RNA Polymerase III (Pol III) is best characterized for its transcription of tRNA molecules. Interestingly, Pol III and its associated complexes have been found to be involved in the regulation of transcription of genes transcribed by RNA Polymerase II (Pol II) (Kleinschmidt, LeBlanc et al. 2011). Extra TFIIIC sites (ETC sites) are chromosomally located sites discovered in S. cerevisiae that bind the Pol III transcription factor TFIIIC only through B-box interactions. These chromosomal locations are not normally transcribed (Dieci and Sentenac 1996). One of the subunits of the TFIIIC complex, TFC6 has an ETC site (ETC6) in its promoter region (Simms, Dugas et al. 2008; Kleinschmidt, LeBlanc et al. 2011). The TFIIIC complex has been shown to directly regulate transcription from the TFC6 promoter. This is a unique example of autoregulation of a Pol II transcribed gene by a Pol III transcription factor. Mutating ETC6 in the TFC6 promoter shows that both it and a mutation in TFC3, results in increased transcription of TFC6. Both mutations inhibit TFIIIC binding to ETC6. TFIIIC binding to ETC6 is inversely proportional to TFC6 transcription levels. Thus, when Tfc6p was overexpressed, promoter activity was inhibited. Via stringent control on Tfc6p levels, this autoregulation is hypothesized to be involved in global regulation of tRNA gene expression and on global regulation of translation. The previous results from Kleinschmidt et al. 2011 demonstrated that TFIIIC binding is altered by overexpressing TFC6. To examine whether the varied expression of Tfc6p has a global effect on TFIIIC binding, and possibly Pol III transcription, we created a set of yeast strains with significant variation in Tfc6p expression: wild type, under-expressed, and two levels of over-expression (two-fold and ~12-fold). By performing a genome wide chromatin immunoprecipitation analysis of TFIIIC binding when TFC6 expression is mis-regulated using high throughput sequencing (ChIP-Seq) technology, we expect that different TFIIIC bound loci will show variations in the ChIP-Seq signals that will differ in magnitude. This analysis will allow us to asses how TFC6 mis-regulation effects tDNA transcription at those loci most sensitive to TFC6 mis-regulation, it may reveal cryptic ETC sites and reveal any changes in the extra-transcriptional functions of Pol III.

Encysted embryos of <i>Artemia franciscana</i> undergo a dramatic respiratory depression upon release from the adult female as they enter a state of hypometabolism termed diapause. The mechanisms by which such a respiratory depression is achieved remain unexplained. Evidence presented here shows that strategic enzymes involved in trehalose catabolism are inhibited during diapause, namely trehalase, hexokinase, pyruvate kinase and pyruvate dehydrogenase. Trehalose is the sole source of fuel in the embryos of <i>A. franciscana</i>, and hence downregulation of trehalose catabolism results in severe limitation of metabolic fuel available to the embryo during diapause. Western blot data demonstrates that pyruvate dehydrogenase becomes phosphorylated during entrance into diapause, and as a consequence, one would predict PDH to be strongly inhibited in this state. Restriction of glycolytic flux will lead to metabolic 'starvation' of the mitochondrion, and in turn will reduce mitochondrial oxidative phosphorylation during diapause. Measurements of ATP, ADP and AMP show that substantial decreases occur in ATP:ADP ratio and in adenylate energy charge during diapause. Respiration studies conducted with embryo lysates document a depression of oxidative phosphorylation during diapause in the case where substrates for respiratory complex I (pyruvate+malate) are used as the fuel source. Reduced respiration through complex I is corroborated by the increased phosphorylation of pyruvate dehydrogenase. When substrates for complexes I and II (pyruvate+malate+succinate) are added simultaneously, the increased electron flow through the electron transport system allows the detection of respiratory inhibition by the phosphorylation system (i.e., the F₁.F_o-ATP synthase, adenine nucleotide transporter, and phosphate transporter). This inhibition of the phosphorylation system is diminished as diapause lysates are diluted, which suggests the presence of an unidentified inhibitor. Finally, measurements of catalytic activity for respiratory complexes extracted from isolated mitochondria in the presence of phosphatase inhibitors reveal a minor decrease in complex I activity during diapause and a drop in activity of complex IV, the latter effect being minimized by COX excess capacity. Taken together, restriction of glycolytic carbon to the mitochondrion appears to the primary mechanism for the in vivo metabolic arrest in <i>A. franciscana</i> embryos during diapause, which is accentuated by inhibitions within the mitochondrion itself.

