<p> Cancer is a leading cause of morbidity and mortality, and one in three individuals in the U.S. will be diagnosed with cancer in their lifetime. At the molecular level, cancer is driven by the activity of oncogenes and the loss of activity of tumor suppressors. The availability of genomic data from large sets of tumor tissue have facilitated the identification of subgroups of patients whose tumors share molecular patterns of expression. These molecular signatures, in turn, can help identify clinically-useful patient subgroups and inform potential therapeutic strategies against cancer.</p><p> In chapter 1, I review the current theories behind carcinogenesis, the molecular factors that regulate gene expression, and statistical methods for analyzing genomic data. In chapter 2, I describe an approach, termed oncomix, developed to identify oncogene candidates from expression data obtained from tumor and adjacent normal tissue. I apply oncomix to breast cancer expression data and identify an oncogene candidate, <i>CBX2</i>, whose expression is gained in a subset of breast tumors. <i>CBX2</i> is expressed at low levels in most normal adult tissue, and the CBX2 protein contains a drug-targetable chromodomain, both of which are desirable properties in a potential therapeutic target. We then provide the first experimental evidence that <i>CBX2</i> regulates the growth of breast cancer cells. In chapter 3, I develop a method for identifying nuclear hormone receptors whose expression is lost in endometrial cancers relative to normal tissue. I report, for the first time, that the loss of expression of Thyroid Hormone Receptor Beta (<i>THRB</i>) is associated with better 5-year survival in endometrial cancer. The loss of <i>THRB</i> expression is independent of the loss of estrogen and progesterone receptor expression, two genes whose loss of expression is known to be associated with poor survival. <i> THRB</i> expression could be considered as a biomarker to risk-stratify endometrial cancer patients. In Chapter 4, I develop a user-friendly application for visualizing chromosomal copy number state obtained from three types of copy number input in single cells &ndash; fluorescence in situ hybridization (FISH), spectral karyotyping (SKY), and whole genome sequencing (WGS). This web application, termed aneuvis, automatically creates novel visualizations and summary statistics from a set of user-uploaded files that contain chromosomal copy number information.</p><p> In this thesis, I develop new computational approaches for identifying candidate molecular regulators of cancer. I also develop a new user-friendly tool to enable biological researchers to identify aneuploidy and chromosomal instability within populations of single cells. Applying these tools to breast and endometrial cancer genomic datasets has highlighted novel aspects of breast and endometrial cancer biology and may inform novel therapeutic strategies based on molecular patterns of genomic heterogeneity. The freely available software developed as part of these projects has the potential to enable other researchers to advance our understanding of cancer genomics and to inform novel therapeutic strategies against cancer.</p><p>

A taxonomic revision of Tatera (Rodentia: Gerbillinae) from tha study area of north-east Africa and Asia is undertaken. The results are discussed in terms of the zoogeography and evolutionary history of the genus Tatera. Five species of Tatera are recognized in north-east Africa; Tatera robusta. T. nlgricauda. T. valida, T.boehmi and T. phillipsi. T. philllpsi isreinstated from the synonymy of T. robusta. T. minuscula from Ethiopia is considered a taxon incertae sedis. A single species, T. indica. is present in Asia. The taxonomic study Is based on the statistical analysis of morphometrlc data, the evaluation of morphological characters and karyological data where available. Intraspecific variation, both non-geographlc and geographic is analysed. Species and their geographical races are defined and their distributions mapped. A matrix of diagnostic characters is included. The palaeontological literature of Tatera is reviewed. The faunal similarities, both present and past, of Africa and Asia are discussed, as are the major climatic and geophysical changes which are known to have occurred in the study area during the late Tertiary and Quaternary. The present day ecological requirements of the relevant Tatera taxa are also assessed. It is concluded that the genus Tatera probably evolved in Africa in the Pliocene and subsequently dispersed into Asia in the Pleistocene, possibly by way of a southern Red Sea landbridge. Evidence cited, includes the presence of fossil Tatera in the Middle Pliocene deposits of East Africa; the occurrence of Asiatic faunal elements in the fossil record of eastern Ethiopia; the presence of subsaharan African mammals in isolated localities in the south-west of peninsular Arabia; sea-bed cores supporting the view that the south of the Red Sea was periodically dry and the absence, to date, of fossil Tatera from palaeontological sites in North Africa and the Near East.

