencing results along with the phylogenetic analysis reduced the number of potential candidates responsible of IR resistance in these isolates. These possible contributors enable at least three unique mechanisms in the seven isolates and the survival data revealed that the related isolates have same levels of IR resistance possibly from the same mechanism. The identified candidates were transferred into the parent strain alone or in combination and the resulting strains were tested for survivability against IR. This experiment identified the possible reasons of IR resistance in these isolates. In fact, a RecA modification has been determined to be a lone contributor for IR resistance in the four isolates. The methods used here, enabled us to separate relevant mutations that accumulated during evolution from non-relevant mutations that do not and thus helped to reduce the number of alleles possibly that could contribute to IR resistance. However, genetic characterization is required to confirm their role in IR resistance due to complexity that exists in allelic selection during evolution.