Recently, we discovered patients with inherited adenosine deaminase (ADA) deficiency are predisposed to pulmonary alveolar proteinosis (PAP). PAP is characterized by the accumulation of surfactant in the alveoli. To overcome ethical issues and limited patient samples, animal models are often utilized. Here, I investigated the lung abnormalities in ADA deficient (ADA -/-) mice, which suffer from severe hypoxia, till their death at 3 weeks. I hypothesized that, similar to ADA-deficient patients, ADA -/- mice demonstrate evidence of PAP. Indeed, electron microscopy showed thickening of type I cells, accumulation of apoptotic foamy alveolar macrophages, cholesterol and lipoproteinaceous material that is periodic-acid Schiff (PAS) positive and diagnostic of PAP. Moreover, the pulmonary abnormalities were corrected with supplementation of ADA. In conclusion, we demonstrated evidence of PAP in ADA -/- mice for the first time and their suitability to study pathogenesis of PAP in ADA deficiency.

Carboxyl esterases, particularly arylesterases, were predicted from 16 microbial genomes, and then expressed in E. coli. Of the more than 175 cloned genes, 86 were expressed in soluble form. These were screened for activity using a range of both commercial and natural substrates. Forty-eight proteins were active on pNP-acetate at pH 8 whereas 38 proteins did not exhibit any activity towards any substrates. Among the 48 active proteins, 20 proteins showed arylesterase activity. To date, 8 bacterial esterases and 2 archaeal arylesterases were characterized in terms of pH stability and optima, thermal inactivation, solvent stability, and kinetics. To our knowledge there is only one other published report of arylesterases from archaea. The synthetic capability of arylesterases can transform phenolic acids to value-added chemicals. Accordingly, this project provides an arsenal of industrially significant activities that can extend the antioxidant properties of lignin-derived molecules in a broader range of renewable products.

Food and nutrition are important for energy balance, reproduction and maintenance of health in all species. Drosophila melanogaster feed on yeast and sugar and food availability affects reproduction. In this thesis, I show that mating frequency and fertility are affected by the composition of food in two D. melanogaster wild-type strains, Canton-S and Oregon-R. Canton-S flies mate multiple times in the presence of yeast and sugar, while Oregon-R only remate in the presence of yeast. However, Oregon-R flies have higher fertility counts on all food types compared to Canton-S. These effects of food do not appear to depend on smell or taste, because both chemosensory mutants and artificial sweeteners tested fail to block the effects of food on reproduction. Moreover, Canton-S, but not Oregon-R flies show an interaction between food and group size. I conclude that genetic differences, social context and nutrition interact to regulate reproduction in flies.

Apolipoprotein B (ApoB) synthesis is partially regulated at the translational level; however, the molecular mechanisms that govern translational control of apoB mRNA remains largely unknown. We imaged intracellular apoB mRNA traffic and determined whether insulin silences apoB mRNA translation by trafficking into translationally-silenced cytoplasmic RNA granules called Processing Bodies (PBS). ApoB mRNA was visualized by using a strong interaction between the bacteriophage MS2 protein and a specific phage RNA sequence that binds MS2 protein. We observed a statistically significant increase in the localization of apoB mRNA into PBs, 4h, 8h, and 16h after insulin treatment. Conversely, acute insulin treatment (1h) did not show any significant effect. Insulin was also found to reduce polysomal association of apoB mRNA 4h and 16h post treatment in HepG2 cells. Overall, our data suggest that chronic insulin treatment silences apoB translation in HepG2 cells by localizing apoB mRNA into PBs and reducing translationally-competent mRNA pools.