<p> Electrophysiological behaviors in cardiomyocytes, such as the action potential and calcium transient, are emergent properties arising from the interaction of an ensemble of ion channels, transporters, and pumps. In this Thesis, I integrate mathematical modeling with experiments to gain new insight into cardiac electrophysiology. Cardiomyocyte models are probed using population-based parameter sensitivity analysis to comprehensively generate quantitative predictions of how key behaviors are determined by the levels of ion channels, transporters, and pumps. Experimental tests ground these predictions in reality and provide opportunities for model improvement when predictions differ from experiments. In Chapter 2, this approach was applied to the determinants of calcium transient amplitude in rat cardiomyocytes. Experiments validated the unexpectedly large predicted effect of the transient outward potassium current on calcium transient amplitude in epicardial cardiomyocytes, but others demonstrated that the sarco/endoplasmic reticulum calcium ATPase had a much larger impact than predicted. Further exploration revealed that model calcium fluxes were inaccurately balanced, which we corrected to yield an improved model accurately reflecting our experiments and previous reports. In Chapter 3, the determinants of action potential duration in guinea pig cardiomyocytes predicted by parameter sensitivity analysis were tested using dynamic clamp, which found generally larger experimental effect sizes than predicted. We adjusted the model using a genetic algorithm to match our results, which led us to show that the overly stable model action potential resulted from higher levels of the slow delayed rectifier current than in our experiments. Subsequent analysis revealed how this current more effectively stabilizes the action potential than a related current, the rapid delayed rectifier. Finally, in Chapter 4 I take a global approach to model analysis, exploring competing models of the rabbit cardiomyocyte by comparing patterns of variability and correlations between behaviors across a population of models with randomly varied parameters. This found key experimentally testable differences between the models, representing a novel potential method for assessing how well these mathematical models represent the electrophysiological system of these cells. Overall, this work adds to our understanding of cardiac electrophysiology and represents a potential new paradigm for combining modeling and experiments to understand complex behaviors.</p><p>

<p> The Lesser Sunda Archipelago, also known as Nusa Tenggara, lies in the southeastern portion of Indonesia and extends between Bali in the west, and New Guinea in the east. While the Lesser Sundas themselves are oceanic islands that have never been land bridged to a continent the islands on either side do. Bali and the other Greater Sunda Islands of Java, Sumatra, and Borneo become periodically land bridged with Asia during glacial maxima forming the Sunda Shelf. New Guinea and Aru become periodically land bridged to Australia during glacial maxima and form the Sahul Shelf. Given their current orientation, the Lesser Sundas may act as &lsquo;stepping stones&rsquo; for animals and plants dispersing between the Sunda and Sahul Shelves and may act as a two-way filter for organisms dispersing between two of the world&rsquo;s great biogeographical realms. Alfred Russel Wallace&rsquo;s discovery of a pattern of clinal mixture of species from different biogeographical realms was a key insight leading to his identification of the Wallace Line and to his creation of the field of biogeography. Even though the Lesser Sundas played a critical role in the development of the field, this region has received little subsequent attention from historical biogeographers and our current understanding of Lesser Sunda biogeography has only modestly improved relative to what was known at the time of Wallace. The reptiles and amphibians of the Lesser Sundas represent a particularly interesting group of vertebrates from a biogeographical standpoint because they appear to show distributional patterns that are most consistent with a stepping-stone model of island colonization caused by the two-way filter zone. In Chapter 1, I review the geological and biogeographical literature for the Lesser Sundas and use these sources to formulate hypotheses concerning the colonization of the archipelago by rafting terrestrial vertebrates. In Chapters 2 through 4, I investigate the possibility that flying lizards, forest skinks, and fanged frogs have colonized the archipelago in a stepping-stone manner using a phylogenomic approach (using sequence data from mtDNA and hundreds of nuclear loci) whereby the relationships among island-specific lineages can be used to infer the sequence of island colonization. Flying lizards of the genus <i>Draco</i> form a monophyletic group that colonized the western Inner Arc islands of Lombok or Sumbawa from the Sunda Shelf around 10 million years ago when Lombok and Sumbawa first became land-positive. <i> Draco</i> continued expanding eastward through the Inner Arc until they reached Lembata, while a series of dispersal events from Flores south to Sumba, east to Timor, north to Wetar, west to Alor, and finally west to Pantar (the island immediately west of Lembata). The islands of Sumbawa and Flores contain multiple non-sister lineages that are parapatrically distributed and are exchanging migrants within an island. Forest Skinks of the genus <i> Sphenomorphus</i> show relatively little morphological divergence across their range yet exhibit large levels of genetic divergence. The oldest lineages of <i>Sphenomorphus</i> within the Lesser Sundas occur on the islands of Lombok and Flores and they expanded eastward through the Inner Arc until they reached Pantar. But rather than reaching Alor from neighboring Pantar, <i> Sphenomorphus</i> dispersed from Flores south to Sumba, then east to Timor, Alor, and Wetar. There are multiple non-sister lineages of <i> Sphenomorphus</i> on Lombok, Flores, and Sumba, and estimates of migration between lineages within each island suggest that these lineages are not interbreeding. Fanged frogs of the genus <i>Limnonectes</i> have colonized the Inner Arc of the Lesser Sundas from the Sunda Shelf. It is possible that <i> Limnonectes kadarsani</i> and <i>L. dammermani</i> diverged <i> in situ</i> on Lombok after which <i>L. kadarsani</i> dispersed east all the way to Lembata. But rather although a tree topology consistent with a stepping-stone pattern of island colonization is suggested by the mtDNA data, the phylogenomic results suggest a leap-frog pattern where Lembata is derived from West Flores, and these two lineages are closer related to Sumbawa than they are to Eastern Flores. The parapatrically distributed lineages on Flores are experiencing asymmetrical gene flow with successful migrants moving from west to east. In summary, the oldest islands of the western Inner Arc tend to harbor the most divergent lineages for all three focal taxa, a pattern expected from lineages originating from the Sunda Shelf. In <i>Draco </i> and <i>Sphenomorphus,</i> the islands of the eastern Inner Banda Arc are colonized by way of the &lsquo;Sumba Route&rsquo; where they disperse into the Outer Banda Arc island of Sumba and then move east to Timor, and finally north into the eastern Inner Arc. All three focal taxa show multiple non-sister lineages on some of the larger islands, suggesting either that multiple colonization events of a single island occurred, or possibly that formerly separated paleo-islands have since merged allowing for secondary contact of lineages that diverged in allopatry. These studies have shown that the biogeography of reptiles and amphibians within the Lesser Sundas is extremely complex. By examining biogeographical patterns across many co-distributed taxa these studies have the potential to provide insights into the geological history of the archipelago. From an evolutionary perspective, these studies highlight the presence of multiple independently evolving lineages within a currently described species occurring on the same island, which suggests that species diversity within reptiles and amphibians of the Lesser Sundas is underestimated.</p>

