<p> While it is interesting to examine the evolutionary history and phylogenetic relationship between species, for example, in a sort of "tree of life", there is also a great deal to be learned from examining population structure and relationships within species. A careful description of phylogenetic relationships within species provides insights into causes of phenotypic variation, including disease susceptibility. The better we are able to understand the patterns of genotypic variation within species, the better these populations may be used as models to identify causative variants and possible therapies, for example through targeted genome-wide association studies (GWAS). My thesis describes a model of local phylogenetic structure, how it can be effectively derived under various circumstances, and useful applications and visualizations of this model to aid genetic studies. </p><p> I introduce a method for discovering phylogenetic structure among individuals of a population by partitioning the genome into a minimal set of intervals within which there is no evidence of recombination. I describe two extensions of this basic method. The first allows it to be applied to heterozygous, in addition to homozygous, genotypes and the second makes it more robust to errors in the source genotypes. </p><p> I demonstrate the predictive power of my local phylogeny model using a novel method for genome-wide genotype imputation. This imputation method achieves very high accuracy&mdash;on the order of the accuracy rate in the sequencing technology&mdash;by imputing genotypes in regions of shared inheritance based on my local phylogenies. </p><p> Comparative genomic analysis within species can be greatly aided by appropriate visualization and analysis tools. I developed a framework for web-based visualization and analysis of multiple individuals within a species, with my model of local phylogeny providing the underlying structure. I will describe the utility of these tools and the applications for which they have found widespread use.</p>

<p> Regulation of gene expression is a fundamental biological process required to adapt the full set of hereditary information (i.e., the genome) to the varied environments that any organism encounters. Here, we elucidate two distinct forms of gene regulation &ndash; of endogenous genes by binding of transcription factors to information-containing genomic sequences and of selfish genes (&ldquo;transposons&rdquo;) by targeting of small RNAs to repetitive genomic sequences &ndash; using a wide array of approaches. </p><p> To study regulation by transcription factors, we used glucocorticoid receptor (GR), a hormone-activated, DNA-binding protein that controls inflammation, metabolism, stress responses and other physiological processes. <i>In vitro</i>, GR binds as an inverted dimer to two imperfectly palindromic &ldquo;half sites&rdquo; separated by a &ldquo;spacer&rdquo;. Moreover, GR binds different sequences with distinct conformations, as demonstrated by nuclear magnetic resonance spectroscopy (NMR) and other biophysical methods. </p><p> <i>In vivo</i>, GR employs different functional surfaces when regulating different genes. We investigated whether sequences bound by GR <i> in vivo</i> might be a composite of several motifs, each biased toward utilization of a particular pattern of functional surfaces of GR. Using microarrays and deep sequencing, we characterized gene expression and genomic occupancy by GR, with and without glucocorticoid treatment, of cells expressing GR alleles bearing differences in three known functional surfaces. We found a &ldquo;sub-motif&rdquo;, the GR &ldquo;half site&rdquo;, that relates to utilization of the dimerization interface and directs genomic binding by GR in a distinct conformation. </p><p> To study repression of tranposons, we characterized the production and function of small RNAs in the yeast <i>Cryptococcus neoformans</i>. We found that target transcripts are distinguished by suboptimal introns and inefficient splicing. We identified a complex, SCANR, required for synthesis of small RNAs and demonstrate that it physically associates with the spliceosome. We propose that recognition of gene products by SCANR is in kinetic competition with splicing, thereby further promoting small RNA production from target transcripts. </p><p> To achieve these results, we developed new bioinformatics tools: twobitreader, a small Python package for efficient extraction of genomic sequences; scripter, a flexible back-end for easily creating scripts and pipeline; and seriesoftubes, a pipeline built upon scripter for the analysis of deep sequencing data. </p>

