The long-term success of transplantation is limited by the need for immunosuppression; thus, tolerance induction is an important therapeutic goal. A 16-day treatment with rapamycin in mice led to indefinite graft survival of fully mismatched cardiac allografts, whereas untreated hearts were rejected after 8-10 days. Specific tolerance was confirmed through subsequent skin grafts and in vitro lymphocyte assays that showed recipient mice remained immunocompetent towards 3rd party antigens but were impaired in responding to donor antigens. Mechanisms that account for this tolerant state were then investigated. Splenic CD8+CD44+ memory T-cells were reduced in tolerant mice but had increased frequencies of the CD62LLO population. CD4+CD25+Foxp3+ regulatory T-cells were increased in tolerant mice. Through multiplex PCR, 4 regulatory T-cell related genes were found up-regulated and 2 proinflammatory genes were down-regulated in accepted hearts. This expression pattern may serve as a putative biomarker of tolerance in patients undergoing transplantation.

Natural compounds have been largely excluded from characterization via high-throughput profiling strategies due to their limited abundance. Herein, I describe the modification of high-throughput yeast chemical genomic (CG) interaction profiling to permit identifying the modes of action of natural compounds. The previous assay proceeded by evaluating the genome-wide yeast deletion collection for drug-hypersensitivity in a volume of 0.7mL. Compound consumption was minimized with the adapted approach by reducing the assay volume 70% through simplifying the complexity of the yeast deletion pool screened. By recreating each yeast mutant in a drug-hypersensitive background, I created a novel resource that increases compound efficiency and further diminishes compound use. Evaluating a series of characterized compounds analyzed previously by the traditional CG approach validated the adaptations incorporated did not negatively affect the quality of data yielded. Ultimately, this modified strategy will be used to screen thousands of natural compounds contained within the RIKEN NPDepo library.

Unlike many amphibians, the paedomorphic axolotl (Ambystoma mexicanum) rarely undergoes external morphological changes indicative of metamorphosis. However, internally, some axolotl tissues undergo cryptic metamorphic changes. A previous study examined interspecific patterns of larval brain gene expression and found that these species exhibited unique temporal expression patterns that were hypothesized to be morph specific. This thesis tested this hypothesis by examining differences in brain gene expression between juvenile (JUV), paedomorphic (PAED), and metamorphic (MET) axolotls. I identified 828 genes that were expressed differently between JUV, PAED, and MET. Expression estimates from JUV were compared to estimates from PAED and MET brains to identify genes that changed significantly during development. Genes that showed statistically equivalent expression changes across MET and PAED brains provide a glimpse at aging and maturation in an amphibian. The genes that showed statistically different expression estimates between metamorphic and paedomorphic brains provide new functional insights into the maintenance and regulation of paedomorphosis. For genes that were not commonly regulated due to aging, paedomorphs exhibited greater transcriptional similarity to juvenile than metamorphs did to juvenile. Overall, gene expression differences between metamorphic and paedomorphic development exhibit a mosaic pattern of expression as a function of aging and metamorphosis in axolotls.

Streptococcus agalactiae is a significant pathogen causing invasive disease in neonates and thus an understanding of the molecular basis of the pathogenicity of this organism is of importance. N-terminal lipidation is a major mechanism by which bacteria can tether proteins to membranes. Lipidation is directed by the presence of a cysteine-containing lsquolipoboxrsquo within specific signal peptides and this feature has greatly facilitated the bioinformatic identification of putative lipoproteins. We have designed previously a taxon-specific pattern (G+LPP) for the identification of Gram-positive bacterial lipoproteins, based on the signal peptides of experimentally verified lipoproteins (Sutcliffe I.C. and Harrington D.J. Microbiology 148: 2065¿2077). Patterns searches with this pattern and other bioinformatic methods have been used to identify putative lipoproteins in the recently published genomes of S. agalactiae strains 2603/V and NEM316. A core of 39 common putative lipoproteins was identified, along with 5 putative lipoproteins unique to strain 2603/V and 2 putative lipoproteins unique to strain NEM316. Thus putative lipoproteins represent ca. 2% of the S. agalactiae proteome. As in other Gram-positive bacteria, the largest functional category of S. agalactiae lipoproteins is that predicted to comprise of substrate binding proteins of ABC transport systems. Other roles include lipoproteins that appear to participate in adhesion (including the previously characterised Lmb protein), protein export and folding, enzymes and several species-specific proteins of unknown function. These data suggest lipoproteins may have significant roles that influence the virulence of this important pathogen.