<p> This study investigates the eIF4E family members in Dinoflagellates. Dinoflagellates are eukaryotic algae with large genomes and a minimal role for transcriptional regulation. All mRNA in dinoflagellates is <i>trans </i>-spliced with a 22-nucleotide 5'-spliced-leader sequence bearing a multi-methylated cap. Like other eukaryotes, dinoflagellates encode multiple eIF4E family members that are anticipated to fulfill a range of functions. Three distinct and novel clades of eIF4E have been recognized in dinoflagellates that are separate from the three metazoan classes of eIF4E. The dinoflagellate <i> Amphidinium carterae</i> encodes eight eIF4E family members while <i> Karlodinium veneficum</i> encodes fifteen eIF4E family members. I assayed six of these family members from <i>A. carterae</i> for expression levels, m⁷GTP binding, yeast knockout complementation and affinity for three mRNA cap analogs using surface plasmon resonance (SPR). Transcripts of each are expressed through a diel cycle, but only eIF4E-1 family members and eIF4E-2a are expressed at the level of protein. Recombinant eIF4E-1 family members and eIF4E-3a, but not eIF4E-2a, are able to bind to m⁷GTP-agarose beads. Of the clade 1 eIF4Es, only eIF4E-1a and -1d1 complement a S. cerevisiae strain conditionally deficient in functional eIF4E, consistent with their function as translation initiation factors. However, only eIF4E-1a can be recovered from <i>A. carterae</i> extracts by m⁷GTP affinity binding. Using SPR analysis, the affinity of <i>A. carterae</i> eIF4E-1a for m⁷GTP is lower than that of murine eIF4E-1A. By the same analysis, <i>A. carterae</i> eIF4E-1a has a higher affinity for m⁷GpppG than m⁷GTP. In addition, <i>K. veneficum</i> eIF4E-1a1 displays many of the same characteristics as <i>A. carterae</i> eIF4E-1a. Four eIF4E-1 and one eIF4E-2 family members from <i>K. veneficum</i> were characterized for m⁷GTP binding capacity, only the eIF4E-1 family members can be pulled down with m⁷GTP. Three eIF4E family members were tested for their ability to interact with a putative eIF4E-interacting protein, although none interacted. Overall, the eIF4E-1a sub-clade emerges with characteristics consistent with the role of a prototypical translation initiation factor. These initial analyses will allow for a better understanding of specific translational control of gene expression through mRNA recruitment in the unique dinoflagellate lineage.</p>