Disturbance is important in maintaining the open structure of savannas. Frequent fires (every 1-3 years) limit the growth of woody plants, and prevent the transition from savanna to forest by maintaining open space for grasses, herbs, and fire-tolerant longleaf pine. Fires are not the only disturbances in southeastern pine savannas, which also experience hurricanes. Hurricane winds mainly affect the overstory, causing treefalls, increasing light, and adding litter to the understory. Hurricane effects were documented after Hurricane Gustav in 2008. More trees fell where the overstory was present, and produced localized effects on the ground below. Increase in light transmittance was similar across different overstory treatments, and responded to scattered treefalls across wide areas of the study site. Defoliation also added pine needles to the groundcover. Increases in fuels may increase fire intensities following hurricanes. I hypothesized that pine needles would augment fires, and therefore decrease survival of woody plants in the understory. The presence of small amounts of pine needles beneath understory hickories led to greater temperatures during fire, longer durations of heating, and more complete combustion of hickory fuels. All hickories survived fire by resprouting, but when pine needles were absent most resprouted from buds aboveground along the stem, rather than from the belowground root crown. These stems may more quickly reach a size that can withstand fire. Therefore, understory woody plants should be larger, and more fire-resistant, away from overstory pines, such as in gaps created by hurricanes. I expected greater numbers and sizes of woody plants in plots where the overstory was experimentally removed. Woody stems were taller in gaps, but there was no increase in species or density. Instead, after the overstory remains absent for several decades, the number of species and density decline. Moreover, woody plants in gaps do not reach a size resistant to fire, and repeated fires continue to top-kill stems. Resprouting stems may be competing with other understory species. Furthermore, many woody plants are dispersed by birds beneath trees. Repeated fires, increased competition, and lack of recruitment may lead to a decline of woody plants in gaps, opening space for longleaf recruitment.

This work describes the crystallographic studies of two enzymes and provides mechanistic insights into their respective catalytic processes. The first study investigates the molecular basis of the fructose-2,6-bisphosphatase reaction of the inducible isoform of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). The bifunctional enzyme is solely responsible for the cellular concentration of a regulator of glucose metabolism, fructose-2,6-bisphosphate. PFKB3 was investigated using the crystal structures of the enzyme in a phospho-enzyme intermediate state (PFKFB3-PF-6-P), in a transition state-analogous complex (PFKFB3AlF4), and in a complex with pyrophosphate (PFKFB3PPi). With these structures, the structures of the Michaelis complex and the transition state were extrapolated. Additionally the C-terminal domain (residues 440-446) was rearranged in PFKFB3PPi, implying that this domain plays a critical role in binding of substrate to, and release of product from, the bisphosphatase catalytic pocket. These findings provide a new insight into the understanding of the phosphoryl transfer reaction. The second study investigates the molecular basis of the reaction catalyzed by the pseudo-glycosyltransferase, VldE. VldE catalyzes non-glycosidic C-N coupling between an unsaturated cyclitol and a saturated aminocyclitol with the conservation of the anomeric configuration to form validoxylamine A 7´-phosphate, the biosynthetic precursor of the antibiotic validamycin A. To study the molecular basis of its mechanism, the three-dimensional structures of VldE from Streptomyces hygroscopicus subsp. limoneus was determined in apo form, in complex with GDP, in complex with GDP and validoxylamine A 7´-phosphate, and in complex with GDP and trehalose. The structures of VldE with the catalytic site in both an open and closed conformation are also described. With these structures, the preferred binding of the guanine moiety by VldE, rather than the uracil moiety as seen in OtsA, could be explained. The elucidation of the VldE structure in complex with the entirety of its products provides insight into the internal return mechanism by which catalysis occurs with a net retention of the anomeric configuration of the donated cyclitol.