<p> The Health Ontology Mapper (HOM) method is a proposed solution to the semantic gap problem. The HOM Method provides the following functionality to enable the scalable deployment of informatics systems involving data from multiple health systems. The HOM method allows a relatively small population of biomedical ontology experts to describe the interpretation and analysis of biomedical information collected at thousands of hospitals via a cloud based terminology server. As such the HOM Method is focused on the scalability of the human talent required for successful informatics projects. The HOM promotes a means of converting UML based medical data into OWL format via a cloud-based method of controlling the data loading process. HOM subscribes to a means of converting data into a HIPAA Limited Data Set format to lower the risk associated with developing large virtual data repositories. HOM also provides a means of allowing access to medical data over grid computing environments by translating all information via a centralized web-based terminology server technology. </p><p> An integrated data repository (IDR) containing aggregations of clinical, biomedical, economic, administrative, and public health data is a key component of research infrastructure, quality improvement and decision support. But most available medical data is encoded using standard data warehouse architecture that employs arbitrary data encoding standards, making queries across disparate repositories difficult. In response to these shortcomings the Health Ontology Mapper (HOM) translates terminologies into formal data encoding standards without altering the underlying source data. The HOM method promotes inter-institutional data sharing and research collaboration, and will ultimately lower the barrier to developing and using an IDR.</p>

<p>Centromeres are the chromosomal loci where microtubule spindles bind, via the kinetochore, during mitosis and meiosis. Paradoxically the centromere, as a functional unit, is essential to guarantee faithful chromosome segregation, whereas its underlying DNA sequences and associated kinetochore proteins are fast evolving. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. In spite of their importance, very little is known about the degree to which centromeric tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from species using publicly available genomic sequence and our own data. We found that despite an overall lack of sequence conservation, centromeric tandem repeats from diverse species showed similar modes of evolution. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond approximately 50 million years of divergence. In addition, we performed a survey of fungi genomes for the presence of high-copy tandem repeats, but found little evidence to suggest that high-copy centromeric repeats are a common feature feature in fungi, with the possible exception of the <i>Zygomycota</i>. phylum. Finally, in most species the kinetochore assembles at a single locus, but in some cases the kinetochore forms along the entire length of the chromosomes forming holocentric chromosomes. Following a literature review we estimate that holocentricity is very common and has evolved at least thirteen times.

<p> Members of the ANL superfamily of enzymes are involved in primary and secondary metabolism throughout all domains of life and identify key pathways that contribute to essential physiological reactions as well as defense mechanisms to evade competition. Specifically, acetyl-CoA synthetases are directly involved in energy metabolism, while NonRibosoaml Peptide Synthetases and some Aryl-CoA Ligases produce secondary natural products that confer virulence for the producing organism. Due to the ANL superfamily's ubiquitous involvement in primary and secondary metabolism, gaining an understanding of how these enzymes work and identifying ways to regulate them could provide an alternative route for antibiotic targets. It is well documented that domain alternation is paramount for the ANL superfamily of enzymes including the adenylation and thioester-forming reactions of NRPS adenylation domains. This thesis utilizes structural and functional analysis in conjunction with computational methods to further our understanding of these unique enzymes. </p><p> In chapter 2 we present the structure of an adenylation:Peptidyl Carrier Protein di-omain NRPS from the cryptic PA1221 biosynthetic operon from <i> Pseudomonas aeruginosa.</i> The PA1221 structure is the second example of an adenylation:PCP in the PDB and validates the chimeric fusion interactions of EntE-B. The similar interacting regions are between the 2^nd PCP helix and a helix in the N-terminal subdomain of the adenylation domain as well as the loop connecting the longest &beta;-strands of the C-terminal subdomains interacting with loop 1 of the PCP. </p><p> Chapter 3 presents the structure of an acetoacetatyl-CoA Synthetase that is a confirmed substrate for a protein acetyltransferase, PatA, for inactivation through acetylation of the catalytic A10 lysine. This <i>Streptomyces lividans</i> acetoacetyl-CoA synthetase is the first structure to fully resolve the loop connecting C-terminal extension helix to the C-terminal subdomain. The C-terminal extension is only present in ACS proteins revealing an interaction where the C-terminal extension stabilizes the dynamic P-loop in the adenylate forming conformation. </p><p> In chapter 4 we further explore the PA1221 operon by functionally identifying the substrate preference of PA1215, the hypothetical fatty-acyl-CoA Ligase, that is proposed to acylate the charge PCP of PA1221. We computationally validate the substrate preference with a homology model and AutoDock to gain insight into the proteins slow kinetics. We also provide further insight into the biochemistry of a subset of ANL superfamily members, the phenylacetic acid CoA ligases, involved in the utilization of aryl-carboxylic acids as a carbon source as well as the derivatization of penicillin. We analyze their unique dimeric structures identifying structural motifs that are contributed through the dimeric interface, but are otherwise located to different sides of the enzyme in a monomeric form. </p><p> Finally, to help identify how the protein moves between the two productive conformations we subject members of the superfamily to computational dynamic simulations including Anisotropic Network Modeling, Interpolative Elastic Network Modeling, all-atom molecular dynamics, and analyze the output from these methods with Principal Component and Normal Mode Analysis. We developed a method to visualize a dynamic reaction coordinate through measuring the Conformation Determining Angle (defined by structural motifs that are present in superfamily members) and use this metric to interrogate all ANL superfamily member PDB entries for domain organization. Finally, we test our hypothesis that domain alternation proceeds through an extended, open conformation with structural comparisons and MD. Here we report functional and structural analysis of ANL superfamily members that are related through bacterial cell metabolism and natural product biosynthesis.</p>