<p>The lack of vaccines for emerging and re-emerging diseases highlights technical gaps and indicates a need for innovative approaches to produce new vaccines. Vaccines may be improved by knowledge of host responses to vaccination, disease pathogenesis, and the effect of age and genetics on vaccine outcome. This study?s purpose was to quantitatively assess the molecular epidemiology of Francisella tularensis (Ft) and Venezuelan Equine Encephalitis Virus (VEEV). Study results support the Epidemiology Nexus model which holds that association of changes in gene expression to vaccination facilitate understanding the mechanisms of immune development and link public health and disease epidemiology. My research questions assessed the relationship between gene expression following vaccination, the relationship between age and vaccine response, and the association between Human Leukocyte Antigen (HLA) allele and vaccine response. The study was a novel secondary analysis of human data subjected to ANOVA to measure association between treatment and outcome, correlation to measure association of age with vaccine outcome, and Mann-Whitney U tests to measure association of HLA allele with vaccine outcome. Both Ft and VEEV vaccination elicited significant changes in gene expression. A highly positive relationship between age and vaccine outcome was shown for VEEV. The results may affect positive social change by contributing to a growing compendium of evidence of vaccine efficacy mechanisms that may function to assure the public of vaccine safety, combat vaccine hesitancy, and promote vaccine acceptance, as well as contribute mechanistic knowledge to reduce developmental costs of novel vaccines.

<p> The genus <i>Ophiocordyceps</i> contains the most diverse assemblage of fungi attacking ants worldwide and are remarkably well adapted to the specific ecologies of their hosts. <i>Desmidiospora myrmecophila</i> is closely related to ant-pathogenic species within <i>Ophiocordyceps</i>, possibly specific to queens, but the sheer infrequency of encounters and previously unsuccessful attempts to culture this fungus has precluded any meaningful assessment until now. A new record of <i>Desmidiospora myrmecophila</i> from Louisiana was found infecting a foundress <i>Camponotus pennsylvanicus </i> queen, the same host species favored by the more common and ubiquitous ant-pathogenic <i>Ophiocordyceps</i> unilateralis found in the same geographic locality. To evaluate a long-held assumption that these fungi represent synanamorphs of a single species, we sampled our Desmidiospora specimen along with the local <i>O. unilateralis</i> population for molecular comparison. We are able to present for the first time the <i>in vitro </i> characteristics and morphology of <i>Desmidiospora myrmecophila</i> as well as a phylogenetic context for this fungus based on combined molecular analysis of representative members of the Ophiocordycipitaceae. Our results place the <i>Desmidiospora myrmecophila</i> lineage within the genus <i>Ophiocordyceps</i> but with a basal affiliation to the ant-pathogen clade. These results further implicate <i>Desmidiospora myrmecophila</i> as an important and quintessential example of cryptic diversity among an already taxonomically diverse and ecologically important group of fungi.</p><p>

<p>North American <i>Speyeria </i>butterflies are a group whose species hypotheses are confounded by shared wing color patterns between sympatric populations of closely related recognized species due to a putatively recent origin in evolutionary time. Previous studies of this group and the closely related Palearctic genus <i>Argynnis</i>, suggest that <i>Speyeria </i>is monophyletic but derived from within <i>Argynnis</i>. Sampling in these studies has either involved few basal <i>Speyeria</i> species, or too few <i>Argynnis</i> species (Simonsen 2006, Simonsen <i>et al. </i>2006). Thus, no comprehensive phylogenetic analysis exists for all members that answers the question of monophyly of <i>Speyeria</i>, or other subgeneric taxa,and their relationship to <i>Argynnis </i>species. A phylogenetic analysis was completed of all North American <i>Speyeria </i>species and nearly all species within <i>Argynnis</i>, using one mitochondrial (CO1) and four nuclear genes (EF1?, WG, GAPDH, and RPS5). The results indicate that North American <i>Speyeria</i> is a monophyletic group, but that Palearctic <i>Argynnis</i> is paraphyletic. Three major lineages are identified within <i>Argynnis sensu lato</i>: two Palearctic and one containing both Palearctic and Nearctic species. <i>Argynnis</i> species representing the subgenera <i>Argyreus</i>, <i>Argyronome</i>, <i>Childrena</i>, <i>Damora</i>,<i> Pandoriana, </i>and <i>Nephargynnis</i>, belong to a well-supported lineage that split early in the evolution of the group and is comprised of species with long branches. <i>Fabriciana</i> and <i>Mesoacidalia</i> were both recovered as strongly supported lineages, except for <i>A. clara</i> which was recovered as sister to <i>Speyeria</i>. In summary, the phylogenetic analyses suggest the need for reorganization into three genera: <i>Argynnis</i>, <i>Fabriciana</i>, and <i>Speyeria</i>. The results have implications for the conservation of these butterflies across the temperate zone by providing a framework for understanding potential gene flow between sympatric species complexes, proper taxonomic validity, and the natural history of threatened populations of <i>Speyeria </i>and <i>Argynnis </i>butterflies.