Recently, we discovered patients with inherited adenosine deaminase (ADA) deficiency are predisposed to pulmonary alveolar proteinosis (PAP). PAP is characterized by the accumulation of surfactant in the alveoli. To overcome ethical issues and limited patient samples, animal models are often utilized. Here, I investigated the lung abnormalities in ADA deficient (ADA -/-) mice, which suffer from severe hypoxia, till their death at 3 weeks. I hypothesized that, similar to ADA-deficient patients, ADA -/- mice demonstrate evidence of PAP. Indeed, electron microscopy showed thickening of type I cells, accumulation of apoptotic foamy alveolar macrophages, cholesterol and lipoproteinaceous material that is periodic-acid Schiff (PAS) positive and diagnostic of PAP. Moreover, the pulmonary abnormalities were corrected with supplementation of ADA. In conclusion, we demonstrated evidence of PAP in ADA -/- mice for the first time and their suitability to study pathogenesis of PAP in ADA deficiency.

The transition from cross-fertilization to predominant self-fertilization is considered the most common evolutionary transition in flowering plants. This change in mating system has profound influences on the amounts and patterns of genetic diversity within and among populations, and on key genetic and demographic processes. The main goal of my thesis is to determine the molecular population genetic consequences of this transition in the annual neotropical aquatic plant Eichhornia paniculata (Pontederiaceae) using DNA sequence from individuals sampled from throughout the species’ geographic range. Populations exhibit a wide range of mating patterns associated the evolutionary breakdown of tristyly facilitating specific contrasts between outcrossing and selfing populations. Analysis of molecular variation supported the hypothesis of multiple origins of selfing, including the evolution of two morphologically distinct selfing variants from Central America and the Caribbean. A survey of 10 nuclear loci from 225 individuals sampled from 25 populations demonstrated the joint influence of mating system, population size and demographic bottlenecks in affecting patterns of nucleotide variation. Small selfing populations exhibited significantly lower genetic diversity compared with larger outcrossing and mixed mating populations. There was also evidence for higher population differentiation and a slower decay of linkage disequilibrium in predominately selfing populations from the Caribbean region. Coalescent simulations of the sequence data indicated a bottleneck associated with colonization of the Caribbean from Brazil ∼125,000 years ago. To investigate the consequences of transitions from outcrossing to selfing across the genome, I used high-throughput, short-read sequencing to assemble ~27,000 ESTs representing ∼24Mbp of sequence. Characterization of floral transcriptomes from this dataset identified 269 genes associated with floral development, 22 of which were differentially expressed in three independently derived selfing lineages compared to an outcrossing genotype. Evidence for relaxed selection in selfing lineages was obtained from an analysis of a subset of ~8000 orthologous sequences from each genotype, as predicted by theory. Selfing genomes showed an increase in the proportion of nonsynonymous to synonymous changes and relaxation of selection for codon usage bias. My thesis represents the most detailed investigation to date of the molecular population genetic consequences of intraspecific variation in the mating systems of plants.

The Hedgehog (Hh) signaling pathway has received attention regarding its important role in embryonic development, however the mechanism by which pathway regulators, such as Suppressor of fused (Sufu), Kinesin family member 7 (Kif7), and primary cilium, mediate Hh signaling transduction is not entirely understood. The work presented here examines the roles of Sufu and Kif7 in regulating Hh signaling in growth plate chondrocytes, as well as how they mediate parathyroid hormone-like hormone (Pthlh) signaling during chondrocyte development. I show here that Sufu and Kif7 are essential regulators of Indian hedgehog (Ihh) signaling. While Sufu negatively regulates Gli transcription factors, Kif7 functions both positively and negatively in chondrocytes. Kif7 plays a role in Sufu protein degradation and the exclusion of Sufu-Gli complexes from the primary cilium. Importantly, halving the dosage of Sufu restores normal Hh pathway activity and chondrocyte development in Kif7-null mice, demonstrating that the positive role of Kif7 is to restrict the inhibitory function of Sufu. Furthermore, Kif7 exerts inhibitory function on Gli transcriptional activity in chondrocytes when Sufu function is absent. Therefore, Kif7 regulates the activity of Gli transcription factors through both Sufu-dependent and Sufu-independent mechanisms. I show that Sufu is crucial for mediating the negative effect of Pthlh on Gli transcriptional activity and chondrocyte hypertrophic differentiation, whereas Kif7 and primary cilium are dispensable in this process. Although primary cilium is required for Hh ligand-mediated activation of Gli transcription, Pthlh negatively controls Gli transcriptional activity in a cilia-independent manner. The results of this work provide insight into how Hh signaling is regulated by Sufu and Kif7 in the context of primary cilium, but also suggest Sufu serves as an important link between Ihh and Pthlh signaling during growth plate chondrocyte development.