The aim of this dissertation is to characterize the physiological roles of E. coli YqjA and YghB, which belong to the ancient and conserved DedA protein family of hitherto unknown functions. YqjA and YghB are inner membrane (IM) proteins with 61% amino acid identity and share partially redundant functions. Such characterization is achieved by addressing the phenotypes exhibited by strain BC202 with in-frame deletions of yqjA and yghB. BC202 exhibits cell division defects, inability to grow at temperatures above 42°C and an altered phospholipid composition. The cell division defect of BC202 is attributed to the inefficient secretion of periplasmic amidases AmiA and AmiC by the twin arginine transport (Tat) pathway into the periplasm. This inefficient secretion by the Tat pathway is likely related to the loss of proton motive force (PMF) as demonstrated by fluorescence microscopy. In addition, several envelope stress response pathways are induced in BC202 under permissive growth conditions as demonstrated using lacZ fusions. The activation of the stress response pathways in BC202 is largely independent of its cell division and temperature sensitive phenotypes, and demonstrates compromised envelope integrity. The temperature sensitivity of BC202 is also related to the loss of PMF and is rescued by overexpression of the multidrug transporter MdfA or by growth in acidic media. The altered phospholipid composition of BC202 is unrelated to its cell division defect and temperature sensitivity and likely results from an adaptive response to compromised IM integrity. Together, these results suggest possible role(s) of YqjA/YghB in cellular PMF homeostasis and IM quality control either directly as proton transporters or indirectly as regulators of extant cellular mechanisms which participate in the homeostasis of PMF.

Ecologists and evolutionary biologists attempt to understand processes that determine distribution, abundance, and diversification of taxa through space and time. Genetic variation can provide insight into mechanisms governing the distribution and structure of natural populations, and population responses to environmental change, which in turn can have important consequences for the maintenance of species diversity. For my dissertation, I studied genetic variation of a seed-dispersing bat, Artibeus lituratus (Chiroptera: Phyllostomidae), in a heterogeneous landscape in South America, with the objective of understanding environmental and spatial forces responsible for the distribution and dynamics of populations. First, I designed thousands of primer pairs to amplify microsatellite loci for A. lituratus, for which no markers were previously available, and tested a subset on A. lituratus and 6 related phyllostomid species. I then used these data as tools to compare genetic structure in highly fragmented Alto Paraná Atlantic forest in eastern Paraguay to that in mostly contiguous forest in neighboring Misiones, Argentina. Results indicated weak genetic differentiation, and no significant correlation between genetic and geographic distance in fragmented or continuous forest. Comparison of empirical and simulated landscapes indicated empirical results were consistent with regular long-distance dispersal and high migration rates. I then determined unique and shared effects of forest configuration (measured at five spatial scales), environmental, and spatial factors on the distribution of genetic variation. Environmental factors reflecting tree composition, climate and forest structure strongly accounted for variation in genetic diversity, as did fragmentation measured at intermediate scales. Almost no variation in genetic structure was explained. Results were consistent with high levels of gene flow, which could be preventing or significantly delaying reduction in population connectivity, and strong influence of environment and fragmentation on genetic diversity, potentially mediated via population size. These findings indicated the importance of accounting for effects of multiple demographic processes, and at multiple spatial scales. My results advance our understanding of how populations respond to landscape alterations and natural environmental heterogeneity, and do so for a relatively poorly studied group of vertebrates, in a highly threatened ecosystem.