<p>There continues to be a lack of agreement concerning the precise phylogenetic placement of Proconsul despite the wealth of fossil material and the extensiveness of its study. The difficulty in resolving the phylogenetic status of this important and well represented Miocene catarrhine is a consequence of its apparent basal position relative to crown catarrhines. This position complicates the inference of character polarities. This dissertation tests three previously proposed hypotheses concerning the phylogenetic position of Proconsul: (1) Proconsul is a stem catarrhine; (2) Proconsul is a stem hominoid; and (3) Proconsul is a basal hominid, most closely related to extant great apes and humans. A phylogenetic analysis based on 719 characters drawn from the skull, forelimb, pelvis and foot, and sampling a diversity of extant anthropoid taxa, offers no compelling support for a hominoid clade that includes Proconsul. The radiation of crown catarrhines involved rapid evolutionary changes from the ancestral catarrhine morphotype, resulting in stem catarrhines appearing much more similar to each other, even where there are key synapomorphies linking them with crown clades. As a result, systematic analyses alone are insufficient to confidently support a single optimal phylogenetic hypothesis. Further exploration of the data, by combining inferred ancestral morphotypes with phenetic visualizations of character evolution, demonstrated that inclusion of Proconsul among Hominoidea or Hominidae pushed the ancestral catarrhine morphotype closer to these clades, respectively. Given a more comprehensive analysis of character evolution under each hypothesis, this dissertation supports the hypothesis that Proconsul is a stem catarrhine. In addition to helping clarify the long-running debate about the phylogenetic status of Proconsul, the results offer fresh insights into the early stages of hominoid evolution and demonstrate the importance of comprehensive phylogenetic analyses in helping to resolve the relationships of problematic stem taxa.

<p> Cystic fibrosis (CF) is the most common lethal autosomal recessive genetic disorder that affects the Caucasian population. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR), and is characterized by a viscous airway surface liquid (ASL) that impairs mucociliary function and facilitates bacterial infection. The molecular mechanisms by which these symptoms result from CFTR malfunction are unclear. We hypothesized that expression and secretion of innate immune proteins is altered in CF ASL. </p><p> We sought to use cell culture models in which the only source of secreted proteins was differentiated airway epithelium. Since CFTR localizes to the apical surface of airway submucosal glands (SMG) and ciliated epithelium, cell culture models that recapitulate two parts of respiratory tract epithelium were studied: 1) SMG acini and 2) mucociliary epithelium. </p><p> We developed a three-dimensional system wherein CF (&Delta;F508/&Delta;F508) and non-CF human bronchial epithelial (HBE) cells differentiated on Matrigel into polarized glandular acini with mature lumens by two weeks with no significant variability in size. Bronchial acini expressed and secreted SMG proteins, MUC5B and lysozyme, at day 22, and exhibited vectorial secretions that were collected along with acinar cell lysates. Proteome profiling demonstrated unique protein signatures for each cellular space. However, abundant contaminating proteins from Matrigel and growth media were identified. Therefore, the ALI cell culture model of airway epithelium was chosen for quantitative proteomic comparison of CF and non-CF HBE apical secretions because the protein-rich media does not contact the apical surface. </p><p> CF and non-CF HBE cells were labeled by stable isotope labeling with amino acids in cell culture and differentiated at ALI. LC-MS/MS and bioinformatic analysis identified seventy-one proteins with altered levels in CF secretions (+/&minus;1.5 fold-change; p-value&lt;0.05). Validation with antibody based biochemical assays demonstrated increased levels of MUC5AC, MUC5B, fibronectin and MMP9, and increased proteolysis/activation of complement C3, in CF secretions. Overall, the function of altered proteins in the CF secretome is indicative of an airway epithelium in a state of repair and altered immunity in the absence of infection, suggesting the downstream consequences of mutated CFTR in CF airways set the stage for chronic inflammation and infection.</p>