<p> Parkinson's disease is a degenerative neurological disease that often impacts older adults, leading to difficulties with transfers, posture, balance, and walking. Approximately half of those with Parkinson's disease develop the symptom of freezing of gait, a condition that makes initiating walking difficult. This leads to loss of independence, fear of falls, injuries, inactivity resulting in social isolation, and increased risk of osteoporosis. Pharmacological therapy provides relief to only some of the neurological symptoms in Parkinson's disease. However, gait, posture, balance, and freezing of gait obtain little benefit from drug treatments. </p><p> Research suggests visual, auditory, and vibratory cueing methods may improve some features of gait. However, visual cueing techniques often require modification of the environment, which is not possible in the community setting. Auditory cueing techniques can be executed with the use of headphones, but this can quickly become a problem, as it becomes a safety concern when it interferes with hearing environmental sounds. Studies of whole body vibration and studies of segment level vibration have demonstrated improvements to walking speed, step duration, step length, and gait variability. However, most studies have only focused on the immediate effects of vibration therapy. </p><p> To assess the impact of vibration as an intervention, we have designed the hard- ware, software, therapy protocol, and conducted a series of studies for this dissertation. The hardware is designed as a segment level device, which can be worn by a user and both provides the vibratory stimulation and measures parameters of gait. We call the device the PDShoe. Three force sensors are embedded into the insole the shoe worn by subjects. Two tactor actuators are placed between the shoe and subject's skin. Upon heel contact with the floor, a tactor placed at the heel begins to vibrate at a predetermined frequency and amplitude. Likewise, when the lateral metatarsal head or hallux of the foot contact the floor, a tactor placed over the metatarsal heads begins to vibrate. Vibration is only provided with the foot is in contact with the floor; this is step synchronized vibration. Data is transferred via a wireless network in real time to a host computer where it is stored for later analysis. Parameters of the vibratory stimulation can be set on the PDShoe from the host computer. Data analysis is performed in a numerical analysis environment, where the time series force data is cut into individual steps. Eight parameters of gait are extracted from each of these steps, providing mean and variability measures for each subject. </p><p> To test the efficacy of step synchronized vibration, we conducted two studies with healthy subjects and four studies with Parkinson's disease subjects. Accuracy and reliability of the device was established and included the use of change point analysis and correlation with the 10-meter Walk Test. Using the PDShoe system without vibratory feedback, we successfully characterized the gait of healthy persons over fifty years old. Subsequently we tested the feasibility, safety, and impact of our step synchronized vibration protocol with healthy subjects. Our protocol was easily implemented, well tolerated, and there were no adverse events. As was expected, no impact from the step synchronized vibration was seen with the healthy subjects. We then performed a feasibility and impact study with Parkinson's disease subjects. Again the protocol was easily implemented, well tolerated, and no adverse events were noted. A difference between pre- and post-intervention clinical scores of the Freezing of Gait Questionnaire, Berg Balance Scale, Timed Up and Go and gait measures of step and stance duration were found. Next, we conducted a clinical study with eight subjects at the All India Institute of Medical Sciences. This study demonstrated that subjects who exhibited the symptom of freezing of gait received the greatest benefit from the step synchronized vibration. These results informed a follow up study on seventeen subjects, all of whom had the symptom of freezing of gait. This last study demonstrated positive improvements in clinical measures of the Freezing of Gait Questionnaire, Berg Balance Scale, Timed Up and Go, Walking Speed, and gait characteristics of step duration, stance duration, swing duration, heel max force timing, and heel contact duration. There is encouraging evidence for further investigation; this data will be used to inform future studies. </p><p> These studies demonstrated the functionality, reliability, validity of the PDShoe device to measure characteristics of gait. Impact was demonstrated in three studies with Parkinson's disease subjects.</p>