Volume is a proxy for biomass in hard bodied arthropods (P < 0.0001). I constructed biomass-volume equations for Neotropical (P < 0.0001), temperate (P < 0.0001) and 12 Neotropical genera of scarabs, of which 11 were highly significant. The effect of short term (< 1 year) alcohol storage on volume was negligable. I found scarab volume on geologically old soils in the Amazon was one-third that of the geologically young Amazon (P < 0.0001). Species richness (P = 0.0002), Chao 1 (P = 0.0003), Fishers  (P = 0.008) and Margalef indices (P = 0.0003) were greater on young soils. Menhinick (P = 0.54), Simpson (P = 0.54) and rarefied species richness (P = 0.24), which correct for sample size differences, indicated no difference in diversity. Young Amazonian soils likely support a higher volume and abundance of scarabs than old soils, but diversity across Amazonia may be similar. I compared beta-diversity of scarab communities across Amazonia. Species rank-abundance curves did not differ among sites whereas volume-abundance distributions and volume-rank abundance curves indicated a larger range of scarab sizes in Brazil than Ecuador. At local levels (<62 km), communities were similar whereas at large distances (>1750 km), they were different. Differences between Brazil and Ecuador may be explained by varied soil age and productivity. I examined the effects of tropical forest fragmentation on scarabs, comparing my results with three other studies from the same site. Among 1, 10, and 100-ha fragments and continuous forest, I found two important trends; beetle biomass and species richness increased with fragment area per sampling effort. Comparing all four studies, diversity increased with fragment size, and by my rarefaction analyses, two studies demonstrated beetle volume increased with fragment size. Average beetle size increased with fragment area, suggesting a shift in dung beetle guild structure and functionality. Tropical-temperate comparsions demonstrated a lower scarab species richness and larger average body size in temperate forests compared with tropical forests. Volume and abundance per trap-day did not segregate along these divisions. The observed latitudinal gradient may be explained by spatial heterogeneity hypotheses and the body size gradient resembles Bergmanns rule.

The gut of insects and plant tissues are productive sources for the isolation of undescribed species of yeasts. In particular, the gut of lignicolous insects is colonized by yeasts that can carry out the fermentation of several sugars. The consistent association between xylose-fermenting (X-F) yeasts and the gut of lignicolous insects has been used as evidence of a symbiotic relationship between them. In general passalid beetles (Passalidae) and wood-roaches (Cryptocercidae) feed on rotted wood where they spend most of their lives. Digestion of the substrate depends on the symbiotic microbiota, which include strict and facultative anaerobic microorganisms such as bacteria, parabasalids, and fungi. The objectives of this study were to describe the yeasts associated with hardwoods in Louisiana (Chapter 2), the gut of the wood roach Cryptocercus collected in the Appalachian Mountains (Chapter 3), passalids collected in Guatemala (Chapter 4) and in Thailand (Chapter 5), and to study selection acting on xylose reductase (XR) in yeasts (Chapter 6). This study confirmed the routine presence of ascomycete yeasts from the clades Scheffersomyces, Spathaspora, Lodderomyces, and Sugiyamaella in the guts of wood roaches and passalid beetles, as well as basidiomycete yeasts in the genera Cryptococcus and Trichosporon in passalids exclusively. In this investigation, four new X-F yeasts, Scheffersomyces illinoinensis, Sc. quercinus, Sc. virginianus, and Sc. cryptocercus, were proposed based on multilocus phylogenetic analyses, molecular, and biochemical characterization. The X-F yeasts in the Scheffersomyces clade were the most abundant species in the gut of both wood-roaches and Guatemalan passalids, results that support and expand the previously described relationship between X-F yeasts and lignicolous insects. This finding, however, was not observed in Thai passalids, where the most abundant yeasts were closely related to Candida insectamans (Spathaspora clade) that does not ferment xylose. In addition, this study determined that the gut of lignicolous insects is a niche rich in undescribed yeasts classified in several clades. The xylose reductase gene (XYL1) has been shown to be useful as a molecular marker for rapid identification of cryptic yeast species, and the xylose reductase enzyme (XR) has been exposed to purifying selection in ascomycete yeasts.