Sperm play a pivotal role in determining the reproductive success of individuals whose sperm must compete directly with that of others. I used sperm precedence assays and experimental evolution to examine the role of sperm traits in the reproductive success of hermaphrodites and males in the androdioecious nematode Caenorhabditis elegans. First, sperm size and the rate of reproduction were analyzed, in the context of male-male sperm competition, for evidence of natural heritable variation. Sperm size proved to be a strong indicator of second-male sperm precedence in the genotypes examined. Second, I tested the theoretically predicted effect of larval development time on the number of self-sperm produced by hermaphrodites. I demonstrated that a short larval development period favored the evolution of fewer sperm, inline with theoretical predictions. These results provide important insights into C. elegans reproductive biology and more generally to our understanding of the evolution of reproductive systems.

The transition from cross-fertilization to predominant self-fertilization is considered the most common evolutionary transition in flowering plants. This change in mating system has profound influences on the amounts and patterns of genetic diversity within and among populations, and on key genetic and demographic processes. The main goal of my thesis is to determine the molecular population genetic consequences of this transition in the annual neotropical aquatic plant Eichhornia paniculata (Pontederiaceae) using DNA sequence from individuals sampled from throughout the species’ geographic range. Populations exhibit a wide range of mating patterns associated the evolutionary breakdown of tristyly facilitating specific contrasts between outcrossing and selfing populations. Analysis of molecular variation supported the hypothesis of multiple origins of selfing, including the evolution of two morphologically distinct selfing variants from Central America and the Caribbean. A survey of 10 nuclear loci from 225 individuals sampled from 25 populations demonstrated the joint influence of mating system, population size and demographic bottlenecks in affecting patterns of nucleotide variation. Small selfing populations exhibited significantly lower genetic diversity compared with larger outcrossing and mixed mating populations. There was also evidence for higher population differentiation and a slower decay of linkage disequilibrium in predominately selfing populations from the Caribbean region. Coalescent simulations of the sequence data indicated a bottleneck associated with colonization of the Caribbean from Brazil ∼125,000 years ago. To investigate the consequences of transitions from outcrossing to selfing across the genome, I used high-throughput, short-read sequencing to assemble ~27,000 ESTs representing ∼24Mbp of sequence. Characterization of floral transcriptomes from this dataset identified 269 genes associated with floral development, 22 of which were differentially expressed in three independently derived selfing lineages compared to an outcrossing genotype. Evidence for relaxed selection in selfing lineages was obtained from an analysis of a subset of ~8000 orthologous sequences from each genotype, as predicted by theory. Selfing genomes showed an increase in the proportion of nonsynonymous to synonymous changes and relaxation of selection for codon usage bias. My thesis represents the most detailed investigation to date of the molecular population genetic consequences of intraspecific variation in the mating systems of plants.

Prior research has suggested that life style factors, such as diet and physical activity, influence prostate cancer (PCa) progression. Metformin intake has been shown to be associated with decreased cancer risk in type II diabetic patients. We hypothesize that a low carbohydrate diet, prolonged aerobic exercise and metformin treatment can all independently slow prostate tumor development and a combination regimen will have an additive benefit. We used LNCaP xenografts to test this hypothesis. Results revealed that a diet low in carbohydrate reduced food consumption and a combination with exercise significantly reduced animal body weights. Ten weeks of metformin did not significantly alter tumor growth rate compared to control animals. Ten weeks of exercise significantly inhibited tumor growth. Out results suggest that dietary carbohydrate alteration or the administration of metformin alone cannot significantly influence prostate tumor progression. A suitable sustained exercise regimen may offer a more protective effect against PCa progression.