<p> Somatic copy number alterations (SCNAs) are a class of alterations that lead to deviations from diploidy in developing and established tumors. A feature that distinguishes SCNAs from other alterations is their genomic footprint. The large genomic footprint of SCNAs in a typical cancer's genome presents both a challenge and an opportunity to find targetable vulnerabilities in cancer. Because a single event affects many genes, it is often challenging to identify the tumorigenic targets of SCNAs. Conversely, events that affect multiple genes may provide specific vulnerabilities through "bystander" genes, in addition to vulnerabilities directly associated with the targets. </p><p> We approached the goal of understanding how the structure of SCNAs may lead to dependency in two ways. To improve our understanding of how SCNAs promote tumor progression we analyzed the SCNAs in 4934 primary tumors in 11 common cancers collected by the Cancer Genome Atlas (TCGA). The scale of this dataset provided insights into the structure and patterns of SCNA, including purity and ploidy rates across disease, mechanistic forces shaping patterns of SCNA, regions undergoing significantly recurrent SCNAs, and correlations between SCNAs in regions implicated in cancer formation. </p><p> In a complementary approach, we integrating SCNA data and pooled RNAi screening data involving 11,000 genes across 86 cell lines to find non-driver genes whose partial loss led to increased sensitivity to RNAi suppression. We identified a new set of cancer specific vulnerabilities predicted by loss of non-driver genes, with the most significant gene being PSMC2, an obligate member of the 26S proteasome. Biochemically, we found that PSMC2 is in excess of cellular requirement in diploid cells, but becomes the stoichiometric limiting factor in proteasome formation after partial loss of this gene. </p><p> In summary, my work improved our understanding of the structure and patterns of SCNA, both informing how cancers develop and predicting novel cancer vulnerabilities. Our characterization of the SCNAs present across 5000 tumors uncovered novel structure in SCNAs and significant regions likely to contain driver genes. Through integrating SCNA data with the results of a functional genetic screen, we also uncovered a new set of vulnerabilities caused by unintended loss of non-driver genes.</p>

<p> Env7 is a vacuole membrane-localized protein kinase that is orthologous to the human serine/threonine protein kinase, STK16. It is evolutionarily well-conserved throughout Eukarya, and it has one ortholog in Bacteria. Phylogenetic analyses of sequences homologous to Env7 revealed clades that are inconsistent with established eukaryotic phylogeny, suggesting that both horizontal and vertical gene transmission are responsible for their conservation. Conserved amino acid residues and motifs that are potentially important to Env7's catalytic activity, localization, and interactions with other proteins were also identified and assessed. Additionally, one such conserved motif&mdash;the glycine-rich loop&mdash;was mutated in an effort to affect ATP binding in Env7. The phenotype resulting from this mutation was a slightly increased number of mutant cells exhibiting multi-lobed vacuoles under normal conditions.</p>