RNA interference (RNAi) is a phylogenetically conserved gene regulation mechanism that modulates a wide variety of biological functions through suppressing gene expression at transcriptional or posttranscriptional levels (Bass 2000; Sharp 2001). One of the major natural functions of RNAi is antiviral defense in cytosol. RNAi directed viral immunity (RDVI) targets viral transcripts for destruction using small interfering RNAs (siRNAs) processed from viral replication intermediates, in the form of double-stranded RNA (dsRNA), as sequence guide (Lu, Maduro et al. 2005). Accumulating evidence suggests RDVI in the nematode worm Caenorhabditis elegans begins with the biogenesis of virus-derived siRNAs (viRNAs) by DCR-1 (Bernstein, Caudy et al. 2001; Duchaine, Wohlschlegel et al. 2006), a type III ribonuclease, and RDE-4, a dsRNA binding protein (Grishok, Pasquinelli et al. 2001; Knight and Bass 2001). Efficient destruction of viral transcripts guided by viRNAs is then orchestrated by several host factors that form distinct classes. Some of the known host factors downstream of viRNA biogenesis include Argonaute proteins (e.g. RDE-1) (Tabara, Sarkissian et al. 1999; Hammond, Boettcher et al. 2001; Parrish and Fire 2001), RNA-dependent RNA polymerases (e.g. RRF-1) and putative RNA helicases (e.g. DRH-1) (Tabara, Sarkissian et al. 1999). To better understand worm RDVI, we have recently performed a genetic screen aiming to isolate novel host factors in the RDVI pathway. RSD-2 is one of our top candidates whose function in RDVI has been confirmed using corresponding genetic mutants in this study. RSD-2 is a novel protein that is not conserved in fungi, plants, insects or vertebrates (Tijsterman and Plasterk 2004). When the level of viral replication was accessed in double mutants that contain both the rsd-2 null allele and null allele corresponding to rde-1, rde-4 or drh-1, enhanced viral replication, as compared to respective single mutants, was observed. Since viRNAs can be readily detected in double mutants corresponding to rde-4;rsd-2, these observations together suggested that RSD-2 functions in a RDE-4-independent pathway for virus silencing. Since RSD-2 appears to be unique to the nematode kingdom, our study on the function and mechanism of RSD-2 may help reveal some unique features of the worm RDVI.

A multidisciplinary study of the factors affecting the potential use of chemical cues by three species of infaunal shrimp was performed in three parts. A set of modified y-maze choice experiments were performed to establish the ability of three species of ghost shrimp, Callichirus islangrande, Callichirus major, and Leptidophthalmus louisianensis, to detect conspecifics via chemical cue. The time budgets for the detection of conspecifics chemical cues differed significantly from controls (no cue) for both animal cues and odor only cues indicating that all three species were able to detect the difference in sex of conspecific chemical cues regardless of cue source. The differences between the trials in which the cue animal was present and odor cue alone was present may indicate the possible use of mechanical or ancillary cues in addition to odor. Odor plume behavior within the burrow environment was characterized using fluorescein dye visualization of an odor mimic in a model burrow for the second series of experiments. Combinations of slow and fast carrier and plume release rates were crossed with the following conditions: no shrimp mimic, 1 shrimp mimic (dye release source), and 2 shrimp mimics (one source and one 6 cm downstream). Release rate was more influential in determining plume structure and direction than carrier flow. Plumes retained their characteristics and did not become turbulent even in the presence of obstructions (shrimp mimcs). Communication between burrows was modeled using two porous burrow mimics built in natural sediment, 3.5 ml min-1 burrow effluent pumping rate, and 5 cm s-1 flow condition in a race track flume. Rhodamine-FWT effluent was tracked using fluorometery. The effect of burrow water density (natural and neutral) and pumping activity of the downstream burrow were crossed. A non-uniform zone of burrow water was established in the sediment surrounding the source burrow. Dilution of 1-2 orders of magnitude occurred between the effluent in the burrow and porewater, but 3-4 orders of magnitude in the surface sediments and downstream burrow. Density affected the concentration of effluent in the porewater around the source and downstream burrow. Pumping activity of the downstream burrow also affected dye distribution.