This work investigates two distinct roles of lysine deacetylases (KDACs) in the budding yeast Saccharomyces cerevisiae. The first part focused on the classical, well characterized role of KDACs as transcriptional regulators and deciphering their role in G1 transcription. I show that two yeast KDACs, Rpd3 and Hos3 are recruited to G1 promoters through their interactions with the negative regulator Whi5 and that these KDACs are necessary for proper Whi5-mediated repression. The second part examines a newly discovered role for KDACs extending their role beyond the chromatin as modifiers of proteins other than the histones. I present here the first systematic approach that comprehensively examines these non-histone targets of KDACs in vivo. I identified 73 non-histone proteins acetylated in vivo involved in diverse cellular processes. Swi4, a component of the G1 transcription factor SBF, was identified in the Rpd3 screen and I show that the interaction between Swi4 and its heterodimeric partner Swi6 was regulated by acetylation. My findings significantly expand the scope of the yeast acetylome and demonstrate the utility of systematic functional genomic screens to explore enzymatic pathways.

All cellular processes depend on the coordinate expression of genes and their interactions. Regulatory sequences encoded in the genome stipulate the necessary instructions interpreted by sequence-specific transcription factors (TFs) to control the spatial-temporal output of gene expression. Detection of cis-regulatory signals is challenging, owing to the lack of distinguishing features such as open reading frames and an overwhelming excess of spurious to functional TF binding site matching sequences embedded within the vast non-coding regions of vertebrate genomes. From an evolutionary standpoint, functional alterations in cis-regulatory architecture are thought to be important in diversifying morphology and physiology in the evolution of vertebrates, which share a similar body plan and complement of genes. Correspondingly, recent studies have highlighted the plasticity of cis-regulatory architecture organization over evolutionary time, finding associations with examples of both diverged and conserved patterns of gene expression. These observations underscore the gap in our collective knowledge with respect to the rules by which TFs recognize and bind their targets in vivo, as well as how this process evolves in vertebrates, and serve as a motivating basis for this thesis work. To begin, I probed the extent of conservation and divergence of sequence and expression profiles across tissues of diverse vertebrate species, identifying thousands of candidate genes with conserved expression by microarray analysis. However, corresponding conservation of non-exonic and potentially regulatory sequence was lacking, suggestive of binding site turnover over evolutionary time. Next, I analyzed the sequence specificity of a wide array of mouse and yeast TFs, finding great diversity and complexity in their binding preferences, with many factors recognizing multiple distinct motifs. Furthermore, comparative analysis of orthologous TFs suggest well conserved binding specificities. I also demonstrate the likely biological relevance of sequences highly preferred by these TFs by revealing distinctive signatures in their distribution and organization within putative regulatory regions in each genome. Lastly, I have begun to explore the organization of cis-regulatory sequences active in vertebrate tissues by high-throughput sequencing of open chromatin. Together, these data help illuminate the organization and evolution of vertebrate regulatory architectures, providing a useful toolkit for the testing of new models and hypotheses.

This work investigates two distinct roles of lysine deacetylases (KDACs) in the budding yeast Saccharomyces cerevisiae. The first part focused on the classical, well characterized role of KDACs as transcriptional regulators and deciphering their role in G1 transcription. I show that two yeast KDACs, Rpd3 and Hos3 are recruited to G1 promoters through their interactions with the negative regulator Whi5 and that these KDACs are necessary for proper Whi5-mediated repression. The second part examines a newly discovered role for KDACs extending their role beyond the chromatin as modifiers of proteins other than the histones. I present here the first systematic approach that comprehensively examines these non-histone targets of KDACs in vivo. I identified 73 non-histone proteins acetylated in vivo involved in diverse cellular processes. Swi4, a component of the G1 transcription factor SBF, was identified in the Rpd3 screen and I show that the interaction between Swi4 and its heterodimeric partner Swi6 was regulated by acetylation. My findings significantly expand the scope of the yeast acetylome and demonstrate the utility of systematic functional genomic screens to explore enzymatic pathways.

Comparative analysis of genomic data investigates the relationship of genome structure and function across different biological species to shed light on their similarities and differences. In this dissertation, we study two important problems in comparative genomics, namely comparative sequence analysis and comparative network analysis. In the comparative sequence analysis, we study the multiple sequence alignment of protein and DNA sequences as well as the structural alignment of multiple RNA sequences. For closely related sequences, multiple sequence alignment can be efficiently performed through progressive techniques. However, for divergent sequences it is very challenging to predict an accurate alignment. Here, we introduce PicXAA, an efficient non-progressive technique for multiple protein and DNA sequence alignment. We also further extend PicXAA to PicXAA-R for structural alignment of RNA sequences. PicXAA and PicXAA-R greedily build up the alignment from sequence regions with high local similarity, thereby yielding an accurate global alignment that effectively captures local similarities among sequences. As another important research area in comparative genomics, we also investigate the comparative network analysis problem. Translation of increasing number of large-scale biological networks into meaningful biological insights requires efficient computational techniques. One such example is network querying, which aims to identify subnetwork regions in a large target network that are similar to a given query network. Here, we introduce an efficient algorithm for querying large-scale biological networks, called RESQUE. RESQUE adopts a semi-Markov random walk model to probabilistically estimate the correspondence scores between nodes that belong to different networks. The target network is iteratively reduced based on the estimated correspondence scores until the best matching subnetwork emerges. The proposed network querying scheme is computationally efficient, can handle any network query with an arbitrary topology, and yields accurate querying results. We also extend the idea used in RESQUE to develop an efficient algorithm for alignment of multiple large-scale biological networks, called SMETANA. SMETANA outperforms state-of- the-art network alignment techniques, in terms of both computational efficiency and alignment accuracy. The accomplished studies have enabled us to provide a coherent framework for probabilistic approach to comparative analysis of biological sequences and networks. Such a probabilistic framework helps us employ rigorous mathematical schemes to find accurate and efficient solutions to these problems.