<p> A Framework for Comparative Analysis of Gene Expressions and Mutations Linked to Cancer Analysis of the aberrations occurring at the functional sites of genes and proteins is essential to understanding the genomic basis of human disease. There are many data sources that offer rich repository of information on sequence features, but their heterogeneity poses a challenge to developing an intuitive and high-confidence workflow for next-generation sequencing (NGS) data analysis. Moreover, the failure of existing repositories to incorporate results from both small-scale and large-scale studies has inhibited the identification of many novel non-synonymous single-nucleotide variations (nsSNVs). The HIVE (High-performance Integrated Virtual Environment) platform offers integrated and curated sources of nsSNVs and gene expression data from trusted genomic and proteomic repositories and publications. We demonstrate a data-driven functional genomics approach primarily leveraging the HIVE framework to identify priority targets for further investigation in the lab. Additionally, we developed the HIVE Genecast mobile app for Android devices that is annotated with our priority target results to provide scientists with access to gene sequence information while away from their workspaces.</p>

<p> It is estimated that at least 10-20% of the mammalian genome is dedicated towards regulating the 1-2% of the genome that codes for proteins. This non-coding, regulatory layer is a necessity for the development of complex organisms, but is poorly understood compared to the genetic code used to translate coding DNA into proteins. In this dissertation, I will discuss methods developed to better understand the gene regulatory layer. I begin, in Chapter 1, with a broad overview of gene regulation, motivation for studying it, the state of the art with a historically context and where to look forward.</p><p> In Chapter 2, I discuss a computational method developed to detect transcription factor (TF) complexes. The method compares co-occurring motif spacings in conserved versus unconserved regions of the human genome to detect evolutionarily constrained binding sites of rigid transcription factor (TF) complexes. Structural data were integrated to explore overlapping motif arrangements while ensuring physical plausibility of the TF complex. Using this approach, I predicted 422 physically realistic TF complex motifs at 18% false discovery rate (FDR). I found that the set of complexes is enriched in known TF complexes. Additionally, novel complexes were supported by chromatin immunoprecipitation sequencing (ChIP-seq) datasets. Analysis of the structural modeling revealed three cooperativity mechanisms and a tendency of TF pairs to synergize through overlapping binding to the same DNA base pairs in opposite grooves or strands. The TF complexes and associated binding site predictions are made available as a web resource at http://complex.stanford.edu.</p><p> Next, in Chapter 3, I discuss how gene enrichment analysis can be applied to genome-wide conserved binding sites to successfully infer regulatory functions for a given TF complex. A genomic screen predicted 732,568 combinatorial binding sites for 422 TF complex motifs. From these predictions, I inferred 2,440 functional roles, which are consistent with known functional roles of TF complexes. In these functional associations, I found interesting themes such as promiscuous partnering of TFs (such as ETS) in the same functional context (T cells). Additionally, functional enrichment identified two novel TF complex motifs associated with spinal cord patterning genes and mammary gland development genes, respectively. Based on these predictions, I discovered novel spinal cord patterning enhancers (5/9, 56% validation rate) and enhancers active in MCF7 cells (11/19, 53% validation rate). This set replete with thousands of additional predictions will serve as a powerful guide for future studies of regulatory patterns and their functional roles.</p><p> Then, in Chapter 4, I outline a method developed to predict disease susceptibility due to gene mis-regulation. The method interrogates ensembles of conserved binding sites of regulatory factors disrupted by an individual's variants and then looks for their most significant congregation next to a group of functionally related genes. Strikingly, when the method is applied to five different full human genomes, the top enriched function for each is reflective of their very different medical histories. These results suggest that erosion of gene regulation results in function specific mutation loads that manifest as disease predispositions in a familial lineage. Additionally, this aggregate analysis method addresses the problem that although many human diseases have a genetic component involving many loci, the majority of studies are statistically underpowered to isolate the many contributing loci.</p><p> Finally, I conclude in Chapter 5 with a summary of my findings throughout my research and future directions of research based on my findings.</p>