Loblolly pine (Pinus taeda L.) is an ecologically and economically important southern pine, distributed across the southeastern United States. Its genetic improvement for breeding and deployment is a major goal of the Western Gulf Forest Tree Improvement Program (WGFTIP) hosted by the Texas A&M Forest Service. Rapid advances in genomics and molecular marker technology have created potential for application of Marker Assisted Selection (MAS) and Genomic Selection (GS) for accelerated breeding in forest trees. First-generation selection (FGS) and second- generation selection (SGS) breeding populations of loblolly pine from east Texas were studied to estimate the genetic diversity, population structure, linkage disequilibrium (LD), signatures of selection and association of breeding traits with genetic markers using a genome-wide panel of 4264 single nucleotide polymorphisms (SNPs). Under- standing the genetic basis of local adaptation is crucial to disentangle the dynamics of gene flow, drift and selection and to address climate change. Bayesian mixed linear models and logistic regression were used to associate SNP variation with geography, climate, aridity and growth season length and markers with strong correlations were investigated for biological functions. Relatively high levels of observed (Ho = 0.178–0.198) and expected (He = 0.180-0.198) heterozygosities were found in all populations. The amount of inbreeding was very low, and many populations exhibited a slight excess of heterozygotes. The population substructure was weak, but FST indicated more pronounced differentiation in the SGS populations. As expected for outcrossing natural populations, the genome-wide LD was low, but marker density was insufficient to deduce the decay rate. Numerous associations were found between various phenotypes and SNPs, but few remained significant after false positive correction. Signatures of diversifying and balancing selection were found in markers representing important biological functions. Strong correlations supported by Bayes factors were found between various environmental variables and several SNPs. Logistic regression found hundreds of significant marker-environment associations, but none remained significant after false-positive correction, which was likely too stringent and will require further investigation. Annotations of significant markers implicated them in crucial biological functions. These results present the first step in the application of MAS to the WGFTIP for loblolly pine genetic improvement and will contribute to the knowledgebase necessary for genomic selection technology. Results from environmental association study provide important information for designing breeding strategies to address climate change and for genetic conservation purposes.

The integrity of the genome is critical to organisms and it is affected by many factors. Radiation, for example, poses a serious threat to genome stability of human beings. While physical monitors for radiation hazard are present, the biological consequences of long term exposure to radiation are not well understood. With the opportunity as part of the International Caenorhabditis elegans Experiment-1 flight project, several approaches using C. elegans were taken to measure mutational changes that occurred during the spaceflight. Among these methods, the eT1 balancer system was demonstrated to be well-suited as an integrating biological dosimeter for spaceflight. The dog-1 gene in C. elegans is required to prevent mutations at poly-G/poly-C tracts, and previous work has described that in the absence of DOG-1, small deletions initiate within these tracts, most likely as a consequence of improperly repaired replication blocks. The eT1 balancer system was adapted to investigate the broad mutational spectrum of dog-1 mutants. Using this system, I was able to determine a forward mutation rate of approximately 1 x 10-3, 10 fold higher than spontaneous. Both small deletions as reported previously and unreported large chromosome rearrangements were observed, and most of mutations analyzed are associated with G/C tracts. Thus, I propose that following dog-1-induced replication blocks, repair leads to a wide range of mutational events and chromosomal instabilities, similar to those seen in human cancers. The existence of the G/C tracts in C. elegans creates a fortuitous but perplexing problem. They are hotspots for genome instability and need enzymatic protection. In the genome of C. elegans, approximately 400 G/C tracts exist and are distributed along every chromosome in a non-random pattern. G/C tracts are also over-represented in another Caenorhabditis species, C. briggsae. However, the positions and distribution differ from those in C. elegans. Furthermore, in C. elegans, analysis of SAGE data showed that the position of the G/C tracts correlated with the level of gene expression. Although being a threat to genome stability, the genomic distribution of G/C tracts in C. elegans and their effect on regional transcription levels suggest a role for G/C tracts in chromatin structure.